Cell Biology

I. NUCLEAR STRUCTURES

A. Nuclear envelope

1. The inner membrane is associated with a network of intermediate filaments (lamins A, B, C) called the nuclear lamina, which plays a role in the re-assembly of the nuclear envelope during telophase of mitosis. The outer membrane is studded with ribosomes and is continuous with the rough endoplasmic reticulum (rER).

2. The inner and outer membranes are separated by a perinuclear cisterna.

3. The nuclear pore complex consists of many different proteins arranged in octagonal symmetry with a central channel. The nuclear pore complex allows passage of molecules between the nucleus and cytoplasm (Table 1-1).

B. Chromatin is double-helical DNA associated with histones and nonhistone proteins.

1. Heterochromatin is condensed chromatin and is transcriptionally inactive. In electron micrographs, heterochromatin is electron dense. An example of heterochromatin is the Barr body, which is found in female cells and represents the inactive X chromosome.

2. Euchromatin is dispersed chromatin and is transcriptionally active.

3. A nucleosome consists of DNA coiled around histones H2A, H2B, H3, and H4, forming an 11-nm-diameter chromatin fiber. A nucleosome that has a "beads-on-a-string" appearance is the basic unit of chromatin packaging. These 11 -nm chromatin fibers can be packaged together into 30-nm chromatin fibers by histone HI.

C. Chromosomes contain some specialized nucleotide sequences.

1. Centromeres are nucleotide sequences that mark the primary constriction along the chromosome. Protein complexes called kinetochores assemble at the centromere and bind microtubules of the mitotic spindle during mitosis.

2. Telomeres are nucleotide sequences (GGGTl A) located at the end of a chromosome that allow replication of DNA to its full length using the enzyme called telomerase.

3. The replication origin is a nucleotide sequence that serves as an origination site of chromosome replication. Human chromosomes contain numerous replication origins to ensure rapid replication. In humans, DNA polymerase « and 8 catalyze DNA replication. Other DNA polymerases exist within the cell; namely DNA

Table 1-1

Molecular Transpon Between Nucleus and Cytoplasm

Direction of Movement

Mechanism

Ions

Small molecules (< 5000 d) Proteins (< 60,000 d)

mRNA

tRNA

rRNA

Proteins (> 60,000 d) such as nucleoplasms, steroid receptors, DNA and RNA polymerases, gene regulatory proteins, RNA-process-ing proteins

Nucleus cytoplasm Nucleus cytoplasm

Nucleus —> cytoplasm

Nucleus cytoplasm

Passive transport (diffusion) No ATP hydrolysis

Active transport Requires ATP hydrolysis Requires binding of RNA to proteins with a signal sequence of 4-8 amino acids for recognition by the nuclear pore complex

Active transport Requires ATP hydrolysis Requires a signal sequence of 4-8 amino acids for recognition by the nuclear pore complex

ATP = adenosine triphosphate; mRNA = messenger RNA; rRNA = ribosomal RNA; tRNA = transfer RNA.

polymerase (i and e, which catalyze DNA repair, and DNA polymerase y, which catalyzes mitochondrial DNA replication.

D. The nucleolus consists of portions of five pairs of chromosomes (i.e., 13, 14, 15, 21, and 22) that contain genes that code for ribosomal RNA (rRNA). In humans, RNA polymerase I catalyzes the formation of rRNA. Other RNA polymerases exist within the cell; namely RNA polymerase II, which catalyzes the formation of messenger RNA (mRNA), and RNA polymerase III, which catalyzes the formation of transfer RNA (rRNA). By electron microscopy, three regions of the nucleolus can be distinguished.

1. The fibrillar center is pale-staining and contains transcriptionally inactive DNA.

2. The dense fibrillar component contains rRNA in rhe process of being synthesized.

3. The granular component contains rRNA hound to ribosomal proteins beginning to mature into ribosomes.

II. CYTOPLASM contains enzymes for glycolysis, fatty acid synthesis (i.e., fatty acid synthase), three reactions of the urea cycle (using argininosuccinate synthetase, argininosuc-cinate lyase, and arginase), glycogen synthesis and degradation, and protein synthesis, as well as intermediates of metabolism and many cofactors.

III. CYTOPLASMIC STRUCTURES

A. Ribosomes

1. Ribosomes consist of 40S (small) and 60S (large) subunits containing rRNA and various proteins (Table 1-2).

2. They are the sites where translation of mRNA into an amino acid sequence (i.e., protein synthesis) occurs.

3. Ribosomes may cluster along a strand of mRNA to form a polyribosome (or

Table 1-2

Ribosomal Subunits

Subunit

rRNA Type

Number of Proteins

Functions

40S

18S

= 33

Has binding sites for mRNA and tRNA Binds to mRNA and finds the start codon AUG

60S

5S, 5.8S, 28S

s 49

Binds to the 40S subunit after 40S subunit finds the start codon AUG Has peptidyl transferase activity

rRNA = ribosomal RNA; mRNA = messenger RNA; tRNA = transfer RNA.

rRNA = ribosomal RNA; mRNA = messenger RNA; tRNA = transfer RNA.

polysome) that is involved in the synthesis of cytoplasmic proteins (e.g., actin, hemoglobulin).

4. They may be directed to the endoplasmic reticulum to form rER if the nascent protein contains a hydrophobic signal sequence at its amino terminal end, which is cleaved in the rER lumen by signal peptidase.

