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.     

Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

ULTRASTRUCTURE OF GLANDULAR TRICHOMES OF LEAVES OF NICOTIANA TABACUM L., cv XANTHI

Electron microscopy confirms previous light microscope observations that tobacco leaf trichomes are glandular and that there are two different types. Both the tall trichome (multicellular stalk, unicellular or multicellular head) and the short trichome (unicellular stalk; multicellular head) exhibit characteristics common to gland cells—a dense cytoplasm, numerous mitochondria, and little vacuolation. The tall trichome contains structurally well developed chloroplasts and an elaborate network of endoplasmic reticulum. The short trichome contains undifferentiated plastids and endoplasmic reticulum which parallels the nucleus and plasmalemma. Few dictyosomes are seen either in the short trichome or in the tall trichome. The short trichome appears to undergo structural changes concurrently with the appearance of secretory product within the cells. The most noticeable change is the formation of the extraplasmic space between the cell wall and the plasmalemma. Electron dense secretory product is observed between the plasmalemma and the cell wall and within the intercellular spaces.     

Prevalence and distribution of four serotypes of SMSV serum neutralizing antibodies in wild animal populations

Serum neutralizing antibodies to four serotypes of San Miguel Sea Lion Virus (SMSV) were demonstrated in a variety of marine and terrestrial species. These results show a wide geographic distribution of SMS viruses in the marine environment and indicate that certain terrestrial mammals have been infected with these so-called marine viruses. Evidence is presented supporting the theory that unidentified submammalian marine species are a reservoir for SMSV.     

Initial Characterization of the Pig Skin Bacteriome and Its Effect on In Vitro Models of Wound Healing

Elucidating the roles and composition of the human skin microbiome has revealed a delicate interplay between resident microbes and wound healing. Evolutionarily speaking, normal cutaneous flora likely has been selected for because it potentiates or, at minimum, does not impede wound healing. While pigs are the gold standard model for wound healing studies, the porcine skin microbiome has not been studied in detail. Herein, we performed 16S rDNA sequencing to characterize the pig skin bacteriome at several anatomical locations. Additionally, we used bacterial conditioned-media with in vitro techniques to examine the paracrine effects of bacterial-derived proteins on human keratinocytes (NHEK) and fibroblasts (NHDF). We found that at the phyla level, the pig skin bacteriome is similar to that of humans and largely consists of Firmicutes (55.6%), Bacteroidetes (20.8%), Actinobacteria (13.3%), and Proteobacteria (5.1%) however species-level differences between anatomical locations exist. Studies of bacterial supernatant revealed location-dependent effects on NHDF migration and NHEK apoptosis and growth factor release. These results expand the limited knowledge of the cutaneous bacteriome of healthy swine, and suggest that naturally occurring bacterial flora affects wound healing differentially depending on anatomical location. Ultimately, the pig might be considered the best surrogate for not only wound healing studies but also the cutaneous microbiome. This would not only facilitate investigations into the microbiome’s role in recovery from injury, but also provide microbial targets for enhancing or accelerating wound healing.     

Association of prolactin and insulin receptors with mammogenesis and lobulo-alveolar formation in pregnant ewes

1. Pregnant, multiparous ewes were sacrificed at d 50 (n = 3), 80 (n = 4), 115 (n = 3), or 140 (n = 4) for biochemical, histological and autoradiographical quantitation of mammary growth. 2. Significant increases in concentration of mammary parenchyma DNA (0.25-24 mg/g tissue), total under DNA (57-1304 mg), and total under RNA (36-1504 mg) were observed by d 115. 3. Mammary tissue at d 115 contained the maximal percent of tissue volume occupied by epithelium (41.2%), number of cells per alveolar cross section (36.6) and percent of [methyl-3H]thymidine labeled epithelial cell nuclei (3.5%). 4. Concentration prolactin binding sites were significantly increased at d 115 of gestation, but serum prolactin and growth hormone concentrations remained low until d 140. 5. Mammary insulin binding sites (per unit membrane protein) progressively decreased during gestation. 6. Results suggest that serum growth hormone concentration and quantity of insulin receptors do not limit mammogenesis but greater concentrations of prolactin binding sites coincident with lobulo-alveolar formation, suggest a primary association with mammogenesis in the ewe.     

A selective somatostatin type-2 receptor agonist inhibits neointimal thickening and enhances endothelial cell growth and morphology following aortic balloon injury in the rabbit

Somatostatin analogs have been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and attenuate neointimal thickening following experimental balloon catheter injury. In this study, the effects of a selective agonist for the somatostatin receptor subtype 2, PRL-2486, on neointimal thickening and endothelial cell regrowth 2 weeks following balloon catheterization of male New Zealand White rabbits were determined. Rabbits treated 2 days prior to and 2 weeks after catheter injury with 10 g/kg/day PRL-2486 (PRL-tx) had decreased I/M ratios (intimal area/medial area × 100; p < 0.05) but had no effect at lower (5 g/kg/day) or higher (20 g/kg/day) doses. PRL-tx had significantly decreased VSMC proliferation compared to untreated animals. PRL-tx increased endothelial regrowth by over 2-fold (p < 0.002) and improved endothelial cell morphology. Endothelial-dependent relaxation responses to acetylcholine were attenuated by catheter injury, and were not improved with PRL-tx. These data suggest that the PRL-2486-mediated inhibition of neointimal thickening exhibits a bell-shaped dose-response curve. This inhibition may be due in part to decreased VSMC proliferation, which may be a function of enhanced endothelial regrowth, but not the return of endothelium-dependent vascular function.