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H Rubin

Publications and source records attributed to H Rubin.

At least 19 recordsLinked to original sources
PubMed1995

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.

3T3 Cells↗
PubMed1995

Identification of lysines within alpha 1-antichymotrypsin important for DNA binding. An unusual combination of DNA-binding elements.

The human serum serine protease inhibitor (serpin) alpha 1-antichymotrypsin (ACT) appears to be unique among serpins in its ability to bind to double-stranded DNA. Using site-directed mutagenesis and chemical modification, a tri-lysine sequence (residues 210-212) falling within a solvent exposed loop and the C-terminal peptide containing two lysines (residues 391 and 396) were shown to be important for DNA binding. Mutation of residues 210-212 from lysines to either glutamates or threonines abolished DNA binding. The Lys210-Thr211-212 and Thr210-Th4(211)-Lys212 variants displayed reduced affinity for DNA, especially at higher ionic strength. Limited acetylation of rACT with acetic anhydride led to loss of DNA binding and, conversely, DNA protected rACT from acetylation. A combination of CNBr digestion, peptide separation, and peptide sequencing identified Lys396, two residues from the C terminus, as the most reactive lysine in rACT. Acetylation of Lys396 is strongly decreased in the presence of DNA. The double mutant K391T/K396T-rACT had very little affinity for DNA. The epsilon-amines of lysines 210-212 are 8-15 A across a cleft from the epsilon-amines in Lys391 and Lys396, and together these two elements may form an unusual DNA binding domain. Attempts to isolate a DNA sequence to which ACT binds specifically have been unsuccessful to date, raising the possibility that nonspecific binding of ACT to DNA suffices to account for the ACT found in certain cell nuclei. ACT variants not binding to double-stranded DNA retain ACT protease inhibitory activity, a potentially important result for the use of ACT variants as therapeutic agents.

Acetylation↗
PubMed1995

Heritable, population-wide damage to cells as the driving force of neoplastic transformation.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.

3T3 Cells↗
PubMed1995

Acute gastroenteritis in three community-based nursing homes.

BACKGROUND: Acute gastroenteritis is a cause of considerable morbidity and mortality in the elderly population. A prospective assessment of acute diarrhea in three community-based long-stay homes is described. METHODS: A cohort study of acute gastroenteritis was performed in three community-based nursing homes, involving 572 residents over an 8-month period. Diarrhea cases were enrolled on the basis of the acute onset of loose stools of > or = 24 hours, as well as one of the following: a rectal temperature of > or = 100 degrees F, dehydration, positive occult blood, > or = 48 hours duration, or as a part of any outbreak. Stool cultures for Clostridium difficile were performed on all NH 1 patients. RESULTS: Fifty-three gastroenteritis cases were ascertained, consistent with incidence rates of 14.6, 36.4, and 6.7 cases/100 patient years in NH 1, NH 2, and NH 3, respectively. Requiring a Foley catheter (OR = 2.57; 95% CI, 0.93, 7.09) increased diarrhea risk. Six Clostridium difficile enteritis cases and an episode attributable to Aeromonas/Pleisomonas species were diagnosed. One C. difficile diarrhea case was imported from hospital to NH 1. Ten of 12 fecal excretors resided in close geographic clusters in NH 1, where a majority of the latter were mobile and incontinent of stool. CONCLUSIONS: Acute gastroenteritis was a common disease in the study nursing homes, for which specific risk factors were identified. A predominant role for Clostridium difficile in the taxonomy of nursing home diarrhea was suggested.

Acute Disease↗
PubMed1995

New strategies for the control of viral infection in organ transplantation.