B. rER. This membranous organelle contains ribosomes attached to its cytoplasmic surface by the binding of ribophorin I and II to the ribosomal 60S subunit.

1. It is the site of synthesis of secretory proteins (e.g., insulin), cell membrane proteins (e.g., receptors), and lysosomal enzymes.

2. It is the site of co-translational modification of proteins:

a. N-linked glycosylation (addition of sugars to asparagine begins in the rER and is completed in the Golgi complex)

b. Hydroxylation of proline and lysine during collagen synthesis C. Cleavage of the signal sequence d. Folding of the nascent protein into three-dimensional configuration e. Association of protein subunits into multimeric complex

C. Smooth endoplasmic reticulum (sER) is a membranous organelle that contains no ribosomes. It is involved in:

1. Synthesis of membrane phospholipids (phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylthanolamine), cholesterol, and ceramide

2. Synthesis of steroid hormones in testes, ovary, adrenal cortex, and placenta

3. Drug detoxification using cytochrome P45o> which is a family of heme proteins (also called mixed-function oxidase system) that participates in hydroxylation of barbiturates, phenytoin, or benzopyrene (a carcinogen found in cigarette smoke), makes them more soluble in water, and allows excretion into the urine a. Activation of cytochrome P450 by one agent enhances the detoxification of other agents, which has clinical implications.

b. In chronic alcoholics or newborns, large amounts of anesthesia agents are needed (which may be dangerous) because cytochrome P450 has been activated by detoxifying either alcohol or breakdown products of fetal hemoglobulin, respectively.

4. Fatty acid elongation

5. Calcium fluxes associated with muscle contraction

D. Golgi complexes are stacks of membranous cisternae with a cis-face (convex) that re-

ceives vesicles of newly synthesized proteins from the rER and a tram-face (concave)

that releases condensing vacuoles of posttranslationally modified proteins.

1. It is the site of posttranslational modification of proteins, such as:

a. Completion of N-linked glycosylation that began in the rER

b. O-linked glycosylation; that is, addition of sugars to serine by the enzyme gly-cosyltransferase

C. Sulfation d. Phosphorylation (phosphorylation of mannose forming mannose-6-phos-phate occurs only in lysosomal enzymes)

2. It is involved in protein sorting and packaging.

a. Secretory proteins (e.g., insulin) are packaged into clathrin-coated vesicles.

b. Cell membrane proteins (e.g., receptors) are packaged into nonclathrin-

coated vesicles.

C. Lysosomal enzymes are packaged into clathrin-coated vesicles after phosphorylation of mannose.

3. It is involved in membrane recycling.

E. Mitochondria

1. Function. Mitochondria are involved in the production of acetyl coenzyme A (CoA), the tricarboxylic acid cycle, fatty acid (3-oxidation, amino acid oxidation, and oxidative phosphorylation [which causes the synthesis of adenosine triphosphate (ATP) driven by electron transfer to oxygen].

a. Substrates are metabolized in the mitochondrial matrix to produce acetyl CoA, which is oxidized by the tricarboxylic acid cycle to carbon dioxide.

b. The energy released by this oxidation is captured by reduced nicotinamide adenine dinucleotide (NADU) and flavin adenine dinucleotide (FADH2). NADH and FADH2 are further oxidized, producing hydrogen ions and electrons.

C. The electrons are transferred along the electron transport chain, which is accompanied by the outward pumping of hydrogen ions into the intermembrane space (chemiosmotic theory), d. The F0 subunit of ATP synthase forms a transmembrane hydrogen ion pore so that hydrogen ions can flow from the intermembrane space into the matrix, where the F, subunit of ATP synthase catalyzes the reaction ADP + Pj —> ATP.

2. Components and contents are listed in Table 1-3.

3. Clinical considerations a. Leder's hereditary optic neuropathy is characterized by progressive optic nerve degeneration and is caused by a mitochondrial DNA mutation in the gene for subunit 4 of the NADH dehydrogenase complex. Mitochondrial diseases are maternally inherited and affect tissues that have a high requirement for ATP (e.g., nerve, muscle).

b. Myoclonic epileptic ragged red fiber disease is characterized by progressive myoclonus (muscle jerking), dementia, and hearing loss. It is caused by a mitochondrial DNA mutation in the gene for tRNA for lysine.

C. Cyanide, carbon monoxide, and antimycin A inhibit the electron transport chain and thus block ATP synthesis, d. Oligomycin and venturicidin are antibiotics that bind to ATP synthase and thus block ATP synthesis.

F. Lysosomes are membrane-bound organelles that contain lysosomal enzymes (also called acid hydrolase enzymes) including cathepsin B and L (proteases), nuclease, 5'-nucleoti-

Cell Biology 5

Table 1-3

Components and Contents

Components

Contents

Outer membrane

Porin (a transport protein that increases permeability to metabolic

sustrates)

Intermembrane space

Hydrogen ions

Inner membrane

Electron transport chain (NADH dehydrogenase, succinate dehydrogenase,

(folded into cristae)

ubiquinone-cytochrome c oxidoreductase, cytochrome oxidase)

ATP synthase (found on elementary particles)

ATP-ADP translocator (moves ADP into the matrix and ATP out of the

matrix)

Matrix compartment

Tricarboxylic acid (TCA) cycle enzymes (except succinate dehydrogenase)

Fatty acid p-oxidation enzymes

Amino acid oxidation enzymes

Pyruvate dehydrogenase complex

Carbamoylphosphate synthetase 1

Ornithine transcarbamoylase (part of urea cycle)

DNA, mRNA, tRNA, rRNA

Granules containing calcium and magnesium ions

NADH = reduced nicotinamide adenine dinucleotide; mRNA = messenger RNA; rRNA = ribosomal RNA; tRNA = transfer RNA; ATP = adenosine triphosphate (ATP); ADP = adenosine diphosphate (ADP).

