The biology and biochemistry of antichymotrypsin and its potential role as a therapeutic agent.
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Animals↗
Biomedical subjects
H Rubin
Publications and source records attributed to H Rubin.
Cost-effectiveness of HA-1A monoclonal antibody for gram-negative sepsis. Economic assessment of a new therapeutic agent.
OBJECTIVE: To assess the cost-effectiveness of the HA-1A monoclonal antibody for the treatment of gram-negative bacteremia. DESIGN: Cost-effectiveness analysis of a randomized, double-blind, placebo-controlled trial using clinical efficacy data reported in the medical literature. SETTING: Hospitalized patients with sepsis. PATIENTS: 543 patients with sepsis and suspected gram-negative infection. Patients enrolled in the study met strict criteria for sepsis, including fever or hypothermia (less than 35.6 degrees C or greater than 38.3 degrees C), tachycardia (greater than 90 beats per minute), tachypnea (greater than 20 breaths per minute), and hypotension or two of six signs of systemic toxicity. INTERVENTION: HA-1A vs placebo in addition to usual care. MAIN OUTCOME MEASURES: We determined the range of possible cost-effectiveness ratios for HA-1A therapy, using modeling techniques when clinical or economic variables were unknown. We subjected the model to rigorous sensitivity analysis. We calculated the incremental cost of care and years of life saved for patients with sepsis and modeled two different treatment strategies: treat all patients with sepsis or test and then treat only patients with positive test results. $24,100 per year of life saved based on the treat strategy and $14,900 based on the test strategy. In sensitivity analysis the ratios ranged from $5200 to $110,200 per year of life gained. Annual costs of care for these two strategies are $1.3 billion for the test strategy and $2.3 billion for the treat strategy. CONCLUSION: Economic assessment of new technologies early in their development can be used to guide their efficient clinical introduction.
Antibodies, Monoclonal↗
Growth in high serum concentrations leads to rapid deadaptation of cells previously adapted to growth in an extremely low concentration of serum.
A subline of NIH 3T3 cells adapted to multiply in 0.25% calf serum (CS) by frequent passage (every 2-3 days) at low population density in 0.25% CS was deadapted by frequent successive passages of the cells in 10% CS for 3 weeks. The cells adapted to 0.25% CS multiplied with an average doubling time of 16.9 hr in 10% CS, and cells that had always been kept in 10% CS multiplied with an average doubling time of 14.6 hr, so there was weak selection for the latter in the higher serum concentration. When adapted cells were subjected to two passagers in 10% CS prior to assay of growth in 0.25% CS, a 4-day lag period was evident before commencement of exponential growth, and there was a decrease in saturation density. Further delay of growth in 0.25% CS developed as the number of passages of cells in 10% CS increased. The marked delay of growth in 0.25% CS of the bulk population after a few days in 10% CS argued against selection in 10% CS of rare nonadapted mutants from the adapted population and for an epigenetic origin of the change. Reconstruction experiments utilizing adapted cells mixed with non-adapted cells in 0.25% CS buttressed this explanation. Eight clones of the adapted population exhibited some loss of growth capacity in 0.25% CS after a single passage in 10% CS, though the extent of loss varied from clone to clone. The results support the idea that all cells in the adapted population respond to the lifting of growth constraints with loss of their growth potential under highly constrained conditions. They are consistent with the concept of progressive state selection in which selection operates on fluctuating metabolic states of individual cells rather than on genetic variants.
3T3 Cells↗
Cellular adaptation in the origin and development of cancer.
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Adaptation, Biological↗
Inhibition of human neutrophil superoxide generation by alpha 1-antichymotrypsin.
Reactive oxygen intermediates and serine proteases are important components of host defense systems but can produce host injury if not tightly regulated. To determine whether these components can be coordinately controlled, we investigated regulation of superoxide generation by physiologically relevant concentrations of a) highly purified serum-derived antichymotrypsin (ACT), b) recombinant, wild-type ACT, c) rACT in which amino acid substitutions were engineered into the reactive center, and d) chymotrypsin/ACT complexes. These proteins and protein complexes inhibited superoxide anion production in neutrophils stimulated by f-Met-Leu-Phe, Con A, or PMA. In contrast, ligand-stimulated degranulation was not inhibited. In addition, using the recombinants and complexes, the region of ACT involved in inhibiting superoxide anion production was shown to be structurally distinct from the reactive center of the protein. The results indicate that functional domains of ACT corresponding to different biological activities can be decoupled and suggest that three species of ACT (intact ACT, a complexed protease/ACT form, and a partially denatured or proteolyzed form of ACT) that can exist in the microenvironment of an activated neutrophil may play an important role in regulating neutrophil function.
