The antagonism of glucocorticoid inhibition of wound healing in rats by growth hormone-releasing factor

Daily therapeutic injections of cortisone to rats will cause weight loss and impaired wound healing. Weight loss is attributed to the catabolic effect of steroid, whereas impaired healing is associated with reductions in fibroplasia and connective tissue deposition. As the major structural protein component of connective tissue is collagen, its absence is responsible for the retarded gain in wound breaking strength. Cortisone also blocks wound closure by inhibiting wound contraction. An anabolic agent such as growth hormone may antagonize the effect of cortisone on the wound healing process. Endogenous GH can be released from the pituitary by exogenous injections of growth hormone-releasing factor (GRF). Two synthetic GRF peptides, a natural 44-amino acid peptide of the human GRF sequence, GRF-44, and an N-terminally substituted analog 29 residues, GRF-29A, were studied. Each was given twice daily with a single daily injection of cortisone for a 7-day period. Concurrent administration of GRF-44 or GRF-29A and cortisone to rats had no effect on restored body weight loss or inhibited wound contraction. While GRF-44 restored collagen deposition and caused restored wound breaking strength, GRF-29A was ineffective in restoring either. GRF-44, a synthetic peptide that stimulates pituitary release of growth hormone, antagonized some of the inhibiting effect of steroid on wound repair by promoting fibroplasia and collagen deposition.     

Stress and immunity: an integrated view of relationships between the brain and the immune system

The old notion that stress exacerbates the progression of physical illness via its corticosteroid-mediated immunosuppressive effects must be revised. Experimental and clinical studies demonstrate that both laboratory and natural stressors alter the activities of lymphocytes and macrophages in a complex way that depends on the type of immune response, the physical and psychological characteristics of the stressor and the timing of stress relative to the induction and expression of the immune event. The influences of stress on immunity are mediated not only by glucocorticoids but also by catecholamines, endogenous opioids and pituitary hormones such as growth hormone. Sensitivity of the immune system to stress is not simply fortuitous but is an indirect consequence of the regulatory reciprocal influences that exist between the immune system and the central nervous system. The immune system receives signals from the brain and the neuroendocrine system via the autonomic nervous system and hormones and sends information to the brain via cytokines. These connections appear to be part of a long-loop regulatory feedback system that plays an important role in the coordination of behavioral and physiological responses to infection and inflammation.     

Beta-oxidation of long-chain fatty acids by human fibroblasts: evidence for a novel long-chain acyl-coenzyme A dehydrogenase.

Fibroblasts from patients with long-chain acyl-CoA dehydrogenase deficiency were found to oxidize [1-14C]linoleate at an average rate of 60% of normal but [9,10(n)-3H]myristate at an average rate of only 37% of normal, a relationship reverse from that predicted by the chain-length specificities of the three known straight-chain mitochondrial acyl-CoA dehydrogenases. The residual long-chain beta-oxidative activity was found to be mitochondrial and associated with the accumulation of tetradecadienoate (C14:2w6) when the mutant fibroblasts were incubated with 100 mumol/L linoleate (C18:2w6) or eicosadienoate (C20:2w6). The results suggest the presence in human fibroblasts of a novel acyl-CoA dehydrogenase with activity toward 15 to 20 carbon-length fatty acids.     

Renaturation of cobra venom phospholipase A2 expressed from a synthetic gene in Escherichia coli.

Cobra venom (Naja naja naja) phospholipase A2 (PLA2) contains 14 cysteines in the form of 7 disulfide bonds amongst its 119 amino acids. A gene encoding the PLA2 was synthesized and inserted into a bacterial expression vector containing the phage lambda pL promoter. In order to obtain protein without the initiating methionine at the N-terminus, a Factor Xa site was engineered upstream from the PLA2 gene. Upon heat-induction of the cells transformed with the expression plasmid, the protein is produced as insoluble inclusion bodies. The enzyme was partially purified by washing the inclusion bodies with Triton X-100 and urea. The expressed protein was first denatured with 8 M guanidine-HCl and 10 mM DTT. After digestion with Factor Xa, formation of disulfide bonds and refolding into the fully active form was carried out in the presence of cysteine and Ca2+. The renatured recombinant protein was purified by Affi-gel blue column chromatography. The purified recombinant enzyme had the same specific activity as the native enzyme when assayed on a variety of substrates and cross-reacted with antisera prepared against the native enzyme. This is the first report of the expression of a recombinant PLA2 from any venom.     