NADH = reduced nicotinamide adenine dinucleotide; mRNA = messenger RNA; rRNA = ribosomal RNA; tRNA = transfer RNA; ATP = adenosine triphosphate (ATP); ADP = adenosine diphosphate (ADP).

dase, (3-galactosidase, (^-glucuronidase, glycosidase, aryl sulfatase, lipase, esterase, and acid phosphatase that function at pH 5. Most lysosomes function intracellularly; however, some cells (e.g., neutrophils, osteoclasts) release their lysosomal contents extracellularly.

1. Golgi hydrolase vesicles bud from the Golgi complex and contain inactive acid hydrolase enzymes.

a. Golgi hydrolase vesicles fuse with a late endosome, which contains an H+' ATPase in its membrane that produces a pH 5 environment, which activates the acid hydrolases.

b. A late endosome may fuse with a phagocytic vacuole forming a phagolysosome, which degrades material phagocytosed by the cell.

C. A late endosome may fuse with an autophagic vacuole forming an au-tophagolysosome, which degrades cell organelles.

2. Residual bodies contain undigestible material and may accumulate within a cell as lipofuscin pigment.

3. Clinical considerations. There are a number of genetic diseases that involve mutations of genes for various lysosomal enzymes (acid hydrolases; Table 1-4).

G. Peroxisomes are membrane-bound organelles.

1. Contents of peroxisomes include:

a. Amino acid oxidase and hydroxyacid oxidase, which produce hydrogen peroxide (H202)

b. Catalase and other peroxidases that decompose hydrogen peroxide to water and oxygen (H202 H20 + 02)

C. Fatty acid P-oxidation enzymes that oxidize long-chain fatty acids (>20 carbons) to short-chain fatty acids, which are transferred to mitochondria for complete oxidation

2. Clinical consideration. Adrenoleukodystrophy is a genetic disease that involves

Table 1-4

Lysosomal Storage Diseases

Table 1-4

Lysosomal Storage Diseases

Major Accumulating

Disease

Enzyme Involved

Metabolite

Hurler's disease

L-iduronidase

Heparan sulfate

Deramatan sulfate

Sanfilippo A

Heparan sulfamidase

Heparan sulfate

Tay-Sachs disease

Hexosaminidase A

GM2 ganglioside

Gaucher's disease

p-glucosidase

Glucosylceramide

Niemann-Pick disease

Sphingomyelinase

Sphingomyelin

Pompe's disease

a-1, 4-Glucosidase (acid maltase)

Glycogen

l-cell disease

Phosphotransferase

Mucopolysaccharide

Krabbe's disease

p-galactosidase

Galactosylceramide

mutation of genes for various peroxisomal enzymes used in fatty acid (3-oxidation that results in abnormal accumulation of lipid in the brain, spinal cord, and adrenal gland and leads to dementia and adrenal failure.

IV. CYTOSKELETON

A. Filamentous actin (F-actin)

1. F-actin comprises microfilaments (6-nm diameter) arranged in a helix of polymerized globular monomers of actin (G'actin).

2. It is in a constant state of polymerization and depolymerization.

3. F-actin functions in exocytosis, endocytosis, cytokinesis, locomotion of cells forming lamellipodia, and movement of cell membrane proteins.

4. Cytochalasin is a toxic fungal alkaloid that causes F-actin to depolymerize.

5. Phalloidin is a toxic substance derived from the Amanita mushroom that binds to F-actin, thereby inhibiting polymerization/depolymerization.

B. Intermediate filaments (10-nm to 12-nm diameter)

1. These function as the cytoplasmic link between the extracellular matrix, cytoplasm, and nucleus.

2. Intermediate filaments demonstrate specificity (Table 1-5) for certain cell types/tumors, and therefore can be used as markers for pathologic analysis.

C. Microtubules are 25-nm-diameter tubules that consist of 1.3 circularly arranged proteins called « and (J tubulin.

1. They are in dynamic equilibrium with a cytoplasmic pool of a and 3 tubulin such that a polymerization end [plus ( + ) end] and a depolymerization end [minus ( —) end] are present on each microtubule.

2. Microtubules are always associated with microtubule-associated proteins (MAPs).

a. Kinesin has ATPase activity for movement of vesicles along microtubules toward the plus end (anterograde transport).

b. Dynein has ATPase activity for movement of vesicles along microtubules toward the minus end (retrograde transport).