Cell Degranulation↗
Effect of interclonal heterogeneity on the progressive, confluence-mediated acquisition of the focus-forming phenotype in NIH-3T3 populations.
Confluence is an agent that promotes the progressive acquisition of the focus forming phenotype in clones of cells within NIH-3T3 populations. This conclusion is based on four results. (a) Even in cultures which have been confluent for more than 2 weeks without making foci, some cells in those cultures have been affected by the confluent state and will make foci if replated and allowed to grow into new saturated cultures. Because replicate dishes are very similar in the number and type of foci formed after replating, we conclude that the progression toward focus formation is substantially completed before replating, while the cells are in their original confluent cultures. (b) Different NIH-3T3 populations were produced by expansion of small or large numbers of starting cells. When plated without exposure to confluence there was little difference in focus production among cells from these different sized starter populations. However, confluence caused foci to arise more frequently in platings of 10(5) cells derived from large starting numbers than from platings of 10(5) cells derived from small starting numbers of cells. This implies that the confluent state successfully promotes the acquisition of the focus forming phenotype in a limited percentage of cells in an NIH-3T3 culture and that those cells are absent from many small starting populations. (c) There is a progressive temporal effect of the confluent state on focus formation; the number and density of foci that emerge from replated confluent cultures increase with the length of time the cells spend in the confluent state. (d) There is heterogeneity among different batches of NIH-3T3 cells in the ability of the confluent state to induce acquisition of the focus forming phenotype. Also, the morphology of the foci that do arise after confluent treatment differs substantially among cell populations. Nonetheless, the foci formed from a single batch of cells are typically similar in morphology, indicating that those foci arose from one clone, or very few clones, of cells.
Cell Aggregation↗
Epigenetic features of spontaneous transformation in the NIH 3T3 line of mouse cells.
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3T3 Cells↗
Language analysis skills of children with mental retardation.
The ability of children with moderate mental retardation to analyze orally presented sentences into words and words into syllables and phonemes was studied. The subjects, aged 10 to 15, were grouped by method of reading instruction. All of them could analyze spoken sentences into words and words into syllables equally well. However, subjects receiving code-emphasis reading instruction performed significantly better on the more difficult phoneme manipulation tasks than did subjects receiving whole-word instruction. Results suggest that code-emphasis reading instruction for some subjects with mental retardation should be used. Further research on linguistic analysis skills and the use of code-emphasis reading instruction in conjunction with linguistic awareness training with this population is warranted.
Adolescent↗
The carboxyl terminus heptapeptide of the R2 subunit of mammalian ribonucleotide reductase inhibits enzyme activity and can be used to purify the R1 subunit.
The heptapeptide, FTLDADF, identical in sequence to the last seven amino acid residues of the carboxyl terminus of the R2 subunit of mouse ribonucleotide reductase (RR), and its N alpha-acetyl derivative both inhibit calf thymus RR. The N alpha-acetyl derivative is considerably more potent, displaying a K1 of 20 microM. The same K1 was found for N-AcFTLDADF inhibition of a reconstituted ribonucleotide reductase from calf thymus R1 and mouse R2, indicating that the C-termini of calf R2 and mouse R2 might be identical. Our results, taken together with previous results of others on inhibition of viral RR, suggest that inhibition of RRs by peptides mimicking the C-terminus of R2 may be a general phenomenon. In addition, we have shown that an affinity column, FTLDADF-Sepharose 4B, can be used to prepare approximately 95% pure calf thymus R1, devoid of contamination with R2, in a very simple procedure that should be generally applicable to R1 purification from many sources.
Acetylation↗
Early "step-down" transfer of low-risk patients with chest pain. A controlled interventional trial.
