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H Rubin

Publications and source records attributed to H Rubin.

At least 109 records · Page 6Linked to original sources
PubMed1993

Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex.

The interactions of bovine pancreatic chymotrypsin (Chtr) and recombinant alpha 1-antichymotrypsin (rACT) and rACT variants were studied by kinetic and gel electrophoretic analyses, leading to the formulation of a general kinetic scheme that accounts for all known results concerning this serpin-protease pair, as well as for results obtained with other such pairs. Incubation of rACT and Chtr leads rapidly to the formation of an inhibited complex, Chtr.rACT*, that is stable toward sodium dodecyl sulfate denaturation and boiling. The extent of release of active Chtr from this complex increases markedly as ionic strength, mu, is raised. The kinetic scheme quantitatively accounts for this effect on the basis of a partitioning of Chtr.rACT* between dissociation of the complex to yield active enzyme and cleaved rACT, and Chtr-catalyzed conversion of the complex to a form that is much more resistant to release of active enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of reaction mixtures of rACT and Chtr are consistent with the scheme. Also consistent are the results of experiments measuring the effects of 1) Chtr.rACT* concentration, 2) uncomplexed Chtr, and 3) added alpha 2-macroglobulin on active Chtr release from Chtr.rACT*. Proteolysis of Chtr.rACT* to give a resistant complex is also catalyzed by human neutrophil elastase, a process with potential physiological relevance. Comparison of the rates of Chtr dissociation from the complexes formed with rACT and with rACT variants mutated at the P1 site suggests that such rates are more sensitive to P1 substitution at low mu than at high mu. Several equivalents of the L358R-rACT variant are required for full inhibition of Chtr. This observation is also quantitatively accounted for by the proposed kinetic scheme, on the basis of another partitioning step between L358R-rACT acting as a substrate or as an inhibitor toward Chtr.

Animals↗
PubMed1993

Reaction of human chymase with reactive site variants of alpha 1-antichymotrypsin. Modulation of inhibitor versus substrate properties.

Inhibition of human chymase by alpha 1-antichymotrypsin produces 3.5 mol of degraded inhibitor for every mol of chymase inhibited, resulting in a stoichiometry of inhibition (SI) of 4.5. In the present study, the substrate versus inhibitor properties of this reaction were examined further using wild type and mutant recombinant antichymotrypsins (rACT). Titration of chymase hydrolytic activity with rACT-L358 (wild type) and reactive site (P1) variants of ACT, L358W, L358M, and L358F revealed that the SI was sensitive to P1 residue replacements. SI values increased in the order of Trp < Met < Leu < Phe where SI values were 1.5, 2, 4, and 7, respectively. Chymase inhibitor complex and cleaved inhibitor were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for all variants; the relative intensities of each band were consistent with SI values established by titration. NH2-terminal sequence analyses of the products formed in the reaction of chymase with rACT-L358F indicated that the P1-P1' bond was the primary site of cleavage resulting in the hydrolysis and inactivation of this variant. The apparent second-order rate constant for chymase inhibition (k'/[I]) by rACT also was affected by P1 substitution. k'/[I] values increased in an order opposite that obtained for SI values (Phe < Leu < Met < Trp). The reactive loop mutant (rACT-P3P3') produced by replacing the reactive site region of ACT (Thr356-Val361) with that of alpha 1-proteinase inhibitor (Ile356-Pro361) revealed a different reaction pattern. Although its SI was near 1, the value for k'/[I] was the lowest among variants. rACT-L358R, another P1 variant, did not inhibit chymase. These results are evaluated with respect to the substrate preferences of human chymase and with respect to partitioning schemes proposed to explain SI values greater than 1.

Amino Acid Sequence↗
PubMed1993

Crystallization and atomic resolution X-ray diffraction analysis of antichymotrypsin variants.