C. Dynamin has ATPase activity for elongation of nerve axons.

Table 1-5

Specifity of Intermediate Filaments for Cell Types or Tumors

Table 1-5

Specifity of Intermediate Filaments for Cell Types or Tumors

Intermediate Filament

Cell or Tumor Specificity

Cytokeratin

Epithelial cells

Epithelial tumors (e.g., squamous carcinoma, adenocarcinoma)

Vimentin

Endothelial cells, vascular smooth muscle, fibroblasts, chondroblasts, and

macrophages

Mesenchymal tumors (e.g., fibrosarcoma, liposarcoma, angiosarcoma,

chondrosarcoma, osteosarcoma)

Desmin

Skeletal muscle, nonvascular smooth muscle

Muscle tumors (e.g., rhabdomyosarcoma)

Neurofilament

Neurons

Neuronal tumors

Glial fibrillar acidic

Astrocytes, Oligodendroglia, microglia, Schwann cells, ependymal cells,

protein (GFAP)

and pituicytes

Gliomatous tumors

Lamins A, B, C

Inner membrane of nuclear envelope

3. Functions of microtubules include maintaining cell shape (polarity), movement of chromosomes (karyokinesis), movement of secretory granules and neurosecretory vesicles, beating of cilia and flagelia, and phagocytosis/lysosomal function.

4. The microtubular organizing center of the cell for the assembly of microtubules is called the centrosome. At the center of the centrosome are two hollow structures oriented perpendicular to each other called the centriole.

5. Clinical considerations a. Chediak-Higashi syndrome is a genetic disease characterized by neutropenia and impaired phagocytosis of bacteria due to a defect in microtubule polymerization that impairs lysosomal function of leukocytes. Large abnormal lysosomes can be observed in the cytoplasm of leukocytes in people with this syndrome.

b. Colchicine is an antimitotic agent that inhibits microtubule assembly. C. Taxol is an antimitotic agent that stabilizes microtubule movement.

V. CELL MEMBRANE. The cell membrane (8~ to 10~nm thick) appears in electron microscopy of osmium-fixed tissue as two electron-dense lines separated by an electron-lucent space. The electron-dense lines are due to the deposition of osmium on the hy-drophilic heads of lipids. The electron-lucent space represents the hydrophobic tails of lipids.

A. The lipid component consists of four phospholipids: phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine. Cholesterol and gly-colipids (e.g., ganglioside GMj) also are present.

1. The lipids that constitute the lipid bilayer are amphiphilic; that is, they have a hy-drophilic (polar) head and a hydrophobic (nonpolar) tail.

2. The lipid component exhibits asymmetry in which phosphatidylcholine and sphingomyelin arc located in the outer leaflet; phosphatidylethanolamine and phosphatidylserine are located in the inner leaflet.

3. The lipid component exhibits fluidity, which means that the phospholipids diffuse laterally within rhe lipid bilayer.

a. Fluidity is increased by increases in both temperature and degree of unsatu-ration of the fatty acid tails.

b. Fluidity is decreased by increases in cholesterol content.

B. The protein component

1. Composition. The protein component consists of peripheral and integral proteins.

a. Peripheral proteins can be easily disassociated from the lipid bilaycr by changes in ionic strength or pH.

b. Integral proteins are difficult to disassociate from the lipid bilayer unless detergents |e.g., sodium dodecyl sulfate or Triton X-100| are used. Transmem-brane proteins are integral proteins that span the lipid bilayer, exposing the protein to both the extracellular space and the cytoplasm. Many transmembrane proteins are now known to be receptor proteins.

2. Receptor proteins a. Ion channel-linked receptors arc proteins that include voltage-gated ion channels, mechanical-gated ion channels, and neurotransmitter-gated ion channels.

(1) Neurotransmitter-gatcd ion channels are receptors that bind neurotransmitters and mediate ion movement.

(2) Some important neurotransmitter-gated ion channels are the nicotinic acetylcholine receptor, the 5-hydroxytryptamine serotonin receptor, the N-methyl-D-aspartate receptor, the "y-aminobutyric acid receptor, and the glycine receptor.

b. G-protein-linked receptors are proteins that span the cell membrane seven times and are linked to trimeric GTP-binding proteins (called G proteins). These receptors activate a chain of cellular events either through the cyclic adenosine monophosphate (cAMP) pathway or calcium ion (Ca2+) pathway.

(1) The cAMP pathway increases or decreases cAMP levels by stimulation or inhibition of adenylate cyclase, respectively.

(2) The calcium ion pathway activates phospholipase C, which cleaves phosphatidylinositol biphosphatc into inositol triphosphate (IP)) and di-acylglycerol (DAG).

(a) Inositol triphosphate causes the release of calcium ions from the endoplasmic reticulum, which activates the enzyme Ca2+/calmodulin-dependent protein kinase (Cam-kinase).

(b) Diacylglycerol activates the enzyme protein kinase C (PKC). Some important G-protein-linked receptors are the muscarinic acetylcholine receptor, the a- and P-adrenergic receptors, the dopamine receptor, and the glucagon receptor.

C. Enzyme-linked receptors are proteins that span the cell membrane one time and are linked to an enzyme (e.g., tyrosine kinase). When the appropriate signal hinds to a receptor, its intrinsic tyrosine kinase autophosphorylates tyrosine residues within the receptor.

(1) This activates a chain of cellular events that includes SH2 (sre [Roux sarcoma virus] homology) domain proteins, Sos (son-of-sevenless protein), Ras protein (a gene product of ras proto-oncogene), Raf protein kinase, and mitogen-activated protein (MAP) kinase, which eventually affects gene transcription within the nucleus.

