Cleavage specificity of human skin type IV collagenase (gelatinase). Identification of cleavage sites in type I gelatin, with confirmation using synthetic peptides

Type IV collagenase (gelatinase) readily cleaves denatured collagen into very small peptides. Large cyanogen bromide fragments (25 kDa) of type I collagen are degraded at the same rate as the complete alpha-chain. A number of the gelatinolytic cleavage sites of alpha 1(I)CB7 and alpha 1(I)CB8, representing 50% of the collagen alpha-chain, were determined by sequence analysis of product peptides. In addition to the expected cleavage between glycine and hydrophobic residues, several other cleavage sites were identified. These sites were Gly-Glu, Gly-Asn, and Gly-Ser. Basic residues were found adjacent to the cleavage site in several cases. Hexapeptides containing these unexpected cleavage sites were synthesized, and Km and kcat values were determined. All but one of the Km values were in the submillimolar range, and turnover numbers for the peptides uncharged at the carboxyl terminus were on the order of 10,000/h. Of particular significance was the finding that hydroxyproline occurs 5 residues from the cleavage site in all carboxyl-terminal product peptides and also occurs 5 residues from the cleavage site in seven of nine amino-terminal product peptides. A requirement for hydroxyproline may be of importance in determining the specificity of this enzyme for denatured collagenous substrates.     

The gelatinolytic activity of human skin fibroblast collagenase

The gelatinolytic activity of human skin fibroblast collagenase was examined on denatured collagen types I-V. All denatured substrates were cleaved, including types IV and V, which are resistant to collagenase in native form. Interestingly, the earliest major cleavage in denatured collagen types I-III occurred at a 3/4-1/4 locus, resulting in products electrophoretically identical with TCA and TCB fragments of mammalian collagenase action on these native collagens. However, in the denatured substrates, multiple additional proteolytic cleavages followed. The propensity for cleavage at a 3/4-1/4 site in denatured collagen, where sequence is the major specifier of enzymatic action, would seem to indicate that the most favorable amino acid sequence of gamma chains for catalysis is located in this region. The peptide bond specificity of human fibroblast collagenase on gelatin was examined by amino acid sequencing of extensively cleaved denatured type I collagen. Analysis of the NH2-terminal amino acid residues from the resultant gelatin peptides showed sequences of "-H2N-Ile-Y-Gly" and "H2N-Leu-Y-Gly" only (where Y indicates that any amino acid can be found in that position), indicating that Gly-Ile and Gly-Leu bonds are the only sites of collagenase cleavage in this substrate. Whereas the gamma1 chains of denatured collagen types I-III were cleaved at similar rates, fibroblast collagenase was a much better gamma2-gelatinase than gamm1-gelatinase on denatured type 1 collagen. This preference for the cleavage of gamma2(I) was the result of both a higher kcat (750 versus 230 h-1) and lower Km (3.7 versus 7.0 microM) than for a gamma1(1), resulting in an overall selectivity (kcat/Km) of greater than 6-fold. Compared to such kinetic parameters on native collagen, these values indicate that gelatinolysis is somewhat slower than collagenolysis.     

Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.     

Correlated Responses in Male Reproductive Traits in Mice Selected for Litter Size and Body Weight

Correlated responses in male reproductive traits were determined at 4, 6 and 8 weeks of age in lines of mice selected for large litter size (L⁺), large 6-week body weight (W⁺), large litter size and small body weight (L⁺W^-) and small litter size and large body weight (L^-W⁺), and in an unselected control (K). Concentration of serum testosterone and weights of testes, seminal vesicles, epididymides and adrenal glands increased with age. Line differences in testosterone concentration were not detected. L⁺ and W⁺ males exhibited positive correlated responses in testes, epididymides and seminal vescile weights. Testis weight adjusted for body weight was significantly larger for L⁺ than controls and approached significance for W⁺. Realized genetic correlation betestis weight and litter size was 0.60 ± 0.04, and the realized partial genetic correlation holding body weight constant was 0.42. Therefore, pleiotropic loci, acting via the hypothalamic-pituitary axis, affect testis weight and litter size independently of body weight. Additionally, genes influencing overall growth have a pleiotropic effect on testis weight and litter size in mice; the realized genetic correlations of body weight with testis weight and with litter size were 0.60 ± 0.03 and 0.52 ± 0.10. Testis weight increased in both L⁺W^- and L^-W⁺ males. The positive correlated response in L⁺W^- may have resulted from changes in frequency of genes controlling reproductive processes; whereas, in L^-W⁺ it could have been the result of changes in the frequency of genes associated with body weight.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

Environmental maternal influences on body composition in mice selected for body weight

Summary The effect of the postnatal maternal environment, simulated by rearing mice in litters of three, six or nine, on body weight and body composition was investigated in three lines of mice differing widely in growth rate. The lines were selected for high (H₆) and low (L₆) 6-week body weight while the control line was maintained by random selection. Body weight and weights and percentages of ether extract, water, ash and protein at 21, 42, 63 and 84 days were recorded. With few exceptions, there were positive correlated responses to selection in body weight and in weights of body components. At 21 and 42 days the correlated responses were larger in L₆ mice than in H₆ mice. Body weight and weights of body components were larger for mice reared in litters of three than for those reared in litters of nine. Also, mice reared in litters of six were intermediate in body weight and weights of some of the body components between those reared in litters of three and nine. Differences in body weight and weights of body components due to postnatal maternal environment were small by comparison with differences due to genetic line. There were significant line by maternal environment interactions in body weight at 21 days and in ether extract weight at 21 and 63 days. Line and maternal environment differences in percentages of body components did not follow any consistent trend. The results for percentages of body components were further complicated by line x maternal environment interactions. In general, both line and postnatal maternal environmental differences in percentages of body components diminished with age.Paper No. 5670 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27650. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned     

Clastogenic effects of methylnitrosourea and ethylnitrosourea on chromosomes from human fibroblast cell lines.

