TGF-β1 induces the expression of type I collagen and SPARC,and enhances contraction of collagen gels,by fibroblasts from young and aged donors

Fibroblasts have a major role in the synthesis and reorganization of extracellular matrix that occur during wound repair. An impaired biosynthetic or functional response of these cells to stimulation by growth factors might contribute to the delayed wound healing noted in aging. We, therefore, compared the responses of dermal fibroblasts from young and elderly individuals (26, 29, 65, 89, 90, and 92 years of age) to transforming growth factor-β1 (TGF-β1) with respect to: (1) the synthesis of type I collagen and SPARC (two extracellular matrix proteins that are highly expressed by dermal fibroblasts during the remodeling phase of wound repair) and (2) the contraction of collagen gels, an in vitro assay of wound contraction. With the exception of one young donor, all cultures exposed for 44 hours to 10 ng/ml TGF-β1 exhibited a 1.6- to 5.5-fold increase in the levels of secreted type 1 collagen and SPARC, relative to untreated cultures, and exhibited a 2.0- to 6.2-fold increase in the amounts of the corresponding mRNAs. Moreover, the dose-response to TGF-β1 (0.1–10 ng/ml), as determined by synthesis of type I collagen and SPARC mRNA, was as vigorous in cells from aged donors as in cells from a young donor. In assays of collagen gel contraction, fibroblasts from all donors were stimulated to a similar degree by 10 ng/ml TGF-β1. In conclusion, cells from both young and aged donors exhibited similar biosynthetic and contractile properties with exposure to TGF-β1. It therefore appears that the impaired wound healing noted in the aged does not result from a failure of their dermal fibroblasts to respond to this cytokine. © 1994 Wiley-Liss, Inc.     

A linkage map of diploid Avena based on RFLP loci and a locus conferring resistance to nine isolates of Puccinia coronata var. ‘avenae’

An F₂ oat population was produced by crossing the diploid (n=7) species Avena strigosa (CI 3815) with A. wiestii (CI 1994), resistant and susceptible, respectively, to 40 isolates of Puccinia coronata, the causal agent of crown rust. Eighty-eight F₂ individuals were used to construct an RFLP linkage map representing the A genome of cultivated hexaploid oat. Two hundred and eight RFLP loci have been placed into 10 linkage groups. This map covers 2416 cM, with an average of 12 cM between RFLP loci. Eighty-eight F₃ lines, derived from F₂ individuals used to construct the map, were screened for resistance to 9 isolates of P. coronata. One locus, Pca, was found to confer a dominant resistance phenotype to isolates 203, 258, 263, 264B, 290, 298, 325A, and 345. Pca also conferred resistance to isolate 276; however, an unlinked second gene may also be involved.Journal Paper No. 15143 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3134 and 2447     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

Agonist Binding to α2-Adrenoceptors Is Elevated in the Locus Coeruleus from Victims of Suicide

Abstract: The binding of an agonist, p-[¹²⁵I]iodoclonidine, and an antagonist, [³H]yohimbine, to α₂-adrenoceptors was measured autoradiographically in the locus coeruleus from 10 pairs of antidepressant-free victims of suicide and age-matched controls. Agonist binding to α₂-adrenoceptors was significantly greater in the locus coeruleus from victims of suicide compared with control subjects. In contrast, antagonist binding to α₂-adrenoceptors in the locus coeruleus did not differ significantly between control and suicide subjects. HPLC analysis of norepinephrine in tissue sections of the locus coeruleus did not reveal any differences between control subjects and suicide victims, suggesting that differences in agonist binding are not a result of differences in retention of the endogenous agonist norepinephrine in tissue sections. The increase in agonist binding to α₂-adrenoceptors in the locus coeruleus of victims of suicide links an altered expression of the high-affinity state of autoinhibitory α₂-adrenoceptors with suicide.     

A mating-type factors of Coprinus cinereus have variable numbers of specificity genes encoding two classes of homeodomain proteins

We have identified the seven genes that constitute the A43 mating-type factor of Coprinus cinereus and compare the organisation of A43 with the previously characterised A42 factor. In both, the genes that trigger clamp cell development, the so-called specificity genes, are separated into and loci by 7 kb of noncoding sequence and are flanked by homologous genes -fg and -fg. The specificity genes are known to encode two classes of dissimilar homeodomain (HD1 and HD2) proteins and have different allelic forms which show little or no cross-hybridisation. By partial sequencing we identified a divergently transcribed HD1 (a1-2) and HD2 (a2-2) gene in the A43 locus. a2-2 failed to elicit clamp cell development in three different hosts, suggesting that it is non-functional. a1-2 elicited clamp cells in an A42 host that has only an HD2 gene (a2-1) in its locus, thus demonstrating that the compatible A mating interaction is between an HD1 and an HD2 protein. The A43 locus contains three specificity genes, the divergently transcribed HD1 and HD2 genes b1-2 and b2-2 and a third HD1 gene (d1-1) that was shown by hybridisation and transformation analyses to be functionally equivalent to d1-1 in A42. An untranscribed footprint of a third A42 HD1 gene, c1-1, was detected between the A43 b2-2 and d1-1 genes by Southern hybridisation.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Analysis of variance of first-year growth in captive garter snakes (Thamnophis elegans) by family and sex

Although body size in reptiles has important consequences for their ecology, it is quite variable in most species, presumably because growth in these animals is influenced by many factors. In this study, we analyse the effects of family membership and sex on first-year growth of garter snakes (Thamnophis elegans) held under identical conditions in the laboratory. Litters, but not the two sexes, differed significantly in mean size at birth. Larger snakes ate more food and had a higher absolute (but not relative) growth rate in length than smaller snakes. Thus, there was significant variation in length increase among litters, but not between the sexes. All else being equal, variance in size at birth could contribute significantly to variance in adult body size.     

Effect of fructose-1,6-bisphosphate on glutamate uptake and glutamine synthetase activity in hypotix astrocyte cultures

Astrocytes are important in regulating the microencironment of neurons both by catabolic and synthetic pathways. The glutamine synthetase (GS) activity observed in astrocytes affects neurons by removing toxic substances, NH₃ and glutamate; and by providing an important neuronal substrate, glutamine. This glutamate cycle might play a critical role during periods of hypoxia and ischemia, when an increase in extracellular excitatory amino acids is observed. It was previously shown in our laboratory that fructose-1,6-bisphosphate (FBP) protected cortical astrocyte cultures from hypoxic insult and reduced ATP loss following a prolonged (18–30 hrs) hypoxia. In the present study we established the effects of FBP on the level of glutamate uptake and GS activity under normoxic and hypoxic conditions. Under normoxic conditions, [U-¹⁴C]glutamate uptake and glutamine production were independent of FBP treatment; whereas under hypoxic conditions, the initial increase in glutamate uptake and an overall increase in glutamine production in astrocytes were FBP-dependent. Glutamine synthetase activity was dependent on FBP added during the 22 hours of either normoxic- or hypoxic-treatment, hence significant increases in activity were observed due to FBP regardless of the oxygen/ATP levels in situ. These studies suggest that activation of GS by FBP may provide astrocytic protection against hypoxic injury.     

The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

The relationship between synthesis and N-liked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (<10g ml^–1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.Abbreviations clp-PRL clipped form of prolactin - DMEM/F12 11 Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12 - G-PRL glycosylated (N-linked) fraction of prolaction - NG-PRL prolactin fraction without N-linked glycosylation - PMSF phenylmethylsulfonylfluoride