(2) Some important enzyme-linked receptors are the insulin receptor, the epidermal growth factor (EGF) receptor, and the fibroblast growth factor (FGF) receptor.

3. The protein component exhibits patching or capping, which means that proteins diffuse laterally with the lipid bilayer.

4. The protein component is studied by electron microscopy (EM) using the freeze-fracture technique, whereby the lipid bilayer is cleaved between the inner and outer leaflets.

a. The P-face is the outer surface of the inner leaflet and contains the majority of integral proteins, which are seen by EM as "bumps."

b. The E'face is the inner surface of the outer leaflet and is seen by EM as a smooth surface with "pits."

5. The protein component is well characterized in red blood cell (RBC) membranes and includes the following proteins (Figure 1-1):

a. Spectrin maintains the biconcave shape of the RBC. The tail ends of spectrin bind to actin and band 4.1 protein.

b. Ankyrin attaches to spectrin and band 3 protein.

C. Band 3 protein is an anion transporter that allows bicarbonate ion (HCO^")

to cross the RBC membrane in exchange for chloride ions (Cl~). d. Glycophorin is the first transmembrane protein for which a complete amino acid sequence was determined. Its hydrophilic amino-terminal end is exposed to the extracellular space, its hydrophobic portion (22 amino acids long arranged in an a-helix) spans the lipid bilayer, and its hydrophilic carboxyl-terminal end is exposed to the cytoplasm.

C. Clinical consideration. Familial hypercholesterolemia is a genetic disease involving a mutation in the low-density lipoprotein (LDL) receptor in which patients have greatly elevated levels of serum cholesterol and suffer myocardial infarctions early in life. The mutation in the LDL receptor blocks a normal process called receptor-mediated endocytosis, which involves the following steps:

1. Circulating serum LDL binds to the LDL receptor located on the cell membrane, and the complex undergoes endocytosis as clathrin-coated vesicles.

2. The clathrin-coated vesicles fuse with cytoplasmic early endosomes, where LDL disassociates from the LDL receptor, and the LDL reccptor is recycled to the cell membrane.

Figure 1-1. A schcmatic diagram of the red blood cell (RBC) membrane depicting the well-characterized protein component.

3. The early endosomes fuse with late endosomes containing active lysosomal enzymes that digest the LDL to cholesterol.

4. Cholesterol inhibits 3-hydroxy-3-methyglutaryl CoA reductase, which suppresses de novo cholesterol synthesis, and therefore maintains normal levels of serum cholesterol.

VI. CELL CYCLE

B. Control factors of the cell cycle

Table 1-6

Phases of the Cell Cycle

Table 1-6

Phases of the Cell Cycle

Phases

Cellular Events

Cell cycle is suspended

Gi (gap) (5 hours)

RNA, protein, and organelle synthesis occurs. Cdk2-cyclin D and Cdk2-cyclin E form.

Gx Checkpoint

Interphase

S (synthesis) (7 hours)

DNA synthesis occurs.

RNA and histone synthesis occurs.

Centrosome (MTOC) duplicates but remains together as a complex on one side of the nucleus.

G2 (gap) (3 hours)

ATP synthesis occurs.

Cdkl-cyclin A and Cdkl-cyclin B form.

G2 Checkpoint

Prophase

Chromatin condenses to form well-defined chromosomes.

Centrosomes (MTOC) move to opposite poles.

Mitotic spindle (microtubules) forms between the centrosomes.

Prometaphase

The nuclear envelope is disrupted.

The nucleolus disappears.

Kinetochores assemble at each centromere.

Kinetochore, polar, and astral microtubules are apparent.

Mitosis (M Phase)

(1 hour)

Metaphase

Chromosomes align at the metaphase plate.

Cells can be arrested in this phase by microtubule inhibitors (e.g., colchicine). Cells can be isolated for karyotype analysis.

Anaphase

Kinetochores separate, and chromosomes move to opposite poles.

Telophase

Chromosomes decondense to form chromatin. The nuclear envelop re-forms. The nucleolus reappears.

Cytokinesis

Cytoplasm divides by a process called cleavage. A cleavage furrow forms around the middle of the cell. A contractile ring consisting of actin and myosin forms at the cleavage furrow.

* Based on a 16-hour cell cycle. MTOC - microtubular organizing center.

1. Cyclin-dependent protein kinase 1 and 2 (Cdkl and Cdk2) induce cell cycle events by phosphorylation of target proteins. Cdk activity is controlled by cyclins.

2. Cyclins are produced by a family of related genes. Cyclins bind to Cdk and control the ability of Cdk to phosphorylate.

a. Cdk2-cyclin D and Cdk2-cyclin E form during G[ and mediate the transition from G, phase to the S phase at the G, checkpoint.

b. Cdkl-cyclin A and Cdkl-cyclin B form during G2 and mediate the transition from G2 phase to the M phase at the G2 checkpoint.

3. Retinoblastoma (Rb) protein is coded for by an anti-oncogene (or tumor suppressor gene). Rb protein binds to gene regulatory proteins such that there is no expression of target genes that stimulate the cell cycle. Therefore, the Rb protein causes suppression of the cell cycle. Mutations in the Rb anti-oncogene cause retinoblastoma tumor, which occurs in childhood and develops from precursor cells in the immature retina.