Crystals of two recombinant antichymotrypsin (rACT) variants have been prepared: variant rACT-T345R crystallizes in space group P2(1) (a = 109.2 A, b = 79.4 A, c = 111.9 A, beta = 116.3 degrees, with 2 molecules in the asymmetric unit), and variant ACT' crystallizes in space group P2(1)22(1) (a = 69.7 A, b = 77.2 A, c = 83.8 A, with one molecule in the asymmetric unit). The latter variant is an engineered dimer having the P3-P3' hexapeptide sequence of the related serpin, alpha 1-proteinase inhibitor, substituted for the corresponding wild-type sequence. Crystals of each variant diffract to a limiting resolution of 2.5 A, which represents the best diffraction yet achieved for a crystalline, inhibitory serpin. The exceptional quality of ACT' crystals probably arises from favorable protein-protein interactions as well as a stabilizing disulfide crosslink engineered between the monomers.

Chymotrypsin↗
PubMed1993

Cloning, sequence determination, and regulation of the ribonucleotide reductase subunits from Plasmodium falciparum: a target for antimalarial therapy.

Malaria remains a leading cause of morbidity and mortality worldwide, accounting for more than one million deaths annually. We have focused on the reduction of ribonucleotides to 2'-deoxyribonucleotides, catalyzed by ribonucleotide reductase, which represents the rate-determining step in DNA replication as a target for antimalarial agents. We report the full-length DNA sequence corresponding to the large (PfR1) and small (PfR2) subunits of Plasmodium falciparum ribonucleotide reductase. The small subunit (PfR2) contains the major catalytic motif consisting of a tyrosyl radical and a dinuclear Fe site. Whereas PfR2 shares 59% amino acid identity with human R2, a striking sequence divergence between human R2 and PfR2 at the C terminus may provide a selective target for inhibition of the malarial enzyme. A synthetic oligopeptide corresponding to the C-terminal 7 residues of PfR2 inhibits mammalian ribonucleotide reductase at concentrations approximately 10-fold higher than that predicted to inhibit malarial R2. The gene encoding the large subunit (PfR1) contains a single intron. The cysteines thought to be involved in the reduction mechanism are conserved. In contrast to mammalian ribonucleotide reductase, the genes for PfR1 and PfR2 are located on the same chromosome and the accumulation of mRNAs for the two subunits follow different temporal patterns during the cell cycle.

Amino Acid Sequence↗
PubMed1993

High level expression of the large subunit of mouse ribonucleotide reductase in a baculovirus system.

The large subunit of ribonucleotide reductase from mouse has been overexpressed in Spodoptera frugiperda cells infected with recombinant baculovirus. The expressed protein was purified by affinity chromatography to apparent homogeneity as determined by SDS-PAGE. The homogeneous protein is recognized in Western blot analysis by a monoclonal antibody raised to the large subunit of ribonucleotide reductase from calf thymus, has the correct N-terminal sequence, and, in the presence of the small subunit of mouse ribonucleotide reductase and nucleoside triphosphate effectors, catalyzes the reduction of both purine and pyrimidine nucleoside diphosphates.

Animals↗
PubMed1993

Phonological and morphological analysis skills in young children.

Twenty-two normally developing five-year-olds were asked to judge, identify, repair and explain phonological and morphological errors. All of the errors involved the addition, substitution or omission of a single phoneme, which in the morphological task, was also an inflectional morpheme. Stimuli were controlled for type of error (omission, addition, substitution), location of the error (word final) and word status of the resulting error (word, non-word). Children performed significantly better on the phonological task than on the morphological task. It is proposed that the results are due to differences in the type and location of linguistic information to be analysed and to differences in memory demands in the tasks.

Child Language↗
PubMed1993

'Spontaneous' transformation as aberrant epigenesis.

