bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissecting the roles of individual interactions in protein stability: Lessons from a circularized protein
Authors: Goldenberg David P.
Published: December 2004

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N‐ and C‐termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide‐coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross‐link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross‐link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross‐linked groups are held rigidly in the native protein in the optimum orientation for forming the cross‐link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.
Purification of human DNA (cytosine‐5‐)‐methyltransferase
Authors: Zucker Keith E. , Riggs Arthur D. , Smith Steven S.
Published: December 2004

We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000‐fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.
12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA)‐induced gene sequences in human primary diploid fibroblasts and their expression in SV40‐transformed fibroblasts
Authors: Angel Peter , Rahmsdorf Hans Jobst , Pöting Annette , Lücke‐Huhle Christine , Herrlich Peter
Published: December 2004

We have isolated cDNA sequences from TPA‐treated primary human fibroblasts, which indicate RNA species that are coordinately regulated after treatment of these cells with either ultraviolet light, mitomycin C, the UV‐induced factor EPIF, or TPA. The levels of RNA are elevated in Bloom syndrome (cells of two out of three patients). After transformation with SV40 one of the sequences is overexpressed while another one is reduced. Both genes maintain their inducibility by the agents mentioned.
The relation between adenosine diphosphate‐ribosylation and mammary gland differentiation
Authors: Bolander Franklyn F.
Published: December 2004

Poly(adenosine diphosphate [ADP]‐ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP‐ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP‐ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP‐ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMP, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP‐ribose) degradation. This reciprocal relationship between poly(ADP‐ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated α‐lactalbumin three‐fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship.
Synthetic potential of Staphylococcus aureus V8‐protease: An approach toward semisynthesis of covalent analogs of α‐chain of hemoglobin S
Authors: Seetharam Ramnath , Acharya A. Seetharama
Published: December 2004

Enzyme‐catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the α‐chain of hemoglobin S with Staphylococcus aureus V8‐protease converts the chain into a fragment‐complementing system by hydrolyzing the peptide bond Glu(30)‐Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of α‐chain could be achieved if conditions for the V8‐protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8‐protease has been now investigated by incubating V8‐protease‐derived fragments of α‐chain, namely α1–30 and α31–47 with the enzyme at pH 6.0 in the presence of n‐propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as α1–47 by amino acid analysis, redigestion with V8‐protease (in the absence of n‐propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of α1–47 is at pH 6.0, 4°C, and 24 hr of incubation with 25% n‐propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of α1–47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8‐protease and the fact that α1–30 and α31–141 interact to form a native‐like complex, opens up an approach for the semisynthesis of covalent analogs of α‐chain of hemoglobin S.
Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase
Authors: Frenkel Krystyna , Blum Fredric , Troll Walter
Published: December 2004

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H2O2) prevents polyspermy in Arbacia punctulata. We found that it is not H2O2 but probably hypochlorous acid/hypochlorite (HOCl/OCl−) derived from H2O2 that is toxic to the supernumerary sperm. The spermicidal activity of H2O2 is potentiated by at least one order of magnitude by cupric ions (Cu2+). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu2+. More‐over, substitution of Cu2+ by ferrous ions (Fe2+), which are known to cause formation of ·OH from H2O2, had no effect on fertilization even at 102−103 times higher concentrations. In contrast, 3‐amino‐1,2,4‐triazole (AT), an HOCl/OCl− scavenger, totally reversed the toxic effects of Cu2+. Furthermore, we found that HOCl/OCl− is generated in solutions of H2O2 and Cu2+ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl−. HOCl/OCl− generated by Cu2+ from H2O2 and Cl−, a low concentration of exogenously added HOCl/OCl−, or increased concentrations of H2O2 has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl− from the Cl− present in sea water through reaction with H2O2 generated by the newly fertilized egg.
Guanidine hydrochloride denaturation studies of mutant forms of staphylococcal nuclease
Authors: Shortle David
Published: December 2004