4. The p53 zinc finger protein is coded for by an anti-oncogene (or tumor suppressor gene). p53 causes expression of target genes whose gene products suppress the cell cycle at G( by inhibiting Cdk2-cyclin D and Cdk2-cyclin E. Therefore, p53 causes suppression of the cell cycle. Mutations in the p53 anti-oncogene play a role in Li-Fraumeni syndrome, which is an inherited susceprihility to a variety of cancers in which 50% of the affected individuals develop cancer by age 30 and 90% by age 70.

5. The BRCA (breast cancer) zinc finger protein is coded for by an anti-oncogene (or tumor suppressor gene). BRCA causes suppression of the cell cycle. Mutations in the BRCA anti-oncogcnc play a role in breast and ovarian cancer.

VII. APOPTOSIS is a distinctive form of cell death that is characterized by chromatin clumping into a distinct crescent pattern along the inner margins of the nuclear envelope and then into a dense body. The chromatin is eventually cleaved by a specific endonuclease into DNA fragments that generate a distinctive 180-bp ladder that is pathognomonic of apoptotic cell death. The bcl-2 gene encodes an intracellular inhibitor of apoptosis. Apop-tosis occurs in hormone-dependent involution of cells during the menstrual cycle, em-bryogenesis, toxin-induced injury (e.g., diphtheria), viral cell death (e.g., Councilman bodies in yellow fever), and cell death via cytotoxic T cells or other immune cells. Apoptosis does not clicit an inflammatory response.

VIII. CELL INCLUSIONS

A. Lipofuscin is a yellow-brown "wear and tear" pigment found predominately in residual bodies, which are the end point of lysosomal digestion.

1. It is composed of phospholipids complexed with proteins, suggesting that it is derived from the lysosomal digestion of cellular membranes.

2. Lipofuscin is a telltale sign of free radical damage and is found prominently within hepatocytes, skeletal muscle cells, and nerve cclls of elderly people or patients with severe malnutrition.

B. Hemosiderin is a golden brown hemoglobin-derived pigment consisting of iron.

1. Iron is absorbed mainly by surface absorptive cells within the duodenum, transported in the plasma by a protein called transferrin, and is normally stored in cells as ferritin, which is a protein-iron complex.

2. Small amounts of ferritin normally circulate in the plasma, making plasma ferritin a good indicator of the adequacy of body iron stores.

a. In iron deficiency, serum ferritin is less than 12 (xg/L.

b. In iron overload, serum ferritin approaches 5000 (xg/L.

(1) Also during iron overload, intracellular ferritin undergoes lysosomal degradation, in which the ferritin protein is degraded and the iron aggregates within the cell as hemosiderin in a condition called hemosiderosis.

(2) Hemosiderosis can he observed in patients with increased absorption of dietary iron, impaired utilization of iron, hemolytic anemias, and blood transfusions.

C. Glycogen is the storage form of glucose and is composed of glucose units linked by a-1,4 glycosidic bonds. Glycogen synthesis is catalyzed by glycogen synthase. Glycogen degradation is catalyzed by glycogen phosphorylase. Liver hcpatocytes and skeletal muscle cells contain the largest glycogen stores, but the function of glycogen differs widely.

1. Liver glycogen functions in the maintenance of blood glucose levels.

a. Synthesis. Liver glycogen is synthesized (using glycogen synthase) during a high-carbohydrate meal due to hyperglycemia and an increase in the insulin :glucagon ratio.

b. Degradation. Liver glycogen is degraded (using liver glycogen phosphorylase isoenzyme) during hypoglycemia (e.g., fasting), exercise, or other stressful situations due to a decrease in the insulin:glucagon ratio and the secretion of epinephrine from the adrenal medulla, which binds to a- and 3-adrcnergic receptors on the hepatocyte.

(1) Liver glycogen is degraded to glucose-6-phosphate, which is catalyzed to free glucose by the enzyme glucose-6-phosphatase.

(2) Glucose-6-phosphatase is found only in the liver and kidney.

2. Skeletal muscle glycogen functions in the formation of ATP through glycolysis.

a. Synthesis. Skeletal muscle glycogen is synthesized (using glycogen synthase) during a high-carbohydrate meal due to hyperglycemia and an increase in the insulin :glucagon ratio.

b. Degradation. Skeletal muscle glycogen is degraded (using muscle glycogen phosphorylase isoenzyme) during exercise or stressful situations due to a decrease in ATP, calcium released during contraction, and secretion of epinephrine from the adrenal medulla, which binds to a- and (3-adrenergic receptors on rhe skeletal muscle cell.

(1) Skeletal muscle glycogen is degraded to glucose-6-phosphate, which enters glycolysis to produce ATP

(2) The absence of glucose-6-phosphatase enzyme in skeletal muscle prevents the degradation of glycogen to free glucose.

3. Glycogen storage diseases are genetic diseases that involve mutations in one of the enzymes of glycogen synthesis or degradation.

a. Von Gierke disease (type 1 glycogenosis) results from a deficiency in the enzyme glucose-6-phosphatase, causing an enlarged liver and severe hypoglycemia.

b. McArdle disease (type V glycogenosis) results from a deficiency in the enzyme muscle glycogen phosphorylase, causing exercise-induced muscle pain and cramps.