The NIH 3T3 line of cells has particular advantages for studying the dynamics of change in cellular phenotype in response to environmental conditions. Similar to stem cell growth during development, the cell line changes its phenotype under growth constraints that elicit differentiation or, alternatively, it maintains its original state over many replication cycles when grown without constraint. Unlike many cell types which respond by undergoing terminal differentiation, the NIH 3T3 cells continue to multiply indefinitely following an induced alteration in phenotype; the heritability of this change may thus be analyzed under stringent conditions of cell culture. During the course of over two years of frequent passage at low density in high calf serum (CS) concentration, a new subline developed which exhibited a consistent capacity to respond quickly and pervasively to growth constraints with an increase in saturation density, development of transformed foci in confluent cultures and altered appearance of isolated colonies. A retrospective study was undertaken, with cells from cryopreserved samples, of the course of changes in responsiveness of the cells to growth constraint leading up to the highly responsive state. Three stages were discerned, the first with an initially high capacity of a small fraction of cells to produce diffuse foci, but with a rapid decline in this capacity with frequent low density passages; the second stage, extending over more than 200 passages, of refractoriness to transformation; and the third stage (which probably arose by mutation) in which there is a consistent transformation-related response by the entire population to growth constraint, a response which has remained relatively constant over some 100 passages. A striking and novel feature of the third stage is seen on cloning the cells. Almost all the colonies obtained by cloning cells from post-confluent, growth-inhibited cultures are distinctly different in morphology from those obtained by cloning cells from the frequent low density passages. The pervasiveness of this morphological change among the clones is unmistakable evidence for a heritable adaptive response to growth constraint by most if not all of the cells in the population. The population-wide response of the cells of the third stage offers the opportunity for a rigorous, quantitative analysis of the nature of this type of persistent cellular change. Although cells of the third stage may be of mutational origin, their pervasive heritable response once the variant population is established supports the concept of progressive state selection which postulates that transformation can arise by the continuous fluctuation of growth states within cells, accompanied by the progressive selection of those states best suited to function under the selecting constraint. Relevance of the concept to the process of differentiation under growth constraint is considered.

3T3 Cells↗
PubMed1993

Shortening of the poly(A) tail at the 3'-end of mRNA requires its translation.

We report here that when alpha- or beta-globin mRNAs were hybridized with their alpha- or beta-complementary DNAs, respectively, and then translated in rabbit reticulocyte lysates, globin synthesis as well as shortening of the poly(A) tail were much less than that observed with nonhybridized mRNA. Furthermore, when the hybrid (alpha-globin mRNA/alpha-cDNA) was translated in the presence of nonhybrid (alpha-globin mRNA), the estimated average shortening of the poly(A) tails of the hybrid and the nonhybrid were 8% and 40%, respectively. Similar results were obtained when a hybridized beta-globin mRNA with beta-cDNA was incubated in the same translation mixture with beta-globin mRNA. No globin synthesis occurred in the mixtures containing the cDNA-mRNA hybrids only. In the mixtures containing both the hybrid and the nonhybridized mRNAs, globin was synthesized at a rate similar to that observed in a translation reaction mixture alone, that is, in the absence of any hybrid.

Animals↗
PubMed1992

Sensitivity of transformation to small differences in population density during serial passage of NIH 3T3 cells.

Early passages of the NIH 3T3 mouse cell line undergo spontaneous neoplastic transformation leading to the development of transformed foci if grown to confluence in 2% (vol/vol) calf serum (CS) and left there for more than a week. Transfer of the postconfluent cultures results in the appearance of large numbers of transformed foci; many of them are larger and denser than those in the original culture. If the cells are continually kept at low population densities by frequent passages in 10% CS, they lose the capacity to undergo spontaneous transformation. If however the low-density passages are made in 2% CS or in 10% (vol/vol) fetal bovine serum, both of which support lower growth rates and saturation densities than does 10% CS, they gain the capacities to grow to high saturation densities and produce more foci when grown to confluence in 2% CS. These increases are proportional to the population densities used in the frequent passages, although the densities are all kept well below confluence. We conclude that the combined constraints of submaximal serum plus those of the limited cell contacts of the low cell densities used here elicit an adaptive response that endows the entire population with increased growth capacity. The increased growth capacity of the heterogeneous population in turn increases the capacity of a fraction of the population to initiate distinctive transformed foci. Similar studies have indicated that the capacity of cells to produce tumors and metastases in mice and rats is enhanced by prior maintenance at high density in culture. We propose the concept of progressive state selection to account for the general increase in the growth capacity of cells that is elicited by moderate constraints on their growth and metabolism.

3T3 Cells↗
PubMed1992

Crystallization, activity assay and preliminary X-ray diffraction analysis of the uncleaved form of the serpin antichymotrypsin.