Control of viral infection in organ transplant recipients requires attention to the following interventions: prevention, whenever possible of viral acquisition; the proper deployment of active and passive immunization, with hyperimmune globulin preparations directed against cytomegalovirus, hepatitis B, varicella, and, perhaps, respiratory syncytial virus, offering significant benefit when used appropriately; and the prescription of antiviral agents at critical points in the post-transplant course. Two important principles should be kept in mind when approaching this problem: prevention is the goal, as treatment of established infection is fraught with difficulty; and effective preventative strategies must be linked to the intensity of the immunosuppressive program being employed. To achieve these goals, the addition of pre-emptive therapy (therapy geared to a laboratory marker of impending disease or to escalation in the immunosuppressive program) to standard prophylactic regimens represents a significant advance.

Antiviral Agents↗
PubMed1994

Incipient and overt stages of neoplastic transformation.

A subline of NIH 3T3 cells was developed that multiplies to low saturation density and produces no transformed foci in a standard assay at confluence if the cells had been kept in continuous exponential growth by passage at extremely low density. Repeated cycles of extended incubation at confluence result in the production of large dense foci with large increases in saturation density of the culture. I investigated the changes preceding overt transformation and the conditions that favor them by making a preliminary 2- or 3-week incubation of cells at high density in 2, 5, or 10% (vol/vol) calf serum (CS) followed by three consecutive assays at 2-week intervals, all in 2% CS. The cell counts on transfer of each consecutive assay constituted their saturation density, and staining of sister dishes exhibited transformed foci and other morphological evidence of transformation. To strengthen the significance of the observations, each CS category consisted of four separate but parallel lineages. The initial incubation in 2, 5, and 10% CS is referred to as the first-round (1 degree) assay. In the second-round (2 degrees) assay in 2% CS after the primary incubation for 2 weeks in different concentrations of CS, there were small but consistent increases in saturation density that were proportional to the CS concentration used in the first round. There were also large numbers of very light foci in the 2 degrees assay that increased in size as a function of the CS concentration used in the 1 degree assay but were uniform within each CS category. In the 3 degrees and 4 degrees successive assays, large dense foci appeared in a sporadic manner but these were also dependent on the CS concentration in the initial incubation. Cultures kept in the 1 degree assay in 5 and 10% CS for 3 weeks produced large dense foci at an earlier stage of the subsequent assays than did those kept for only 2 weeks in the 1 degree assay. The barely perceptible nature of the early morphological changes suggests an analogy to incipient neoplasia. Their graded nature, relatively high frequency, regularity of occurrence, and uniformity of appearance are characteristic of an epigenetic process, which Foulds [Foulds, L. (1969) Neoplastic Development, Vol. I (Academic, New York), preface, pp. 41-89] cited as the most plausible basis for incipient neoplasia [corrected]. In contrast, the low-frequency and sporadic occurrence of the large dense foci, which have the main features of overt neoplasia, are consistent with both genetic and epigenetic processes.

3T3 Cells↗
PubMed1994

A critical test of the role of population density in producing transformation.

Cells of the NIH 3T3 line gain the capacity to produce neoplastically transformed foci when they are maintained at high density for more than 1 week and transferred in a standard assay for focus formation. This change in cell behavior has been variously attributed to an adaptive response to the constraint of the high population density or to a spontaneous genetic change that increases in probability for a culture with the increase in the total number of cell divisions. To distinguish between these alternatives, 200 cells of the 28H subline were seeded in many culture dishes of two size classes differing 6-fold in surface area and allowed to multiply for 1, 2, and 3 weeks. At each weekly interval, 18 dishes of each class were assayed for focus formation, and two of the original dishes were stained for focus formation. The cells in the small (S) and large (L) dishes multiplied to the same extent at 1 week and produced only a few small light foci in some of the assay dishes. At 2 weeks, cells in the S dishes had become confluent and had only one-third the number of cells as those in the nonconfluent L dishes. Upon assay, 14 of the 18 S cultures produced some foci whereas only 9 of the L cultures did so. In addition, 4 of the S cultures produced large dense foci while none of the L cultures did. By 3 weeks, the L cultures were confluent and had four times as many cells as the S cultures. When assayed at this time, both sets produced dense foci in many of the cultures and light foci in the remaining ones, indicating a narrowing of the differences between the S and L cultures between 2 and 3 weeks of incubation. There were differences in the morphology of the foci produced in parallel assays from different cultures. The results showed that transformation is a diverse graded response to the growth constraint of high population density and not a spontaneous event dependent on the number of cell divisions in a cell culture. Transformation thus is basically an epigenetic process since it represents a response to physiological restraint, but the final form of response may be modulated by genetic alterations.