IX. SELECTED PHOTOMICROGRAPHS

A. Nuclear envelope and nuclear pore complex (Figure 1-2; see I A)

Figure 1-2. (A) Electron micrograph of nucleoplasm»! labeled with colloidal gold particles. Nucleoplasms is a large protein synthesized in rhe cytoplasm and transported into the nucleus. Brackets denote a nuclear pore complex. Note that the gold particles are localized specifically at the nuclear pore complex as nucleoplasmin moves from the cytoplasm to the nucleus. (Rcproduccd with permission from Feldherr C, Kallenbach E, Schultz N: J Cell Biol 99:2216, 1984 by copyright permission of the The Rockefeller University Press.) (B) A freeze-fracture replica of the nuclear envelope is shown. Note the nuclear pore complex (arrow 1). In addition, the outer membrane of the nuclear envelope has been stripped away (arrow 2), exposing the perinuclear cisterna. (Reproduced with permission from Stafstrom J, Stahelin L: ) Cell Biol 98:699, 1984 by copyright permission of the Rockefeller University Press.) (C) Electron micrograph of octagonal protein complexes isolated from the nuclear envelope and negatively stained. Note the nuclear pore complex (circles) and a central channel or central pore (arrow 1). (Reproduced with permission from Unwin P: J Cell Biol 93:63, 1982 by copyright permission of The Rockefeller University Press.)

Figure 1-2. (A) Electron micrograph of nucleoplasm»! labeled with colloidal gold particles. Nucleoplasms is a large protein synthesized in rhe cytoplasm and transported into the nucleus. Brackets denote a nuclear pore complex. Note that the gold particles are localized specifically at the nuclear pore complex as nucleoplasmin moves from the cytoplasm to the nucleus. (Rcproduccd with permission from Feldherr C, Kallenbach E, Schultz N: J Cell Biol 99:2216, 1984 by copyright permission of the The Rockefeller University Press.) (B) A freeze-fracture replica of the nuclear envelope is shown. Note the nuclear pore complex (arrow 1). In addition, the outer membrane of the nuclear envelope has been stripped away (arrow 2), exposing the perinuclear cisterna. (Reproduced with permission from Stafstrom J, Stahelin L: ) Cell Biol 98:699, 1984 by copyright permission of the Rockefeller University Press.) (C) Electron micrograph of octagonal protein complexes isolated from the nuclear envelope and negatively stained. Note the nuclear pore complex (circles) and a central channel or central pore (arrow 1). (Reproduced with permission from Unwin P: J Cell Biol 93:63, 1982 by copyright permission of The Rockefeller University Press.)

B. Chromatin (exons and introns) and nucleosomes (Figure 1-3; see I B)

Figure 1-3. (A) Electron micrograph of DNA containing the gene for ovalbumin hybridized with ovalbumin messenger RNA (mRNA). Linear regions of the gene (.bracket I) that hybridize to mRNA are called exons because the processed mRNA "exits" rhe nucleus into the cytoplasm to participate in translation. Looped regions of the gene (arrow 2) that do not hybridize to mRNA arc called introns. (Reprinted with permission from Chambon P: Sci Am 244:60, 1981.) (B) Electron micrograph of DNA isolated and subjected to treatments that unfold its native structure. This "beads-on-a-string" appearance is the basic unit of chromatin packing called a nucleosome. The globular structure ("bead"; arrow 1) is a histone octamer that is composed of specific proteins (H2A, H2B, H3, and H4). The linear structure ("string"; arrow 2) is DNA. (Reprinted with permission from McKnight S, Miller OL: Cell 8:305, 1976.) Inset: A diagram of a nucleosome depicting the histone octamer (arrow 1) and DNA (arrow 2).

Figure 1-3. (A) Electron micrograph of DNA containing the gene for ovalbumin hybridized with ovalbumin messenger RNA (mRNA). Linear regions of the gene (.bracket I) that hybridize to mRNA are called exons because the processed mRNA "exits" rhe nucleus into the cytoplasm to participate in translation. Looped regions of the gene (arrow 2) that do not hybridize to mRNA arc called introns. (Reprinted with permission from Chambon P: Sci Am 244:60, 1981.) (B) Electron micrograph of DNA isolated and subjected to treatments that unfold its native structure. This "beads-on-a-string" appearance is the basic unit of chromatin packing called a nucleosome. The globular structure ("bead"; arrow 1) is a histone octamer that is composed of specific proteins (H2A, H2B, H3, and H4). The linear structure ("string"; arrow 2) is DNA. (Reprinted with permission from McKnight S, Miller OL: Cell 8:305, 1976.) Inset: A diagram of a nucleosome depicting the histone octamer (arrow 1) and DNA (arrow 2).

C. Nucleus, rER, sER, Golgi complex, and mitochondria (Figure 1-4; see 111 B-E)

Figure 1-4. Electron micrographs of various cell organelles. (A) Nucleus containing predominately euchro-matin and a conspicuous nucleolus (arrow). (B) Rough endoplasmic reticulum (rER), which shows membrane cisternae that are dotted with ribosomes. (C) Smooth endoplasmic reticulum (sER). (D) Golgi (gol) complex surrounded by many secretory granules (sg) and clathrin-coated vesicles (arrows) budding from the trans-face (concave). Inset: Isolated clathrin showing a distinctive three-legged structure called a triskelion. (E) Mitochondria and cristae.