Characteristics and N-terminal amino acid sequence of a manganese peroxidase purified from Lentinula edodes cultures grown on a commercial wood substrate

Summary Extracellular culture filtrates from ligninolytic cultures of the lignin-degrading basidiomycete Lentinula (syn. Lentinus) edodes (Berk.) Pegler contained one major peroxidase when grown on a commercial oak-wood substrate. The peroxidase was purified by polyethylenimine clarification, anion-exchange chromatography, and hydrophobic-interaction HPLC. The enzyme (MnP1) was a heme-iron protein with an apparent molecular weight of 44 600 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and an isoelectric point of pH 3.2. The native enzyme had an absorption maximum at 407 nm, which shifted to 420 nm upon H₂O₂ addition. The pyridine-hemochrome-absorption spectrum indicated that one heme group was present per enzyme as protoporphyrin IX. N-terminal amino acid sequencing showed that MnP1 had higher sequence homology with manganese peroxidases than with lignin peroxidases reported from Phanerochaete chrysosporium. L. edodes MnP1 was capable of oxidizing lignin and lignin-model compounds in the presence of manganese and H₂O₂.On leave from the Department of Biochemistry, University of Otago, P. O. Box 56, Dunedin, New Zealand.Research carried out while a visiting scientist at the USDA Forest Products Laboratory from the National Chemistry Laboratory, Pune, India 41 1008 Offprint requests to: I. T. Forrester     

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions

Summary A serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbecco's modified Eagle's medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated. This work was supported by grants CA 33240 and CA 35641 and by core grant CA 24538 from the National Institutes of Health, Bethesda, MD.     

Controlled experiments of habitat fragmentation: a simple computer simulation and a test using small mammals

Habitat fragmentation involves a reduction in the effective area available to a population and the imposition of hard patch edges. Studies seeking to measure effects of habitat fragmentation have compared populations in fragments of different size to estimate and area effect but few have examined the effect of converting open populations to closed ones (an effect of edges). To do so requires a shift in spatial scope-from comparison of individual fragments to that of fragmented versus unfragmented landscapes. Here we note that large-scale, controlled studies of habitat fragmentation have rarely been performed and are needed. In making our case we develop a simple computer simulation model based on how individual animals with home ranges are affected by the imposition of habitat edges, and use it to predict population-level responses to habitat fragmentation. We then compare predictions of the model with results from a field experiment on Peromyscus and Microtus. Our model treats the case where home ranges/territories fall entirely within or partially overlap with that of sample areas in continuous landscapes, but are restricted to areas within habitat fragments in impacted landscapes. Results of the simulations demonstrate that the imposition of hard edges can produce different population abundances for similar-sized areas in continuous and fragmented landscapes. This edge effect is disproportionately greater in small than large fragments and for species with larger than smaller home ranges. These predictions were generally supported by our field experiment. We argue that large-scale studies of habitat fragmentation are sorely needed, and that control-experiment contrasts of fragmented and unfragmented microlandscapes provide a logical starting point.     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm² of growth flask). Results show that the apparent expression of extended Le^a-Le^x, Le^a and Le^x, sialyl Le^a, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.Abbreviations Mab monoclonal antibody - FIT fluorescein isothiocyanate - TACA tumor associated carbohydrate antigen     

Black tea,green tea,and tea polyphenols

Previous studies have shown that tea consumption can impair trace element metabolism, particularly iron status, and increase the risk of anemia in humans and animals. More recently, however, evidence has been accumulating to show that, in animals, consumption of green tea or its polyphenols is associated with a reduction of the incidence and severity of a variety of experimentally induced cancers. In this study we have monitored the growth, trace element status, including hematological parameters of weanling rats given either (1) water, (2) 1% black tea, (3) 1% green, tea, or (4) 0.2% crude green tea extract as their sole drinking fluid while consuming diets containing either adequate or low amounts of iron. With the exception of manaanese, none of the trace elements studied (iron, copper, zinc, and manganese) or the hematological indices measured were affected by the type of beverage supplied, even though the polyphenol extract was shown to chelate metals in vitro and all the animals fed the low iron diet were shown to be anemic. There appeared to be an effect of black and green teas on manganese balance in, both the first and last weeks of the study. A lower level of brain managanese was associated with green tea consumption, and a higher level of this element in the kidneys of animals fed black tea. The results demonstrate that both black and green teas and a green tea polyphenol extract do not represent a risk to animals consuming the beverages as their sole fluid intake with respect to iron availability, although the interactions with manganese deserve further study.