Activation of 72-kDa Type IV Collagenase/Gelatinase by Normal Fibroblasts in Collagen Lattices Is Mediated by Integrin Receptors but Is Not Related to Lattice Contraction

The matrix metalloproteinase 72-kDa type IV collagenase (also known as gelatinase A) is thought to be involved in both normal connective tissue remodeling and invasive pathological processes. Like other matrix metalloproteinases, 72-kDa type IV collagenase is secreted by fibroblast monolayers as an inactive proenzyme, but is unique among this enzyme family in that it is not activated by serine proteinases such as plasmin. However, when fibroblasts are cultured in a collagen lattice, a situation thought to better approximate in vivo conditions, we have invariably found much of the secreted 72-kDa type IV collagenase in its enzymatically active 62-kDa form. Although collagen lattice contraction appeared to be required for the activation of 72-kDa type IV collagenase, we have found that the process of contraction can be dissociated from proenzyme activation. Both cytochalasin D and α-methylmannoside completely blocked lattice contraction, but not proenzyme activation. Furthermore, the monoclonal antibody M-13, which is directed against the β₁ integrin chain, blocked collagen lattice contraction but not 72-kDa type IV procollagenase activation. At concentrations significantly higher than required to block lattice contraction or cell adhesion to collagen, M-13 was able to inhibit proenzyme activation. A second monoclonal antibody to the β₁ integrin, P5D2, had little effect on collagen lattice contraction at low concentrations, but could significantly inhibit the activation of 72-kDa type IV procollagenase. Antibodies to the integrin α₂ chain also inhibited proenzyme activation. These data show that the activation of 72-kDa type IV collagenase proenzyme, like collagen lattice contraction, is mediated by β₁ integrin receptors, possibly α₂β₁. Although both anti-β₁ antibodies used are directed to the same site on the integrin chain, the fact that each antibody preferentially blocks a different event, either lattice contraction or activation of 72-kDa type IV collagenase, suggests the existence of branch points in the receptor-mediated signal transduction pathway.     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

Novel bacteriophage lambda mutation affecting lambda head assembly.

A novel phage lambda mutation, called dc10, which interferes with proper lambda head assembly has been isolated and characterized. Phage lambda carrying this mutation is (i) unable to form plaques at 30 or 37 degrees C but does so at 42 degrees C and (ii) unable to form plaques at 42 degrees C on pN-constitutive hosts. Both properties are due to dc10 since all phage revertants for one phenotype simultaneously lose the other phenotype and vice versa. The dc10 mutation has been mapped in the B gene and has been shown to be dominant over the corresponding wild-type product. At 30 degrees C the dc10 mutation results in the formation of abnormal petit lambda heads made up of pE, pB, pC, and pNu3. Under pN-constitutive conditions, the dc10 mutation results in the formation of abnormal petit lambda heads made of pE, X1, and X2 only. A model to explain the data is presented.     

Soluble trinitrophenylated proteins and trinitrophenylated cells as specific inhibitors of target cell lysis by cytotoxic T lymphocytes.

Bovine serum albumin (BSA) substituted in 12 to 15 amino groups with 2,4,6-trinitrophenyl (Tnp-BSA) or carbobenzoxy (Cbz-BSA) or acetyl (Ac-BSA) groups was tested as inhibitor of the reaction in which anti-Tnp cytotoxic T lymphocytes (CTLs) lysed syngeneic ⁵¹Cr-labeled Tnp-modified spleen cells [concanavalin A (Con A) blasts]. Inhibition was observed with some consistency only with Tnp-BSA at extremely high concentration (50 mg/ml). To explore the significance of this observation, inhibition of anti-Tnp CTLs was also tested with Tnp-modified cells on which products of the major histocompatibility loci H-2K and H-2D were lacking or different from those on the stimulator cells used to elicit the CTLs. Only those Tnp cells with the same H-2 products as the stimulators were inhibitory, even though all the Tnp cells tested had essentially the same surface density of Tnp (ca 1 × 10⁸ groups/cell). It is concluded that effective specific inhibitors of anti-Tnp CTLs have both Tnp groups and the correct H-2 products on the same particle and that the specific inhibitory activity of soluble Tnp-BSA was probably due to its adsorption onto cells in the suspensions used to assay cytotoxicity.     

Microfluorometry analysis II. A Bayesian approach.