Several mutant forms of Staphylococcal nuclease with one or two defined amino acid substitutions have been purified, and the effects of the altered amino acid sequence on the stability of the folded conformation have been analyzed by guanidine hydrochloride denaturation. Two nuc‐ mutations, which greatly reduced the level of enzyme activity accumulated in Ecoli colonies carrying a recombinant plasmid with the mutant nuc gene (i.e., a NUC‐phenotype), both result in protein unfolding at significantly lower guanidine hydrochloride concentrations than the wild‐type protein, whereas three sup mutations isolated on the basis of their ability to suppress partially the NUC‐phenotype of the above two mutations result in unfolding at significantly higher guanidine hydrochloride concentrations. Characterization of nuclease molecules with two different amino acid substitutions, either nuc − + sup pairs or sup + sup pairs, suggests that the effect of an amino acid substitution on the stability of the native conformation, as measured by the value of ΔΔGD, may not be a constant, but rather a variable that is sensitive to the presence of other substitutions at distant sites in the same molecule. Surprisingly, the slopes of the log Kapp vs guanidine hydrochloride concentration plots vary by as much as 35% among the different proteins.
Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins
Authors: Stoscheck C. M. , King L. E.
Published: December 2004

Epidermal growth factor (EGF) is a peptide which effects the growth and/or differentiated functions of many cell types. Several pieces of evidence indicate that EGF and its receptor may play a role in carcinogenesis. Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins are discussed. EGF has extensive homology with alpha‐transforming growth factor (alpha‐TGF), which may actually be an embryonic form of EGF. Nevertheless, both EGF and alpha‐TGF elicit transformation‐associated phenotypes in target cells under certain conditions.

EGF effects are mediated by a receptor present on the plasma membrane. The EGF receptor is a highly complex protein having several functions in addition to binding EGF in a highly specific manner. One of these functions is to phosphorylate tyrosyl residues on certain proteins. This activity is similar to that expressed by the src family of oncogene‐encoded proteins. Besides sharing functional homology the EGF receptor also exhibits structural homology to several oncogene‐encoded proteins. The v‐erb‐B‐transforming protein has a striking extent of homology (95%) to the cytoplasmic portion of the EGF receptor. These data support the concept that some aspect of EGF‐stimulated metabolism is involved in cellular transformation.
Liver autophagy in the influenza B virus model of Reye's syndrome in mice
Authors: Woodfin Beulah M. , Davis Larry E.
Published: December 2004

Biochemical evidence is presented for the autophagic destruction of liver mitochondria in the influenza B virus model of Reye's syndrome in mice. Separation of lysosomes and autophagic vacuoles from mitochondria was accomplished by prior treatment of the mice with Triton WR‐1339, resulting in uptake of detergent by these organelles (tritosomes), reducing their densities. The organelles were banded in a discontinuous sucrose gradient. Total protein in the heavy tritosomal fraction increased from 1–2% in controls to 7–8% in virus‐treated animals. Ornithine carbamoyl transferase (OCTase), a mitochondrial marker, increased from 2–3% (controls) to 11–15% (virus‐treated), and glucose‐6‐phosphatase, a marker for endoplasmic reticulum, increased from 1–2% (controls) to 8–10% (virus‐treated). β‐Galactosidase, a soluble enzyme in the lysosome, and OCTase also increase in the cell extract fraction following virus treatment, indicating that there was turnover of heavy lysosomal contents.
The receptor for urokinase‐plasminogen activator
Authors: Blasi Francesco , Stoppelli M. Patrizia , Cubellis M. Vittoria
Published: December 2004

Many human cells and cell lines possess a specific receptor that binds urokinase plasminogen activator (uPA) with an affinity of about 10−10 M. Bound enzyme is not internalized, is slowly dissociated, and retains its enzymatic activity. The amino acid sequence of uPA responsible for receptor binding is located within the first 35 aminoterminal residues, ie, in the growth factor domain. Binding, however, is not competed for by other proteins that contain the growth factor domain (including epidermal growth factor). Cells that produce uPA secrete the pro‐uPA form, which subsequently binds to the receptor. A431 cells, in fact, have their receptors completely saturated with pro‐uPA. It is proposed that uPA:uPA‐receptor interaction plays a direct role in physiological and pathological processes that require cell migration.
Calcimedins: Purification and characterization from chicken gizzard and rat and bovine livers
Authors: Mathew J. K. , Krolak J. M. , Dedman J. R.
Published: December 2004