3T3 Cells↗
PubMed1994

Experimental control of neoplastic progression in cell populations: Foulds' rules revisited.

Foulds introduced six rules of tumor progression based on his observations of spontaneous mammary cancer in mice and generalized them to all forms of neoplasia [Foulds, L. (1954) Cancer Res. 14, 327-339 and Foulds, L. (1969) Neoplastic Development (Academic, New York), Vol. 1, preface and pp. 72-74.] Rules III, IV, and V are considered controversial, and research in animals seems inadequate to resolve the controversies. A subline of NIH 3T3 cells undergoes progressive transformation to produce foci of increasing population density when repeatedly constrained by sequential rounds of growth to and maintenance at confluence. Analysis of the results provides a cellular basis for rules III, IV, and V. Rule III states that progression is independent of the growth of the tumor and occurs in tumors that are arrested. Cell culture shows that progression is actually favored by constraint of growth, a result inconsistent with a major role for point mutations in progression. Indeed, there is a suggestion that the transformation may arise from chromatin changes preceding apoptosis. Rule IV states that progression can be gradual or abrupt but the latter conclusion has been frequently criticized. Cell culture exhibits both forms of progression but, in particular, eliminates the doubt about the abrupt form. Rule V, which is in a sense an extension of rule IV, states that progression follows one of alternative paths of development. The results in culture indicate that every independent transforming event gives rise to foci of unique morphology. Thus, even for the single characteristic of transformed focus morphology, many alternative paths to neoplasia are available to cells. In addition to clarifying the rules of progression, a method is described for pinpointing the time of the occurrence of events that are only expressed as dense foci after a variable lag time. The results in culture reinforce Foulds' conclusion that neoplastic development is primarily an epigenetically driven process and identify some of the cellular interactions that underlie that process.

3T3 Cells↗
PubMed1994

Conversion of alpha 1-antichymotrypsin into a human neutrophil elastase inhibitor: demonstration of variants with different association rate constants, stoichiometries of inhibition, and complex stabilities.

Despite the homology with alpha 1-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibitor of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutrophil elastase (HNE) with wild-type recombinant ACT and recombinant variants of ACT were studied. ACT variants were generated where (1) the primary interaction site, the P1 position, was replaced with the P1 residue of alpha 1PI, (2) the residues corresponding to P3-P3' were replaced with those of alpha 1PI, and (3) the residues corresponding to the canonical recognition sequence as well as flanking residues encompassing the exposed reactive loop of the inhibitor were replaced with the corresponding residues of alpha 1PI. Each variant was analyzed to determine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate constant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiometry of inhibition, SI), and (3) the stability of the enzyme-serpin complex. Replacing Leu with Met in the P1 position (rACT-L358M) was sufficient to convert rACT into an inhibitor of HNE with an apparent second-order rate constant (k'/[I]) of 4 x 10(4) M-1 s-1 and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in the reactive loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions.

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2- and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.

Amino Acid Sequence↗
PubMed1994

Selective nature of phorbol 12-myristate 13-acetate-induced neoplastic transformation in NIH 3T3 cells.