OBJECTIVE: To determine whether providing private practitioners with triage criteria for their low-risk chest pain patients would safely enhance bed utilization efficiency in coronary and intermediate care units. DESIGN: Prospective, controlled, interventional trial using an alternate month study design. SETTING: A large teaching community hospital. PATIENTS: Cohort of 404 low-risk patients with chest pain for whom a diagnosis of myocardial infarction has been excluded and who have not sustained complications, required interventions, or developed unstable comorbidity. INTERVENTIONS: During intervention months, private practitioners caring for low-risk patients in the coronary and intermediate care units were contacted 24 hours after admission. Physicians were informed that the transfer of low-risk patients to nonmonitored beds could probably be done safely, based on the results of a pilot study. The practitioner had the option of agreeing to or deferring patient transfer. During control months, physicians were not contacted in this way. MEASUREMENTS AND MAIN RESULTS: Use of the triage criteria by private practitioners reduced lengths of stay in the intermediate and coronary care units by 36% and 53%, respectively. Bed availability increased by 744 intermediate and 372 coronary care unit bed-days per year. Charges decreased by $2.6 million per year and profits improved by $390,000 per year. There were not significant differences in complications between control and intervention patients and in no case (95% CI, 0% to 1.6%) did the triage criteria adversely affect quality of care. CONCLUSIONS: The early transfer triage criteria may be a safe and efficacious decision aid for improving bed utilization in intermediate and coronary care units. In addition, this study shows the feasibility of and potential benefits from applying practice guidelines at a community hospital.
Aged↗
Progressive state selection of cells in low serum promotes high density growth and neoplastic transformation in NIH 3T3 cells.
A subline of NIH 3T3 cells maintained by frequent passage (every 2 to 3 days) in 10% calf serum (CS) at low population density reached a low saturation density in 2% CS and produced no transformed foci on prolonged incubation at confluency in 2% CS. Within 3 frequent low density passages in 2% CS, the saturation density and focus-forming capacity in that serum concentration began an increase which was continued in subsequent passages. The saturation density and focus-forming capacities of the cells in both 2% and 1% CS were further enhanced by passage in 1% CS. The cells could then be passaged in 0.5% CS and then in 0.25% CS, which would support no multiplication of cells previously passaged only in 10% CS. The cells passaged in 0.25% CS gradually increased their saturation density and focus-forming capacity in that extremely low serum concentration during 24 low density passages, although their initial growth rate did not increase. They also attained a colony-forming efficiency in 0.25% CS of about 30%, as compared to less than 1% for cells passaged in 10% CS. Cells passaged, cloned, and passaged again in 2% CS yielded clonal populations which differed from one another in saturation density and focus-forming capacity in 2% CS. We conclude that NIH 3T3 cells diversify phenotypically at a high rate in their capacity to multiply and produce foci in limiting concentrations of serum, and we propose that progressive selection of these heterogeneous states accounts for the acquired capacity to function effectively in low concentrations of serum growth factors. Since lymph and presumably extracellular fluid in vivo contain low concentrations of growth factors which govern the multiplication of normal cells, the adaptation we observe in vitro may be related to tumor production in the animal.
Animals↗
Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins.
Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.
Amino Acid Sequence↗
The significance of biological heterogeneity.
Heterogeneity of expression for a variety of characteristics is found among malignant cells in the organism and in culture. Normal cells are relatively uniform when organized in a tissue, but become heterogeneous for many characteristics when they are dispersed and grown in monolayer culture. The heterogenizing effect of growth in culture indicates that the morphology and behavior of normal cells is ordered by their topological relations in tissues and other homeostatic influences of the organism. Weakening of these ordering relations may contribute to malignant transformation, as it usually does in rodent cell culture. Although phenotypic differences among cells of a given type may be transient, they can be perpetuated by protracted exposure to selective conditions. Examples are cited of selection which leads to an adapted state that is heritable for many cell generations after removal of the selective conditions. Such heritable adaptations are analogous to the Dauermodifikationen, or lingering changes, first described in ciliated protozoa and shown there to be under cytoplasmic control. The concept of progressive state selection is introduced to account for heritable adaptation at the cellular level. It depends on the spontaneous occurrence of transient, variant states and their successive selection to progressively higher levels of adaptation to an altered microenvironment. Although the process is basically epigenetic, it may be stabilized by genetic change. The concept is consistent with our present knowledge of tumor development, including progression to metastasis, and with epigenetic aspects of normal development.