A procedure for the simultaneous extraction and purification of four calcimedins from chicken gizzard, rat liver, and bovine liver is described. These proteins bind to hydrophobic resins in a calcium‐dependent manner similar to calmodulin and troponin C. The four calcimedins purified had molecular weights 67,000 (67K), 35,000 (35K), 33,000 (33K), and 30,000 (30K) as determined by SDS polyacrylamide gel electrophoresis. Their ability to bind calcium was demonstrated using the Hummel‐Dreyer method. Their tissue concentration ranged between 1–4 mg/ 100 g wet weight in the three tissues studied. During gel filtration, calcimedins 67K and 35K, had Rf (Ve‐Vo/Vt‐Vo) values of 0.46 and 0.74, respectively, indicating monomeric structure. However, the 33K and 30K calcimedins had Rf values of 0.26 (molecular weights > 90,000) suggesting that they occur as subunit complexes in their native state. Antibodies raised against the 67K and 35K calcimedins showed cross reactivity suggesting possible common origin. However, peptide mapping studies showed that they are independent proteins with considerable peptide homology. Antibodies to 30/33K calcimedins did not cross‐react with either 67K or 35K calcimedins. Moreover, their peptide maps were strikingly different from those of 67K and 35K calcimedins indicating that they are unique. At present, the regulatory function of this group of proteins is not clear. Indirect evidences support the possibility that they are involved in membrane associated events, such as endocytosis and secretion.
Fibrinolytic system of cultured endothelial cells: Regulation by plasminogen activator inhibitor
Authors: Loskutoff David J. , Ny Tor , Sawdey Michael , Lawrence Daniel
Published: December 2004

Cultured bovine aortic endothelial cells have a relatively complex flbrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The flbrinolytic activity of these cells results from the production of both urokinase‐type and tissue‐type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these flbrinolytic components will be reviewed, and their respective roles in initiating and regulating the flbrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.
Regulation of the ubiquitin‐mediated proteolytic pathway: Role of the substrate α‐NH2 group and of transfer RNA
Authors: Ciechanover Aaron
Published: December 2004

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP‐requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell‐cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem f central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free α‐NH2 group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteo‐lytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptile to ubiquitin‐mediated degradation.
Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles
Authors: Palotie Aarno , Salo Tuula , Vihko Kimmo K. , Peltonen Leena , Rajaniemi Hannu
Published: December 2004

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)‐primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG‐stimulated follicles gradually increased within 6 h to 3.3‐fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8‐fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9‐fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG‐stimulated ovulatory follicles.
Heterologous regulation of EGF receptors in fibroblastic cells
Authors: Olashaw Nancy E. , Pledger W. J.
Published: December 2004

Platelet‐derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C‐3T3. PDGF‐treated BALB/C‐3T3 cells manifest a reduced capacity to bind 125I‐labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding.

12‐0‐tetradecanoylphorbol 13‐acetate (TPA) also reduces EGF binding in BALB/C‐3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine–654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF‐treated WI‐38 cells are phosphorylated at threonine‐654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density‐arrested BALB/C‐3T3 cells.

Both PDGF and TPA caused a rapid, transient, cycloheximide‐independent loss of 251‐EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide‐dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.
Use of anti‐idiotypic antibodies as probes for the interaction of TSH subunits with its receptor
Authors: Briones‐Urbina Rosario , Islam M. N. , Ivanyi J. , Farid Nadir R.
Published: December 2004