Crystals of recombinant wild-type antichymotrypsin have been prepared by the method of vapor diffusion with polyethylene glycol 4000 as a precipitant at pH 5.7. Two crystal forms are observed. One form belongs to tetragonal space group P4(3)2(1)2 (or P4(1)2(1)2) and has unit cell dimensions a = b = 126 A, c = 243 A, with two molecules in the asymmetric unit. The other crystal form belongs to orthorhombic space group P2(1)2(1)2(1) and has unit cell parameters of a = 73 A, b = 78 A and c = 80 A, with one molecular in the asymmetric unit. Diffraction intensity measurements have been made on the tetragonal crystal form to a limiting resolution of 4.1 A, and reflections have been observed on X-ray still photographs to a limiting resolution of 2.5 A for the orthorhombic form. An activity assay of redissolved tetragonal form crystals indicates that the uncleaved, functional serpin has been crystallized.

Chymotrypsin↗
PubMed1992

Characterization of acute myocardial infarction by magnetic resonance imaging.

The T2-weighted spin-echo technique is currently the most frequently used magnetic resonance imaging (MRI) method to visualize acute myocardial infarction. However, image quality is often degraded by ghost artifacts from blood flow, and respiratory and cardiac contractile motion. To enhance the usefulness of this technique for detailed characterization of infarction, a velocity-compensated spin-echo pulse sequence was tested by imaging a flow phantom, 6 normal subjects and 17 patients with acute myocardial infarction. After preliminary studies were performed in 7 patients to determine optimal imaging parameters, a standardized imaging protocol was used in the next 10. The location of myocardial infarction identified by the electrocardiogram and coronary anatomy was correctly identified in 10 of 10 patients. Distribution of the injury within the left ventricle was clearly visualized, and showed that patients often had a mixture of transmural and nontransmural injury. Heterogenous distribution of signal intensity within the infarction suggested the presence of hemorrhage. Papillary muscle involvement was readily apparent. Signal intensity of the infarction (brightest segment) was increased by 89 +/- 31% compared with the mean of the remote segments. The myocardial/skeletal muscle ratio was significantly (p less than 0.001) increased for the infarction segments compared with that for remote myocardium, allowing quantitative analysis of segmental signal intensity. The MRI wall motion study obtained as part of this protocol demonstrated wall thickening in 58% of the infarction segments and in 6 of 10 patients. This finding suggested the presence of reversibly injured myocardium. In conclusion, the results demonstrate the potential of MRI for detailed tissue characterization after acute myocardial infarction.

Adult↗
PubMed1992

High rate of diversification and reversal among subclones of neoplastically transformed NIH 3T3 clones.

NIH 3T3 cells undergo neoplastic transformation when exposed to conditions of moderate physiological growth constraint. One of several characteristics of this transformation that indicates its adaptational nature is its gradual reversibility under conditions of unconstrained growth. We explored the origins of reversibility by isolating cells from each of three highly transformed foci and comparing their focus-forming capacity with that of derivative clones and subclones. A high proportion of the parental cells made dense foci. Six of the nine clones obtained from the three foci produced foci, though the percentage varied widely. The other three clones produced no foci at all. The transformed clones were subcloned and analyzed to evaluate the possibility that the negative clones were genuine revertants, rather than being derived from a small minority of nontransformed cells surrounding or underlying the original foci. In each case the subclones varied widely in the percentage of focus-forming cells and the average was much lower than the parental clone from which they were derived. Indeed, 15 of the 53 subclonal populations produced no foci. The high degree of heterogeneity, including complete reversal of focus-forming capacity, provides additional support for the hypothesis that "spontaneous" transformation is driven by an adaptive response to moderate growth constraint rather than by one or more effectively irreversible mutations.

3T3 Cells↗
PubMed1992

Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes.

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.

Arachidonic Acid↗
PubMed1992

Adaptive evolution of degrees and kinds of neoplastic transformation in cell culture.