Animals↗
Grammatical awareness in the spoken and written language of language-disabled children.
Experiments examined grammatical judgement, and error-identification deficits in relation to expressive language skills and to morphemic errors in writing. Language-disabled subjects did not differ from language-matched controls on judgement, revision, or error identification. Age-matched controls represented more morphemes in elicited writing than either of the other groups, which were equivalent. However, in spontaneous writing, language-disabled subjects made more frequent morphemic errors than age-matched controls, but language-matched subjects did not differ from either group. Proficiency relative to academic experience and oral language status and to remedial implications are discussed.
Attention↗
Relation of spontaneous transformation in cell culture to adaptive growth and clonal heterogeneity.
Cell transformation in culture is marked by the appearance of morphologically altered cells that continue to multiply to form discrete foci in confluent sheets when the surrounding cells are inhibited. These foci occur spontaneously in early-passage NIH 3T3 cells grown to confluency in 10% calf serum (CS) but are not seen in cultures grown to confluency in 2% CS. However, repeated passage of the cells at low density in 2% CS gives rise to an adapted population that grows to increasingly higher saturation densities and produces large numbers of foci in 2% CS. The increased saturation density of the adapted population in 2% CS is retained upon repeated passage in 10% CS, but the number and size of the foci produced in 2% CS gradually decrease under this regime. Clonal analysis confirms that the focus-forming potential of most if not all of the cells in a population increases in response to a continuously applied growth constraint, although only a small fraction of the population may actually form foci in a given assay. The acquired capacity for focus formation varies widely in clones derived from the adapted population and changes in diverse ways upon further passage of the clones. We propose that the adaptive changes result from progressive selection of successive phenotypic variations in growth capacity that occur spontaneously. The process designated progressive state selection resolves the apparent dichotomy between spontaneous mutation with selection on the one hand and induction on the other, by introducing selection among fluctuating states or metabolic patterns rather than among genetically altered cells.
Animals↗
Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.
NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration.
Animals↗
On the nature of enduring modifications induced in cells and organisms.
The clarity of Mendelian genetics and the elegance of the molecular mechanisms of replication and readout of DNA have tended to obscure a solid body of evidence demonstrating that nongenetic, enduring modifications can be induced in the behavior of cells, modifications that continue to be expressed for many divisions after withdrawal of the inducing stimulus. The most prosaic case is the differentiated state of metazoan cells, which persists throughout the lifetime of the organism. Much less widely known but well-characterized examples are also cited for bacteria, protozoa, and cultured cells of higher plants and animals. The spontaneous neoplastic transformation of cultured mouse NIH 3T3 cells is introduced as an enduring adaptive response to moderate growth constraints. Evidence in support of the thesis that physiological adaptation is the driving force for chemically induced carcinogenesis in the intact animal is also presented. The cases described here involve integrated responses of many if not all of the regulatory components of the cell, rather than singular molecular mechanisms. The continuous generation of phenotypic heterogeneity, a process observed readily in cell culture, provides the basis for a model that accounts for enduring modifications. This model, designated progressive state selection, makes no attempt at a detailed biochemical explanation of heterogeneity, but uses it as a fundamental postulate to represent the adaptive behavior of the cells.
Animals↗
Limited proteolysis of C1-inhibitor by chymotrypsin-like proteinases.
Limited proteolysis of C1-inhibitor was observed with human skin chymase, human cathepsin G, and bovine chymotrypsin. In each case, the inhibitor was degraded to one major product migrating slightly faster than the native inhibitor in an SDS-polyacrylamide gel. The inhibitory activity of C1-inhibitor against human plasma kallikrein was not altered by the modification with chymase. Edman degradation of the proteolyzed inhibitor revealed two sequences in a 1:1 ratio: NPNATSSSQ, the N-terminus of native C1-inhibitor, and VEPILEVSSL. This second sequence showed that the Phe33-Val34 bond was hydrolyzed. Our results provide another example of the susceptibility of the N-terminal region of C1-inhibitor to proteolytic cleavage.
Amino Acid Sequence↗