TSH is a heterodimeric glycoprotein hormone, whose dissociated subunits are without biological activity. This has precluded the assessment of the relative contribution of each subunit to hormone action. We have raised anti‐idiotypes to monoclonal antibodies specific, respectively, for the α and β hTSH subunits. The anti‐β anti‐idiotype inhibited l25I‐hTSH binding to the β subunit‐specific monoclonal quantitatively, whereas 125I‐hTSH binding to the α subunit‐specific monoclonal was not inhibited by anti‐α anti‐idiotypes, suggesting that only the former is an “internal image” anti‐idiotype. Neither of the two anti‐idiotypes nor equimolar mixtures thereof inhibited 125I‐bTSH binding to thyroid membranes, even though radiolabelled anti‐idiotypes showed saturable binding to thyroid plasma membrane which was inhibited 41–65% by bTSH. Each anti‐idiotype alone caused 9% inhibition (compared to 50% by NRIgG) of thyroid plasma membrane adenylate cyclase. Equimolar mixtures (125 μg/ml IgG of each anti‐idiotype) induced enzyme activity equivalent to 85% of that of 250 mU/ml of TSH. The TSH‐like action of the two anti‐idiotypes was also reflected in their capacity to increase (450% by 250 μg/ml IgG compared to normal rabbit IgG) the uptake of 131I into isolated thyrocytes and to promote the organization of such cells into follicular structures. At 250 μg/ml, anti‐β anti‐idiotype promoted the organization of small follicles and only at a concentration of 500 μg/ml did it enhance 131I uptake. Anti α idiotype was without effect in both assays. Lastly, mixture of anti‐idiotypes bound to the ∼ 197,000 Mr band (TSH holoreceptor) on protein blots of thyroid plasma membranes resolved on NaDSO4‐polyacrylamide gel electrophoresis under non‐reducing conditions. Individual anti‐idiotypes were without effect.

The TSH α and β subunits apparently deliver two cooperative signals to the receptor and that specificity is associated with the β subunit, while the α subunit is important in enhancing receptor affinity for the heterodimer and in stablizing TSH‐receptor complex.
Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo
Authors: Galcheva‐Gargova Z. , Dessev G. N.
Published: December 2004

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.
Microtubule‐associated proteins present in different developmental stages ofDrosophila melanogaster
Authors: Wandosell Francisco , Avila Jesús
Published: December 2004

Microtubule‐associated proteins (MAPs) have been isolated from different development stages ofDrosophila melanogasterand characterized by their association to tubulin, but not to tubulin lacking its 4‐kD carboxy terminal region (S‐tubulin), and by their ability to promote tubulin polymerization. Following these criteria some peptides of Mr 255, 205, and 180 kD were identified as MAPs.

By means of immunological analogy we have identified a peptide related to mammalian brain MAP known as tau factor.
Developmentally regulated primary glucocorticoid hormone induction of chick retinal glutamine synthetase mRNA
Authors: Patejunas Gerald , Young Anthony P.
Published: December 2004

We have characterized the glucocorticoid hormone induction of glutamine synthetase mRNA in embryonic chick retinal organ cultures by quantitative dot hybridization using a cDNA clone derived from chick retinal RNA. Hydrocortisone (Kapp = 3–4 nM) and dexamethasone (Kapp = 1–2 nM) produce an approximate 30‐fold increase in glutamine synthetase mRNA after incubation of organ cultures derived from embryonic day 12 retinae with either hormone for 3 hr. Progesterone is a poor inducer. The glucocorticoid‐mediated rise is rapid (t½ = 2–3 hr) and occurs in the presence of either of the protein synthesis inhibitors cycloheximide or puromycin, indicating that the induction is a primary or direct response to the hormone. However, the magnitude of the hormonal response observed in culture increases markedly during retinal development. These observations, coupled with the previously reported absence of a hormonal induction in embryonic liver, raise the possibility of a synergistic mechanism, involving tissue‐specific regulatory molecules in addition to the glucocorticoid hormone receptor, to explain the retinal‐specific primary glucocorticoid hormone induction of glutamine synthetase mRNA.
Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant‐activated human polymorphonuclear leukocytes
Authors: Dillon Susan B. , Murray John J. , Uhing Ronald J. , Snyderman Ralph
Published: December 2004

