Mapping of a restriction fragment length polymorphism within the human aldolase B gene

Summary Peripheral blood DNA was hybridized to the full-length cDNA and the cloned structural gene of human aldolase B. With PvuII endonuclease a restriction fragment length polymorphism was detected that was present in the heterozygous state in about 21% of the individuals tested. A map of the human aldolase gene was constructed for the two groups of individuals found to produce different fragments after PvuII digestion. This allowed the localization of the polymorphic site within the gene, which was found to be due to the loss of a PvuII site in the last intron upstream from the 3 end. This polymorphism may be used as a genetic marker to study individuals affected by hereditary fructose intolerance.     

Pancreatic islets of variable size--insulin secretion and glucose utilization

Glucose metabolism and insulin secretion were studied in isolated rat pancreatic islets of different sizes and the amount of tissue was quantitated by the measurement of DNA. It was found that larger islets (140-210 ng DNA/islet) utilized more glucose (based on the conversion of 3H-5-glucose to [3H]20) per ng of DNA than islets containing less DNA (60-120 ng/islet). However, the insulin secreted per ng of DNA in response to a given glucose concentration was the same in islets of all sizes. Also, the islet insulin and glucagon content when expressed in terms of DNA did not depend upon islet size. Thus, although glucose utilization rates expressed as a function of islet DNA content were greater in larger islets, no such relationship was found for glucose-induced insulin release or insulin and glucagon content.     

Post-embryonic genesis of bursal follicles in the duck

The structural and histological characteristics of the limiting layer between the cortex and medulla of the bursa of Fabricius in the duck are described in relation to different functional moments of the organ. On E.M. study the limiting layer, in duck having 30 days of age, is constituted of reticular-epithelial cells palisades in shape with many cytoplasmics extension. Lymphatic migration occur in interstitial spaces.     

8-Azaguanine versus 6-thioguanine: influence on frequency and expression time of induced HGPRT- mutations in Chinese hamster V79 cells

Chinese hamster V79 cells were mutagenized with ethyl methanesulfonate at various concentrations. Clones resistant to 8-azaguanine (20 and 80 micrograms/ml) or 6-thioguanine (4 micrograms/ml) were selected at different times after the treatments. The total yield of induced mutations was only slightly affected by the kind and concentration of purine analog used in the selection. However, full phenotypic expression of the mutants selected with 8-azaguanine was achieved earlier than that of mutants resistant to 6-thioguanine. This result seems to be best explained by the reported lower affinity of 8-azaguanine for the wild-type HGPRT enzyme, thus providing evidence that, in this gene-mutation assay, the phenotypic expression time has a physiological component.     

Effect of a biofilm on the adsorption of 4-chlorophenol on activated carbon

  The adsorption of 4-chlorophenol (4-CP) on activated carbon was studied experimentally both in the presence and in the absence of an inactivated anaerobic biofilm on the surface of carbon pellets. The presence of the biofilm markedly decreased the rate of 4-CP adsorption. However, the final near-equilibrium state (at 27 h) was not affected, and the incremental amount of material adsorbed on the pellets was similar both in the presence and in the absence of the biofilm. The biosorption of 4-CP by a biofilm coating non-adsorbing pellets was also determined. It appears that the biofilm also has some adsorption capability. Freundlich-type equations were used to correlate all data, and transient and near-equilibrium isotherms were obtained for 4-CP adsorption on different adsorbing materials at different times. Received: 30 April 1996 / Received revision: 17 July 1996 / Accepted: 4 August 1996     

Matrigel® invasion by the prostate cancer cell lines,PC3 and DU145, and cathepsin L+B activity

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel®. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel®. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.     

Localization of Mitochondrial Carnitine/Acylcarnitine Translocase in Sensory Neurons from Rat Dorsal Root Ganglia

The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.     

Size,sex and social experience: Experimental tests of multiple factors mediating contest behaviour in a rockpool fish

Aggressive contests amongst conspecifics are important to understand from an ecological and evolutionary perspective as contest dynamics can directly influence individual fitness. For some species, individual attributes such as relative body size closely predict the outcome, intensity and duration of contests, whereas for others, prior social experiences play a key role. However, disentangling the relative effects of individual attributes and social experiences is challenging from an experimental perspective, and because of this, few studies have investigated relative effects of multiple factors. Rockpool fishes have been well studied in terms of factors governing abundance, distribution and community structure, but much less so in terms of contest behaviour. This is surprising because contest dynamics are likely to directly affect the distribution of fishes along the rocky shore, and hence indirectly govern population and community composition. Here, we explored multiple factors potentially influencing contest behaviour in a numerically dominant, resident intertidal fish species, Bathygobius cocosensis (Gobiidae). Using a series of staged pairwise contest trials, we investigated the effect of size, sex and social experiences (namely prior residency and winner–loser experiences) on contest dynamics. We found no evidence that prior residency influenced contest outcome, suggesting social experiences play a minor role in contest dynamics. Previous winner/loser experience also did not influence contest outcome, although this is likely a product of low sample size. In addition, the likelihood of winning was unrelated to contestant sex, and the combination of sexes in paired contests did not influence contest intensity or duration. Instead, body size was related to contest outcome, intensity and duration in the majority of experimental trials. These results suggest that body size rather than sex or social experiences is the key determinant of contest dynamics in this species. We suggest that the dynamic biotic and abiotic environment inhabited by intertidal fish may reduce the influence of prior social experiences in modulating contest dynamics.     

ZMAT3 hypomethylation contributes to early senescence of preadipocytes from healthy first‐degree relatives of type 2 diabetics

Senescence of adipose precursor cells (APC) impairs adipogenesis, contributes to the age‐related subcutaneous adipose tissue (SAT) dysfunction, and increases risk of type 2 diabetes (T2D). First‐degree relatives of T2D individuals (FDR) feature restricted adipogenesis, reflecting the detrimental effects of APC senescence earlier in life and rendering FDR more vulnerable to T2D. Epigenetics may contribute to these abnormalities but the underlying mechanisms remain unclear. In previous methylome comparison in APC from FDR and individuals with no diabetes familiarity (CTRL), ZMAT3 emerged as one of the top‐ranked senescence‐related genes featuring hypomethylation in FDR and associated with T2D risk. Here, we investigated whether and how DNA methylation changes at ZMAT3 promote early APC senescence. APC from FDR individuals revealed increases in multiple senescence markers compared to CTRL. Senescence in these cells was accompanied by ZMAT3 hypomethylation, which caused ZMAT3 upregulation. Demethylation at this gene in CTRL APC led to increased ZMAT3 expression and premature senescence, which were reverted by ZMAT3 siRNA. Furthermore, ZMAT3 overexpression in APC determined senescence and activation of the p53/p21 pathway, as observed in FDR APC. Adipogenesis was also inhibited in ZMAT3‐overexpressing APC. In FDR APC, rescue of ZMAT3 methylation through senolytic exposure simultaneously downregulated ZMAT3 expression and improved adipogenesis. Interestingly, in human SAT, aging and T2D were associated with significantly increased expression of both ZMAT3 and the P53 senescence marker. Thus, DNA hypomethylation causes ZMAT3 upregulation in FDR APC accompanied by acquisition of the senescence phenotype and impaired adipogenesis, which may contribute to FDR predisposition for T2D.     

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.