Effects of plasmid presence on growth and enzyme activity of Escherichia coli DH5α

Summary The effects of recombinant DNA propagation and gene expression on the physiology of the host cell was investigated using a series of copy number mutant plasmids. The plasmids at copy numbers of 30, 57, 115 and 501 per chromosome equivalent encoded constitutive production of the enzyme -lactamase. Ribose phosphate isomerase activity was relatively unaffected by plasmid presence, and glucose-6-phosphate dehydrogenase, fructose 1,6-diphosphate aldolase and fructose 1,6-diphosphatase activities were lower in plasmid-containing cells than in the plasmid-free host strain. Increasing copy number resulted in increased depression of enzyme activity levels. The results indicate that plasmid presence mediates subtle changes in the net expression of host enzymes involved in carbon metabolism. Responses of Escherichia coli DH5 in Evans medium to these plasmids differed substantially from responses of E. coli HB101 in rich medium.Offprint requests to: J. E. Bailey     

Actions of extracellular matrix on Sertoli cell morphology and function

Sertoli cells were isolated and cultured in the absence or presence of extracellular matrix (ECM) to determine whether ECM may influence Sertoli cell function on a molecular level. As previously described, a morphological analysis of the cells indicated that ECM allows the expression of a columnar histotype and the formation of junctional complexes. The combined actions of ECM and hormones were found to have a profound effect in promoting the expression of a polarized Sertoli cell morphology. In our investigation of the effects of ECM on Sertoli cells, we used transferrin and androgen-binding protein (ABP) production as biochemical markers of Sertoli cell function. The presence of ECM was found to cause a 25% increase in the basal level of transferrin production; however, ECM had no effect on the basal level of ABP production by Sertoli cells. Regulatory agents such as follicle-stimulating hormone (FSH) and a combination of FSH, insulin, retinol, and testosterone stimulated the production of both transferrin and ABP. The ability of hormones to stimulate these Sertoli cell functions was not influenced by the presence of ECM. Similar results were obtained with 2-microns- or 50-microns-thick ECM and with a seminiferous tubule biomatrix preparation. ECM was found to increase the maintenance of long-term Sertoli cell cultures; however, the decline in Sertoli cell functional integrity, which occurs during cell culture, was not affected by the presence of ECM. An additional functional parameter examined was the radiolabeled proteins secreted by Sertoli cells. ECM did not promote the production or affect the electrophoretic profile of Sertoli cell-secreted proteins under basal or hormonally stimulated conditions. Combined results indicated that although ECM allowed the expression of a normal Sertoli cell histotype, ECM had no major effects on the Sertoli cell functions analyzed nor on the hormonal regulation of these functions. The inability of ECM to affect Sertoli cell function on a molecular level is discussed with regard to environmental as opposed to regulatory cellular interactions. Our observations imply that dramatic effects of ECM on cell morphology do not necessarily correlate to subsequent effects on cellular function.     

Hypoxia as a risk factor for doxorubicin-induced cardiotoxicity: a NMR evaluation

Previous studies suggested that one possible mechanism of doxorubicin (DXR)-induced cardiomyopathy involves the depletion of high-energy phosphate stores. In this study, we used 31P nuclear magnetic resonance to assess the high-energy phosphate content in Langendorff perfused rat hearts. Hearts were perfused in normoxic conditions (spontaneous flow) or in partially hypoxic conditions obtained by perfusing at 50% of the spontaneous flow. DXR was used at the subtoxic conditions of 50 mg/l for 15 min and at the cardiotoxic concentration of 100 mg/l for 60 min. Left ventricular pressure (dP/dt), heart rate, myocardial ATP and PCr levels and PCr/ATP ratio were measured. We found that, in normoxic conditions, DXR (50 mg/l, 15 min) does not impair cellular high-energy phosphate metabolism. However, in mild hypoxic conditions, DXR induces a significant decrease in PCr/ATP ratio, due to a decrease in PCr and to a simultaneous increase in ATP. Similar results are obtained after 60 min perfusion with the cardiotoxic dose of DXR. This study suggests that hypoxia may represent a risk factor for the development of DXR-induced acute cardiotoxicity.     

Nucleotide sequence of the Methylobacterium extorquens AM1 moxF and moxJ genes involved in methanol oxidation

The nucleotide sequence has been determined for two genes involved in methanol oxidation in the facultative methylotroph, Methylobacterium extorquens AM1. The two genes are moxF, encoding the 66-kDa subunit of the methanol dehydrogenase and moxJ, located immediately downstream from moxF, which encodes a 30-kDa protein with unknown function. This information completes the sequence of the 5.86-kb XhoI-SalI fragment containing the moxFJGI region in M. extorquens AM1, and the structure of this gene cluster is presented. Evidence is presented that moxJ is also present in Paracoccus denitrificans. The aa sequence of MoxJ has provided little information concerning its function, but it does appear to contain a signal sequence suggesting a periplasmic location.     

Inhibited enzyme electrodes. Part 3: A sensor for low levels of H2S and HCN

It is shown that an inhibited enzyme electrode, using cytochrome oxidase, will respond to H₂S, HCN and azide ion. For all three inhibitors the kinetics of the inhibition and recovery processes have been analysed using the theoretical model presented previously (Albery et al., 1990a). Rearrangement of the differential equation describing inhibition and the development of the necessary software has enabled us to obtain values of the concentration of inhibitor in a matter of seconds after exposure of the sensor. The sensor will measure concentrations of H₂S down to 1 ppm in the gas phase and concentrations of HCN and azide ion down to 0·4 μmol dm⁻³ in the solution     

Developmental appearance of the Ca2+-binding proteins parvalbumin,calbindin D-28K,S-100 proteins and calmodulin during testicular development in the rat

Summary Calcium and intracellular Ca²⁺-binding proteins are possibly involved in hormone production and spermatogenesis in rat testis. Parvalbumin, calbindin D-28K, S-100 proteins and calmodulin were localized in the Leydig cells, which are sites of testosterone synthesis. Only the appearance of parvalbumin-immunoreactivity is closely correlated to testosterone production during development of the testes. Calbindin D-28K-immunoreactivity persisted in foetal-type Leydig cells and in adult-type Leydig cells at all stages of development. S-100-immunoreactivity was low during all foetal stages, absent between birth and puberty, and increased thereafter. Calmodulin staining is most prominent in the cytoplasm of developing spermatocytes and of maturing spermatids. All four proteins co-exist in the seminiferous tubules. The distinct localization and developmental appearance of these proteins suggests different regulatory roles in Leydig cell function and spermatogenesis.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

Social organization and ecology of proboscis monkeys (Nasalis larvatus) in mixed coastal forest in Sarawak

Data are presented from a 16-month study of proboscis monkeys in an area of mixed coastal forest in Sarawak. The population density, social organization, and feeding and ranging behavior are described in detail. Results are compared with those from other primates in an attempt to understand why females of certain species (including proboscis monkeys) transfer between social groups. The scarcity of available food and reasons for the limited habitat preferences of proboscis monkeys are also discussed.