Binding of chemoattractants to specific cell surface receptors on polymorphonu‐clear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor‐mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5‐bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol‐1,4,5‐trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2‐diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor‐mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet‐Leu‐Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet‐Leu‐Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN paniculate fractions and with partially purified fMet‐Leu‐Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet‐Leu‐Phe stimulates high‐affinity binding of GTPγS to PMN membranes as well as GTPase activity. A Ga subunit has been identified in phagocyte membranes which is different from other Ga subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5‐IP3 proceeds via two distinct pathways: (1) sequential dephosphorylation to 1,4‐IP2, 4‐IP1 and inositol, or (2) ATP‐dependent conversion to inositol 1,3,4,5‐tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4‐IP3, 3,4‐IP2, 3‐IP1 and inositol. Receptor‐mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5‐IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus,the two pathways for 1,4,5‐IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability ofthe activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.
Lipid polarity and sorting in epithelial cells
Authors: van Meer Gerrit , Simons Kai
Published: December 2004

Apical and basolateral membrane domains of epithelial cell plasma membranes possess unique lipid compositions. The tight junction, the structure separating the two domains, forms a diffusion barrier for membrane components and thereby prevents intermixing of the two sets of lipids. The barrier apparently resides in the outer, exoplasmic leaflet of the plasma membrane bilayer. First data are now available on the generation of these differences in Madin‐Darby canine kidney (MDCK) cells, grown on filter supports. Experiments in which fluorescent precursors of apical lipids were introduced into the cell have demonstrated that upon biosynthesis apical lipids are sorted from basolateral lipids in an intracellular compartment. In this paper we present a model for the sorting process, the central point of which is that the two sets of lipids laterally segregate into microdomains that bud to form vesicles delivering the lipids to the apical and the basolateral plasma membrane domains, respectively.
Characterization of yeast clathrin and anticlathrin heavy‐chain monoclonal antibodies
Authors: Lemmon Sandra K. , Lemmon Vance P. , Jones Elizabeth W.
Published: December 2004

Clathrin‐coated vesicles (CVs) were isolated from Saccharomyces cerevisiae by using procedures developed by Mueller and Branton [17]. Triskelions were purified from this material by extraction of CVs to release clathrin and by subsequent fractionation on Sepharose CL‐4B. Triskelions were composed of ∼ 180,000 Mr heavy chains and a single light‐chain type of ∼ 38,000 Mr and were able to undergo self‐assembly into polyhedral cages. Trypsin digestion of such reassembled cages showed a peptide pattern very similar to that obtained for mammalian clathrin with two fragments of 125,000 and 110,000 Mr, which represent the major portion of the heavy‐chain arm, and a polypeptide of ∼ 43,000 Mr, which is the presumptive terminal domain.

Eight monoclonal antibodies reacting with yeast clathrin heavy chains were produced. All eight bind to the major portion of the heavy‐chain arm, and none bind to the terminal domain fragment. Peptide digestion experiments also indicated that at least three major regions on the arm are recognized by these antibodies. These will be useful in further structural and functional studies of clathrin from yeast.
Correlation of immunomodulatory and therapeutic activities of interferon and interferon inducers in metastatic disease
Authors: Black Paul L. , Phillips Hamblin , Tribble Henry R. , Pennington Robin , Schneider Mark , Talmadge James E.
Published: December 2004

The mechanism of therapeutic activity of recombinant murine interferon‐gamma (rMu IFN‐γ) and the IFN inducer polyinosinic‐polycytidylic acid solubilized with poly‐L‐lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16‐BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN‐γ or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine‐activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN‐γ and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN‐γ and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN‐γ, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN‐γ. LAK activity at any site did not correlate with the therapeutic activity of either agent.
Increased content of chondroitin sulfate proteoglycan in human colorectal carcinoma metastases compared with the primary tumor as determined by an anti‐chondroitin‐sulfate monoclonal antibody
Authors: Yamori Takao , Ota David M. , Cleary Karen R. , Irimura Tatsuro
Published: December 2004