The ability of phorbol 12-myristate 13-acetate (PMA) at 0.02 microgram/ml to induce neoplastic transformation in NIH 3T3 cells is highly dependent on the culture conditions. Optimal transformation, indicated by the saturation density and extent of focus formation in transferred cultures raised under standard conditions, was observed when the original cells were grown in 2% calf serum (CS) and exposed continually to PMA for at least 4 weeks before transfer into the assay. Transformation of stationary cultures in 10% CS occurred later and to a lesser degree than in 2% CS. The same cells subjected to thrice-weekly transfer in 2% or 10% CS at low cell density so that they were in a constant state of exponential growth exhibited no evidence of transformation in response to PMA. This strong condition-dependence of PMA-enhanced transformation is indicative of a selection process similar to that described for spontaneous transformation. In both cases, transformation is promoted by inhibiting multiplication and prevented by maximizing multiplication. Therefore, it has the earmarks of an epigenetic rather than a mutational process and requires phenotypic rather than genotypic variation to supply the states for selection. The concept of "progressive state selection," originally proposed to account for spontaneous transformation, can also account for PMA-enhanced transformation.

3T3 Cells↗
PubMed1994

Hepatotoxicity related to total parenteral nutrition: comparison of low-lipid and lipid-supplemented solutions.

PURPOSE: Because it is unclear whether or not the lipid or carbohydrate component of total parenteral nutrition solutions determines the development of cholestasis during total parenteral nutrition, a prospective randomized clinical trial of a predominantly carbohydrate solution (group I) versus one with isocaloric substitution of 30% nonprotein calories with lipid (group II) was performed. METHODS: Twelve patients in group I, 4 female and 8 male, and 14 patients in group II, 8 female and 6 male, were studied. Each subject received 100% to 150% of the basal energy needs, determined by the Harris-Benedict equation. Amino acids were supplied to provide a protein:kilocalorie ratio of 1:150 to 200. There were no differences between the groups in terms of mortality, nitrogen balance, and maintenance of weight. Weekly serum aspartate aminotrasferase (AST) and alkaline phosphatase (AP) levels were obtained. The change from baseline values was calculated. RESULTS: The observed difference between the two groups for AST was 5.7 (higher for group II), P = .55, and for AP, 39.1 (higher for group I), P = .23. A power calculation was performed to determine the number of patients required for a statistically significant difference in AP between the two groups with an alpha of 0.05 and a power of 0.85:67 patients. CONCLUSIONS: With these statistical considerations, we conclude that there was probably no statistically significant difference between the groups for an increase in AST or AP levels during total parenteral nutrition.

Adult↗
PubMed1994

Crystal structure of an uncleaved serpin reveals the conformation of an inhibitory reactive loop.

The three-dimensional structure of an uncleaved serpin, a variant of human antichymotrypsin engineered to be an inhibitor of human neutrophil elastase, has been determined by X-ray crystallographic methods and is currently being refined at 2.5 A resolution. It contains an intact reactive loop in a distorted helical conformation. A comparison of the current model with that of its cleaved counterpart suggests that the conformational 'stress' of the serpin in its uncleaved and uncomplexed state may not be confined solely to the reactive loop or beta-sheet A. It is intriguing that strand s4A is not pre-inserted into beta-sheet A of the native serpin, and this has profound implications for the mechanism of serpin function.

Amino Acid Sequence↗
PubMed1994

Isolation of ribonucleotide reductase from Mycobacterium tuberculosis and cloning, expression, and purification of the large subunit.

Ribonucleotide reductase, an allosterically regulated, cell cycle-dependent enzyme catalyzing a unique step in the synthesis of DNA, the reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, was purified 500-fold from Mycobacterium tuberculosis Erdman strain through cell disruption, ammonium sulfate fractionation, and dATP-Sepharose affinity column chromatography. As in eucaryotes and certain bacteria and viruses, the M. tuberculosis enzyme consists of two nonidentical subunits, R1 and R2, both of which are required for activity. R1 has a molecular mass of 84 kDa, as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with dATP. The amino acid sequences of the N-terminal peptide and two internal peptides were determined, and a partial R1 gene was isolated by PCR with primers designed from these amino acid sequences. Additional coding sequences were isolated by screening size-selected libraries, and a full-length form of M. tuberculosis R1 was generated by PCR amplification of high-molecular-weight M. tuberculosis DNA and expressed in Eschericnia coli. This coding sequence is 2,169 nucleotides long and contains no introns. The predicted molecular mass of R1 from the DNA sequence is 82,244 Da. Recombinant M. tuberculosis R1, purified to homogeneity, was biochemically active when assayed with extracts of M. tuberculosis enriched for R2.