Human cancers undergo protracted complex development from benign to malignant states, as most thoroughly documented in the mole-to-melanoma sequence. The early stages of the sequence tend to redifferentiate into normal tissues; the later stages progress to ever increasing multiplication and malignancy. When placed under the growth constraint of either crowding or low serum concentrations, the NIH 3T3 line of mouse cells readily undergoes transformation, expressed in the development of foci of cells that continue to multiply at confluence when the rest of the population has stopped. If the nontransformed cells are maintained for 3 months by frequent low-density passages in high concentrations of calf serum, they gradually lose the capacity to undergo transformation when the constraints are applied. The same conditions of passage have been used to reverse the transformation, both processes resembling in principle the reversal of the early stages of the mole-to-melanoma sequence. When the frequent low-density passages are made in high concentrations of fetal bovine serum, which supports a slightly lower growth rate than an equal concentration of calf serum, the degree of transformation is gradually increased, so that the foci become more numerous, broader, and thicker, reaching a maximum in successive assays at about 3 months of passaging. A diversity of focal morphologies is sporadically generated in the calf serum passage by exposing the cells to various concentrations of calf serum for 14 days of growth and confluency before assaying them. The dependence of the number, density, and morphology of foci on the environment in which the cells had been grown before assay reinforces the evidence that the transformation is an epigenetic process. The fact that these effects develop in culture gradually over an extended period of time suggests parallels to the characteristically long-term early regression and later progression, as well as the diversity of the mole-to-melanoma sequence, and may also be representative of other cancers.

3T3 Cells↗
PubMed1992

Individual transforming events in long-term cell culture of NIH 3T3 cells as products of epigenetic induction.

The NIH 3T3 line of mouse fibroblasts undergoes "spontaneous" transformation in culture, exhibited in the development of foci of transformed cells overgrowing the confluent monolayer. Evidence is provided here to support the proposition that the spontaneous generation of individual transformed variants is a product of epigenetic induction by various types of growth inhibition. Novel transformed variants generally arise after prolonged confluence and cessation of net growth, with these new types of foci appearing during a second round of confluence, although not in the first round. Few or no transformed variants exist in the cultures prior to growth constraint. The susceptibility of NIH 3T3 cells to induction of transformation is itself shown to be subject to epigenetic influence, with capacity for transformation reflecting passage histories. Cell sublines maintained in long-term subconfluent passage that allows unimpeded growth become more refractory with time to spontaneous transformation, while sublines passaged in more growth-restricting conditions maintain their sensitivity. The differing capacities of the sublines are reflected in the degree of growth inhibition required to induce individual events of spontaneous transformation, and in the frequency at which such new variants arise. Thus growth inhibition not only induces individual transforming events, but even increases cell susceptibility to further induction of transformation. These phenomena are consistent with the progressive state selection model of heritable change, which postulates a self-regulating selection of better adapted states from among those made available to a biological system during heterogeneous fluctuations in its total pattern of chemical equilibria.

3T3 Cells↗
PubMed1992

Adaptation and selection as factors in the spontaneous transformation of NIH-3T3 cells.

The effect of calf serum (CS) concentration on the spontaneous transformation of NIH-3T3 cells was investigated. Maintenance of NIH-3T3 cell populations in media containing a low concentration of calf serum (2% CS) increased the proportion of spontaneously transformed cells appearing within a population. Higher CS concentration (10% CS) decreased the proportion of transformed cells in the population. In searching for the reasons for the effect of serum concentration on transformation frequency, three conclusions were reached. (i) Non-focus-forming NIH-3T3 cells exhibit heterogeneity in progression toward transformation. (ii) Low serum concentration can increase the frequency of focus-forming cells within an NIH-3T3 population by decreasing the proliferation of non-focus-forming cells relative to focus-forming cells. (iii) The reproductive selection described in (ii) cannot completely account for the focus-enhancing effects of low serum. Evidence for point (iii) is drawn from focus-forming assays, in 2% CS, for a clone of cells maintained in 10% CS before assay. Cells from this clone made no foci in 50 assays, performed on 5 x 10(6) cells, over a 21 week period. However, maintenance of those same cells in 2% CS for as little as 2 days before assay caused these consistent non-focus formers to make foci. We conclude that the basis for this progression to the focus-forming phenotype is an adaptation to the metabolically constraining 2% CS environment. We propose that this adaptation results from progressive selection of physiological states.

3T3 Cells↗