To determine if the amount of chondroitin sulfate proteoglycan (CSPG) in human colorectal tumor tissue correlates with the tumor's aggressiveness we immunochemically determined the CSPG levels in colorectal carcinomas at different stages. A total of 50 specimens—4 polyps, 15 stage B tumors, 9 stage C tumors, 12 stage D tumors, 7 liver metastases, and 3 lymph node metastases—were examined. Tumor tissues were extracted with 4 M guanidine hydrochloride containing protease inhibitors. The extracts were serially diluted and blotted onto nitrocellulose membranes. Reactivity of a chondroitin sulfate‐specific mouse monoclonal antibody (CS‐56) was determined by biotinylated goat antimouse Ig and avidin‐biotin‐peroxidase complex. After comparing tissues from tumors at different stages (classified by the presence or absence of metastasis), we could not find a positive or negative correlation between the amount of CSPG in primary colorectal carcinoma tissues and the tumor's metastatic potential. However, the metastatic foci in the liver or lymph node contained higher amounts of CSPG than the primary tumors did. Immunohistochemical staining of colon carcinoma tissue with CS‐56 revealed that CSPG is predominantly localized in fibrotic portions in the tumor tissues. Two‐year follow‐up studies indicated that a high level of CSPG in primary tumors was not predictive of recurrence.
Inhibition of metastatic potential by fucosidase: An NMR study identifies a cell surface metastasis marker
Authors: Wright Lesley C. , May George L. , Gregory Patricia , Dyne Marlen , Holmes Kerry T. , Williams Philip G. , Mountford Carolyn E.
Published: December 2004

NMR spectroscopy is able to detect subtle changes to the surface chemistry of cells. We have previously shown that high‐resolution 1H NMR methods can identify tumor cells with the capacity to metastasize, and we now report that the long T2 relaxation value (500–800 ms) observed in metastatic rat mammary adenocarcinoma cells is removed by treatment with fucosidase. Two‐dimensional scalar‐correlated NMR (COSY) spectra of fucosidase‐treated cells show that a cross peak, consistent with scalar coupling between the methyl and methinè groups on fucose and usually associated with malignancy and metastatic ability, is absent. Metastases were observed in only two out of ten rats injected subcutaneously with enzyme‐treated cells compared to eight out of ten with untreated cells. NMR studies on isolated cellular lipids identified the long T2 relaxation value only in the ganglioside fraction. This fraction accounts for 51% of the total 14C‐labelled fucose incorporated into the cells. We propose that fucogangliosides are an indicator of metastatic potential in rats. The observation that a cell surface metastasis marker has an NMR signal with a characteristically long relaxation value has important consequences for the future use of magnetic resonance imaging and spectroscopy in the cancer clinic.
Cell surface heparan sulfate proteoglycan and the neoplastic phenotype
Authors: Iozzo Renato V.
Published: December 2004

Cell surface proteoglycans are strategically positioned to regulate interactions between cells and their surrounding environment. Such interactions play key roles in several biological processes, such as cell recognition, adhesion, migration, and growth. These biological functions are in turn necessary for the maintenance of differentiated phenotype and for normal and neoplastic development. There is ample evidence that a special type of proteoglycan bearing heparan sulfate side chains is localized at the cell surface in a variety of epithelial and mesenchymal cells. This molecule exhibits selective patterns of reactivity with various constituents of the extracellular matrix and plasma membrane, and can act as growth modulator or as a receptor. Certainly, during cell division, membrane constituents undergo profound rearrangement, and proteoglycans may be intimately involved in such processes. The present work will focus on recent advances in our understanding of these complex macromolecules and will attempt to elucidate the biosynthesis, the structural diversity, the modes of cell surface association, and the turnover of heparan sulfate proteoglycans in various cell systems. It will then review the multiple proposed roles of this molecule, with particular emphasis on the binding properties and the interactions with various intracellular and extracellular elements. Finally, it will focus on the alterations associated with the neoplastic phenotype and will discuss the possible consequences that heparan sulfate may have on the growth of normal and transformed cells.
Tissue‐specific analogues of erythrocyte protein 4.1 retain functional domains
Authors: Anderson Richard A. , Correas Isabel , Mazzucco Charles , Castle J. David , Marchesi Vincent T.
Published: December 2004