Adenosine Triphosphate↗
PubMed1993

R2 C-terminal peptide inhibition of mammalian and yeast ribonucleotide reductase.

Eucaryotic ribonucleotide reductases (RR) catalyze the reduction of ribonucleoside diphosphates to 2'-deoxyribonucleoside diphosphates. Each has an R1(2)R2(2) quaternary structure with each subunit playing a critical role in catalysis. Separation of the subunits results in loss of activity. Previous studies have demonstrated that peptides corresponding to the C-terminus of R2 disrupt subunit association by competion with R2 and have potential usefulness as therapeutics. Extensive structure-function studies have been carried out on peptide inhibition of herpes simplex RR in an effort to develop antiviral agents based on the observation that the herpes simplex R2 C-terminus, YAGAVVNDL, is quite different from the corresponding mammalian sequence. In this work we report a detailed structure-function analysis of peptide inhibition of mammalian and, to a more limited extent, Saccharomyces cerevisiae RRs. Our results for mammalian RR support the following conclusions with regard to the effect of substitution on inhibitory potency: (a) the N-acetylated R2 C-terminal heptapeptide N-AcPhe384Thr385Leu386Asp387Ala388Asp389Phe390 (N-AcF7TLDADF1) is the minimal core peptide length required; deletion of the N-terminus or of middle positions (resulting in penta- and hexapeptides) results in large losses in inhibitory potency; (b) a free carboxylate is required on the C-terminal Phe; (c) Phe is strongly preferred to Leu in positions 1 and 7 and a bulky aliphatic group is preferred in position 5; (d) neither negative charge in positions 2 or 4 nor a polar side chain in position 6 are required for peptide binding, contrary to what evolutionary patterns in the R2 C-terminus of RR would suggest. S. cerevisiae RR displays a similar length dependence on the corresponding N-acetylated R2 C-terminal heptapeptide, N-AcFTFNEDF. This peptide has a 4-fold higher inhibitory potency toward S. cerevisiae RR than toward mammalian RR. Such selectivity raises the possibility that peptide analogs related to R2 C-termini can be developed as therapeutic agents even against organisms having R2 C-terminal sequences similar to that of mammalian RR.

Acetylation↗
PubMed1993

Automatic enumeration and characterization of heterogeneous clonal progression in cell transformation.

Most human tumors are clonal in origin, although the cells may be diverse in their properties. Since the tumors evolve through progressive stages over decades of time, it is possible that the conditions that induced the tumor transform many cells, but that selective overgrowth of the fastest growing lead to a clonal population of identified tumor cells. We studied the progression of neoplastic transformation in clones from a population in which about 10% of the cells formed well-defined transformed foci. A few of the clones produced many large foci, but most of the clones produced no foci or only one focus. Maintenance of the nonproducing and low-producing clones under the growth constraint of confluence and low serum concentration, which promotes transformation, led to the production of large numbers of small foci by all of them. Visual inspection revealed considerable heterogeneity in size and density among the foci from each clone, and this was quantitated by computer scanning. Subclonal analysis of focus formation was done on one of the clones after it had undergone further growth constraint to promote transformation. As in the original cloning, some of the subclones produced many large foci, but most produced none. Another round of growth constraint was imposed on the nonproducing subclones, which then became producers of many small foci varying in size and density. The results indicate that most if not all cells in the population respond to growth constraint by undergoing transformation. Though there is wide variation in the degree of transformation, the results are consistent with the view that an entire field of cells exposed to carcinogenic conditions in an animal undergoes some progression toward neoplasia but that heterogeneity of the response followed by selective neoplastic growth may lead to a clonal origin of the clinically detected tumor.

3T3 Cells↗