Human fibroblast cell lines were pulse-treated for 1 h with either methylnitrosourea (MNU) or ethylnitrosourea (ENU) at various time intervals before harvesting for chromosome analysis. Cells treated with 1 X 10(-3) M, 5 X 10(-4) M, and 1 X 10(-4) M final concentrations of MNU and ENU during the G2 or M phases of the cell cycle showed a significant increase in chromatid-type abnormalities over controls. Cells exposed to MNU or ENU 23 h before harvest showed some chromosome-type abnormalities, reflecting probable damage induced during the G1 phase of the cell cycle or derived from chromatid damage induced during the previous cell cycle. The mitotic indices and incidences of abnormalities suggested a dose response effect when cells were treated with the two higher concentrations and the three concentrations, respectively, of MNU or ENU. Chromatid abnormalities were observed in MUN and ENU-treated cells from each of four cell lines. From this investigation, it was concluded that MNU and ENU treatment of human diploid cell lines in vitro induced both chromatid and chromosome aberrations. MNU and ENU, both of which had previously been shown to be mutagenic in experimental animals, are, therefore, also considered to be mutagenic at the chromosome level in human fibroblasts grown and treated in cell culture.     

Genetic analysis of nurse dams selected for six-week body weight or postweaning gain in mice.

A modified crossfostering technique was developed to compare the performance of nurse dams in selected and control populations of mice. The H6 and M16 populations were selected for increased 6-week body weight and 3- to 6-week postweaning gain, respectively, while the C2 and ICR populations were the respective controls. Crossfostering was performed using H6, M16 and their reciprocal F1 crosses as nurse dams in the selected crossfostering group and C2 ICR and their reciprocals in the control group. Measurements recorded for nurse dams included mean body weight of 8 young within a nursed litter at birth (MWB) and 12 days of age (MW12). The latter was used as a measure of postnatal maternal performance. Other traits recorded for nurse dams were number born (NB), body weight at parturition (DWP) and 12 days postpartum (DW12), and weight gain (DWG), feed intake (FED) and efficiency (EFF = DWG/FED) for the first 12 days of lactation. The correlated response in MW12 was negative (P less than .01) for M16 and essentially zero for H6. Both lines exhibited positive (P less than .01) correlated responses in DWP and DW12 and no change in EFF. Only the H6 line increases significantly in DWG and FED as a result of selection. NB increased in M16 and H6, but was significant for the latter population only. Population differences in selection response [(M16-ICR)-(H6-C2)] were significant for FED only, primarily due to average direct genetic effects. Direct comparisons of M16 and H6 indicated that M16 was larger in DWP and DW12 but smaller in DWG and EFF. Average direct genetic effects favored M16 for NB, DWP, and DW12, whereas average maternal genetic effects favored H6 for NB, DWP, DW12 and FED. Percent direct heterosis, in F1 crosses of selected populations was significant for MW12 (13.7%) ,FED (10.8%) and NB (11.4%). Direct heterosis in F1 crosses of the controls was significant for MW12 (9.4%), NB (6.6%), DWP (3.5%), DW12 (3.3%) and FED (4.4%). The effects of MW12, DWG and metabolic body size (MBS) accounted for 47% of the variation in FED, pooled within populations. Of these variables, MW12 accounted for the highest proportion (32%) of variation in total feed intake.     

Population Size and Selection Intensity Effects on Long-Term Selection Response in Mice

Long-term response to within full-sib family selection for increased postweaning gain was evaluated in lines having different effective population sizes (N_e) and selection intensities (i). Line designations were I4(4), I8(2), I16(2), M4(4), M8(2) and M16(2), where I and M indicate selection of the top 50% and 25%, respectively; 4, 8 and 16 represent the number of parental pairs per replicate and number of replicates is given in parentheses. Realized within full-sib family heritabilities (h_R²) in the first phase of selection (0-14 generations) were larger in 16-pair lines than in 4- and 8-pair lines. In the second phase of selection (>14 generations), h_R² declined significantly (P<.01) in all lines, and only the I16 and M16 lines had h_R² values significantly (P<.01) greater than zero. Realized genetic correlations involving number born, 12-day litter weight, weaning weight and six-week weight tended to decline in the second phase of selection. The I16, M16 and control (C16) replicates were crossed in all combinations at generation 14. Crosses were then selected within litters for high postweaning gain. The h_R² values in the crossbred lines were all larger than those in the second selection phase for M16-1, M16-2 and I16-1, but not for I16-2. Within each N_e level, total response was significantly (P<.01) less for I lines compared with M lines. Total response increased as N_e increased, within each level of i. Relatively small differences in realized i values among N_e lines could not account for this result. The difference in total response among the N_e lines at a given selection intensity may be due to inbreeding depression and a combination of interactions involving "drift" and selection. By crossing replicates of the M lines with the C16 control, the effects of inbreeding depression were removed. Inbreeding depression and genetic drift, as defined herein, were equally important in accounting for differences among N_e lines in total response.