Analogues of the human erythroid membrane skeletal component protein 4.1 have been identified in perfused rat tissues and human T and B lymphocyte cell lines, olyclonal antibodies were used which are specific for all domains of protein 4.1, the spectrin‐actin‐promoting 8‐Kd peptide, the membrane‐binding 30‐Kd domain, and the 50‐Kd domain. Antibody reactivity, by Western blotting of tissue homogenates, shows reactivity with proteins varying in molecular weight from 175 Kd to 30 Kd. Further, these protein 4.1 analogues appear to be expressed in a tissue‐specific fashion. Of the analogues detected there appear to be at least three classes: analogues containing all erythroid protein 4.1 domains, analogues containing all domains but with modified antigenic epitopes, and analogues containing only some domains. Chemical cleavage at cysteine linkages indicates that in analogues containing the 30‐Kd region the location of cysteine is highly conserved. This datum suggests that in nonerythroid 4.1 isoforms of higher molecular weight the additional protein mass is added to the amino terminal end (30 Kd end).
Relationship of pseudopod extension to chemotactic hormone‐induced actin polymerization in amoeboid cells
Authors: Hall Anne L. , Schlein Anthony , Condeelis John
Published: December 2004

Aggregation‐competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells.

Changes in cell behavior were correlated with F‐actin content by using an NBD‐phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D‐sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F‐actin assembled by 60 sec is localized in these new pseudopods.

These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.
Insulin and IGF‐1 receptors contain covalently bound palmitic acid
Authors: Magee A. I. , Siddle K.
Published: December 2004

We have studied the biosynthesis of the insulin receptor in a human hepatoma cell line, HepG2. As previously reported, these cells synthesize a disulphide–bonded α2 β2 tetrameric insulin receptor. Labelling of HepG2 cells with [3H]palmitate or [3H]myristate followed by immunoprecipitation with a polyclonal antireceptor antibody revealed the incorporation of palmitate, but not myristate, into the β‐subunit and αβ‐precursor of the receptor in a hydroxylamine‐sensitive linkage. The extracellular α‐subunit was not labelled, demonstrating the specificity of incorporation. Acylation of the insulin receptor was an early event as judged by fatty acid incorporation into the αβ‐precursor and prevention by protein synthesis inhibitors. Pulse‐chase studies demonstrated the expected processing of the αβ‐precursor to mature α‐ and β‐subunits, but no evidence for preferential turnover of the fatty acid moiety was found. The site of acylation appears to be in the transmembrane or cytoplasmic domain since proteolytic treatment of intact cells produced a truncated β‐subunit still containing label. Binding studies showed that HepG2 cells contain approximately half as many insulin‐like growth factor‐1 receptors as insulin receptors, raising the possibility that this receptor may also be acylated. Indeed, immunoprecipitation with the antiinsulin receptor serum of MDCK cells expressing IGF‐1 receptors, but not insulin receptors, revealed bands corresponding to the αβ‐precursor, α‐ and β‐subunits, of which the αβ‐precursor and β‐subunits incorporated [3H]palmitate but the α‐subunit did not.
A search for EGF‐elicited degradation products of the EGF receptor
Authors: Stoscheck Christa M. , Gates Ronald E. , King Lloyd E.
Published: December 2004

Epidermal growth factor (EGF) induces the degradation of EGF receptors in both human foreskin fibroblasts and A‐431 cells. Similar degradation products of 125I‐EGF covalently linked to its receptor appeared at the same times in both A‐431 cells and fibroblasts when the cells were exposed to a concentration of 10 ng/ml EGF. Although the products between the two cell types differed in molecular weight, this was at least partly caused by an actual difference in the receptor proteins from the two cell types (as shown by partial proteolysis) rather than from different pathways of receptor degradation. However, when EGF receptors were biosynthetically labeled, no receptor degradation products could be observed, even when the receptor was labeled with radioactive mannose or phosphate, molecules which would predominantly label the outside or inside face of the receptor, respectively. At 20°C, degradation of the receptor slowed and a 150,000‐dalton degradation product was observed. This degradation product has previously been observed in cell homogenates produced in the presence of calcium, mediated by calpain. Thus, calpain may play a role in the intracellular degradation of the EGF receptor.
Epidermal growth factor‐stimulated DNA synthesis requires an influx of extracellular calcium
Authors: Hill Timothy D. , Kindmark Henrik , Boynton Alton L.
Published: December 2004