In flow microfluorometry (FMF) analysis cells stained with a fluorescent dye that binds specifically to DNA are passed through the instrument. The number of cells in the population having a fluorescence intensity is recorded in a single channel of a multichannel pulse height analyzer. The result is a DNA fluorescence histogram for the population.A method for decomposing an FMF histogram into its G₁, S and G₂ + M components, corresponding to similarly designated phases of the cell cycle is given. This technique can also be applied to find the parameters in all of the previous approaches. The parameters are calculated by iteration which eliminates the need for non-linear optimization procedures.     

Correlated responses in growth and body composition of replicated single-trait and index selected lines of mice

Summary Correlated responses in growth, body composition and efficiency were evaluated in lines of mice selected in the following ways: W⁺T _i ^o , increased six-week body weight (WT6); W ° T _i ⁺ , increased six-week tail length (TL6); W⁺T _i ^– , increased WT6 and decreased TL6; W^–T _i ⁺ , decreased WT6 and increased TL6; M16, increased three-to six-week postweaning gain (PWG). Each of the first four selection treatments had two replicate lines (i = 1, 2) selected for 13 generations and the fifth treatment had one line selected for 30 generations. All lines were derived from a randombred ICR albino population which served as a control. Additional traits studied were three-week body weight and tail length, postweaning gain in tail length, percent body composition (ash, fat, moisture and protein) at six weeks of age, and three-to six-week feed consumption (CONS) and efficiency (EFF = PWG/CONS). Efficiency of body constituent gains (ash, fat, protein and caloric value) were determined by dividing each constituent by CONS. Relative to selection treatments, replicate variation in the array of traits was small and was primarily attributable to the effects of genetic drift; more frequent significant replicate differences among traits in W⁺T^– were associated with a replicate difference in cumulative selection differentials. Selection for different criteria involving WT6 and TL6 did not change the allometric relationship between tail length and body weight in the three-to six-week age interval. The significant divergence between W⁺T ° and W °T⁺ and between W⁺T^– and W^–T⁺ was as expected for WT6 and TL6. Significant asymmetry of selection response between W⁺T^– and W^–T⁺ for WT6 and TL6 was attributed to maternal effects. In agreement with theory, antagonistic index selection generally yielded smaller genetic responses than single trait selection. Positive correlated responses in CONS and EFF were found for M16 and W⁺T °. Significant correlated changes in CONS (positive in W °T⁺ and negative in W^–T⁺) were not accompanied by a significant change in EFF. In contrast, W⁺T^– evinced an increased EFF and no change in CONS. Percent fat increased significantly in W⁺T ° and M16. For W⁺T^o, W⁺T^– and M16, an increased energetic, fat and ash efficiency was observed, whereas M16 exhibited a positive increment in protein efficiency as well. Among selection treatment means, there were high positive correlations between WT6 and fat weight, protein weight, percent fat, CONS and EFF and a high negative correlation between WT6 and percent protein.Paper No.4916 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. 27607. 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.     

An IS4 transposition causes a 13-bp duplication of phage lambda DNA and results in the constitutive expression of the cI and cro gene products

We have examined the nature of the additional DNA present in lambda hyp- mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage lambda at nucleotide position 39,139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right lambda-IS4 DNA junctions in phage lambda hyp15rev4. The IS4 integration event resulted in the duplication of 13 bp of lambda DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS4 integrations elsewhere (Klaer et al., 1981).     

Enteric nervous system modulation of luminal pH modifies the microbial environment to promote intestinal health

The enteric nervous system (ENS) controls many aspects of intestinal homeostasis, including parameters that shape the habitat of microbial residents. Previously we showed that zebrafish lacking an ENS, due to deficiency of the sox10 gene, develop intestinal inflammation and bacterial dysbiosis, with an expansion of proinflammatory Vibrio strains. To understand the primary defects resulting in dysbiosis in sox10 mutants, we investigated how the ENS shapes the intestinal environment in the absence of microbiota and associated inflammatory responses. We found that intestinal transit, intestinal permeability, and luminal pH regulation are all aberrant in sox10 mutants, independent of microbially induced inflammation. Treatment with the proton pump inhibitor, omeprazole, corrected the more acidic luminal pH of sox10 mutants to wild type levels. Omeprazole treatment also prevented overabundance of Vibrio and ameliorated inflammation in sox10 mutant intestines. Treatment with the carbonic anhydrase inhibitor, acetazolamide, caused wild type luminal pH to become more acidic, and increased both Vibrio abundance and intestinal inflammation. We conclude that a primary function of the ENS is to regulate luminal pH, which plays a critical role in shaping the resident microbial community and regulating intestinal inflammation.