Figure 1-4. Electron micrographs of various cell organelles. (A) Nucleus containing predominately euchro-matin and a conspicuous nucleolus (arrow). (B) Rough endoplasmic reticulum (rER), which shows membrane cisternae that are dotted with ribosomes. (C) Smooth endoplasmic reticulum (sER). (D) Golgi (gol) complex surrounded by many secretory granules (sg) and clathrin-coated vesicles (arrows) budding from the trans-face (concave). Inset: Isolated clathrin showing a distinctive three-legged structure called a triskelion. (E) Mitochondria and cristae.

D. Protein-secreting cell (Figure 1-5)

Figure 1-5. Electron micrograph of a protein-secreting ccll containing prominent rough endoplasmic reticulum (rER; arrows), Golgi (gal) complex, and secretory granules (sg), which are conspicuous organelles in protein-secreting cells. (Courtesy of Dr. Jack Brinn, East Carolina University School of Medicine.)

Figure 1-5. Electron micrograph of a protein-secreting ccll containing prominent rough endoplasmic reticulum (rER; arrows), Golgi (gal) complex, and secretory granules (sg), which are conspicuous organelles in protein-secreting cells. (Courtesy of Dr. Jack Brinn, East Carolina University School of Medicine.)

E. Steroid-secreting cell (Figure 1-6)

Figure 1-6. Electron micrograph of a steroid-secreting cell containing prominent smooth endoplasmic reticulum (sER), mitochondria with tubular cristae (mit), and lipid droplets (Ip), which are conspicuous organelles in steroid-secreting cells. (Courtesy of Dr. Jack Brinn, East Carolina University School of Medicine.)

Figure 1-6. Electron micrograph of a steroid-secreting cell containing prominent smooth endoplasmic reticulum (sER), mitochondria with tubular cristae (mit), and lipid droplets (Ip), which are conspicuous organelles in steroid-secreting cells. (Courtesy of Dr. Jack Brinn, East Carolina University School of Medicine.)

F. Apoptosis (Figure 1-7; sec VII)

Figure 1-7. (A, B) Electron micrographs ofhumanT cells treated with a lipid hydroperoxide that is toxic to cells and induces cell death. Note the chromatin clumping and mitochondrial changes (arrows in B). These cells are in the process of cell death, called apoptosis. The chromatin of an apoptotic cell condenses into a distinctive crescent-shaped pattern (see A) along the inner margins of the nuclear envelope and then into a dense body that eventually breaks into fragments (see B). During these morphologic changes, the DNA is cleaved to generate a distinctive 180-bp ladder, which is pathognomonic of apoptotic cell death. The facl-2 gene has been cloned and encodes for an intracellular inhibitor of apoptosis. (Courtesy of D. Whitehead, East Carolina University School of Medicine.)

Figure 1-7. (A, B) Electron micrographs ofhumanT cells treated with a lipid hydroperoxide that is toxic to cells and induces cell death. Note the chromatin clumping and mitochondrial changes (arrows in B). These cells are in the process of cell death, called apoptosis. The chromatin of an apoptotic cell condenses into a distinctive crescent-shaped pattern (see A) along the inner margins of the nuclear envelope and then into a dense body that eventually breaks into fragments (see B). During these morphologic changes, the DNA is cleaved to generate a distinctive 180-bp ladder, which is pathognomonic of apoptotic cell death. The facl-2 gene has been cloned and encodes for an intracellular inhibitor of apoptosis. (Courtesy of D. Whitehead, East Carolina University School of Medicine.)

G. Transformation of normal cell to cancer cell (Figure 1-8)

Figure 1-8. Scanning electron micrographs of fibroblasts in culture infected with Roux sarcoma virus (src) that carries a temperature-sensitive mutation in the gene responsible for transformation (v-src oncogene). (A) When cultured at 39°C, the oncogene product is inactive, and these cells appear normal. Normal fibroblasts in nonconfluent cultures appear flat, attach finnly to substratum, spread under tension by the activity of lamellipodia and filopo-dia (locomotion organelles), and demonstrate contact inhibition of locomotion and cell division when the cells contact each other. (B) When cultured at 34°C, the oncogene product is active, and the cells are transformed, or "cancerous." Transformed fibroblasts in culture appear round, attach poorly to substranim, are capable of cell division to an unusually high density, do not demonstrate contact inhibition of locomotion or cell division, and cause tumors when injected into susceptible animals. (Courtesy of Dr. G. S. Martin, University of California at Berkeley.)

Figure 1-8. Scanning electron micrographs of fibroblasts in culture infected with Roux sarcoma virus (src) that carries a temperature-sensitive mutation in the gene responsible for transformation (v-src oncogene). (A) When cultured at 39°C, the oncogene product is inactive, and these cells appear normal. Normal fibroblasts in nonconfluent cultures appear flat, attach finnly to substratum, spread under tension by the activity of lamellipodia and filopo-dia (locomotion organelles), and demonstrate contact inhibition of locomotion and cell division when the cells contact each other. (B) When cultured at 34°C, the oncogene product is active, and the cells are transformed, or "cancerous." Transformed fibroblasts in culture appear round, attach poorly to substranim, are capable of cell division to an unusually high density, do not demonstrate contact inhibition of locomotion or cell division, and cause tumors when injected into susceptible animals. (Courtesy of Dr. G. S. Martin, University of California at Berkeley.)

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