The dependency of normal cell proliferation on adequate extracellular Ca2+ levels was further investigated by determining the role of Ca2+ influx in epidermal growth factor (EGF)‐induced rat liver epithelial (T51B) cell DNA synthesis. Fura‐2‐loaded T51B cells responded with an increase in [Ca2+]i to EGF (5–50 ng/ml) that was blocked by low (25 μM) extracellular Ca2+ or by pretreatment with 50 μM La3+ to inhibit plasma membrane Ca2+ flux. Confluent T51B cells treated for 24 h with EGF (0.1–50 ng/ml) dose‐dependently incorporated [3H]‐thymidine into cell nuclei. Low extracellular Ca2+ or addition of La3+ prevented the EGF stimulated rise in labeled nuclei, indicating that a movement of Ca2+ into the cell was required for DNA synthesis. This was supported by our findings that bradykinin, which induced a rise in [Ca2+]i by opening plasma membrane Ca2+ channels in T51B cells (but not A23187, thrombin or ATP, which raise [Ca2+]i primary through mobilization of intracellular Ca2+ stores), potentiated DNA synthesis stimulated by submaximal doses of EGF. Potentiation of the action of EGF by the tumor promoter 12‐0‐tctradecanoyl‐phorbol‐13‐acetatc (TPA), indicates that activation of protein kinase C and an influx of Ca2+ share a common mechanism for initiating DNA synthesis.
Defection of minor immunological differences among human “universal‐type” alkaline phosphatases
Authors: Goseki Masae , Oida Shinichiro , Sasaki Satoshi
Published: December 2004

Two clones of monoclonal antibodies against swine alkaline phosphatase (ALPase; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1), which were useful in distinguishing human kidney and bone ALPases from liver ALPase, were successfully raised in mice. On the other hand, polyclonal antibody cross‐reacted not only with human kidney ALPase but also with all other human universal type ALPases. The difference in cross‐reactivitiy of monoclonal and polyclonal antibodies may be caused by the specific antigenicity of human enzymes. The monoclonal antibodies were able to recognize minor heterogeneity that could not be distinguished by their enzymatic properties. The present monoclonal antibody preparations will be utilized for clinical as well as basic investigations to detect minor heterogeneity among universal‐type ALPases.
Activation and membrane binding of carboxypeptidase E
Authors: Fricker Lloyd D.
Published: December 2004

Carboxypeptidase E (CPE) is a Carboxypeptidase B‐like enzyme that is thought to be involved in the processing of peptide hormones and neurotransmitters. Soluble and membrane‐associated forms of CPE have been observed in purified secretory granules from various hormone‐producing tissues. In this report, the influence of membrane association on CPE activity has been examined. A substantial amount of the membrane‐associated CPE activity is solubilized upon extraction of bovine pituitary membranes with either 100 mM sodium acetate buffer (pH 5.6) containing 0.5% Triton X‐100 and 1 M NaCl, or by extraction with high pH buffers (pH > 8). These treatments also lead to a two‐ to threefold increase in CPE activity. CPE extracted from membranes with either NaCl/Triton X‐100 or high pH buffers hydrolyzes the dansyl‐Phe‐Ala‐Arg substrate with a lower Km than the membrane‐associated CPE. The Vmax of CPE present in extracts and membrane fractions after the NaCl/Triton X‐100 treatment is twofold higher than in untreated membranes. Treatment of membranes with high pH buffers does not affect the Vmax of CPE in the soluble and particulate fractions. Pretreatment of membranes with bromoacetyl‐D‐arginine, an active site‐directed irreversible inhibitor of CPE, blocks the activation by NaCl/Triton X‐100 treatment. Thus the increase in CPE activity upon extraction from membranes is probably not because of the conversion of an inactive form to an active one, but is the result of changes in the conformation of the enzyme that effect the catalytic activity.