Macromolecular Syntheses During Germination and Outgrowth of Bacillus subtilis Spores

Alanine and glucose used jointly are known to be necessary and sufficient for spore germination in Bacillus subtilis 168. By testing them separately, we have verified that alanine provokes optimal phase-darkening of the spores but inhibits macromolecular syntheses, while glucose is specifically needed for initiating those syntheses. By using them in succession we obtained evidence suggesting that: (i) sporal modifications which lead to phase-darkening must occur before macromolecular synthesis can start; (ii) the amino acid pool, on which the early protein synthesis is solely dependent, expands during incubation in alanine which allows degradative but prevents synthetic activities; and (iii) progression of degradations in alanine not promptly followed by syntheses in glucose produce a metabolic imbalance in the germinating spore. A sharp transition in the origin of building blocks was shown by using a tryptophan-defective mutant. At first the synthesis of proteins depended on pre-existing amino acids from turnover of sporal material since it occurred in the absence of any exogenous amino acid and its rate remained unaltered by supplying either all amino acids except tryptophan or tryptophan alone. Eventually, protein synthesis became dependent strictly on exogenous tryptophan and strongly on the supply of several other amino acids, not required later during vegetative growth. Clearly, by the start of outgrowth, all building blocks must be provided either by endogenous de novo synthesis or by exogenous supply.     

Quantitative determination of methanogenic bacteria in the feces of different mammals

In this research on fresh human, cattle, swine, and rabbit feces, methanogenic bacteria were found in all samples examined, at the following concentrations per gram dry weight: swine, 10⁸; human, 10⁷; cattle, 10⁶; and rabbit, 10⁴. Anaerobic heterotrophic bacteria were found in the following concentrations per gram dry weight: human, 10¹¹; swine, 10¹¹; cattle, 10¹¹; and rabbit, 10¹⁰. The total number of O₂-intolerant was higher than that of O₂-tolerant bacteria: about 10–100 times for methanogenic and 100–1000 times for anaerobic heterotrophic bacteria.     

Cooperative 3-epimerization of chenodeoxycholic acid byClostridium innocuum andEubacterium lentum

In the course of an in vitro screening of intestinal microorganisms from human feces to assess their ability to perform 3-hydroxy epimerization of chenodeoxycholic acid, we isolated aClostridium innocuum strain with 3-hydroxysteroid dehydrogenating activity; this, when cocultured with a strain ofEubacterium lentum able to produced small amounts of the 3-epimer from chenodeoxycholic acid, was able to markedly strengthen the transformation. The activity of both strains has been tested on 3-oxo-CDCA and on 3-hydroxy-CDCA in an effort to clarify the cooperative epimeric conversion.     

Subclinical Myocardial Dysfunction and Cardiac Autonomic Dysregulation Are Closely Associated in Obese Children and Adolescents: The Potential Role of Insulin Resistance

Background

The prevalence of obesity is increasing among children/adolescents. Subtle cardiovascular abnormalities, responsible for a higher mortality later in life, have been reported in obese children/adolescents. The aims of the study were to evaluate cardiovascular autonomic regulation, by means of spectrum analysis of R-R interval variability, and myocardial function, by means of standard and tissue Doppler echocardiography, in a group of non-hypertensive asymptomatic obese children and adolescents; furthermore, the influence of insulin resistance was tested.

Subjects and Methods

R-R interval variability was analyzed during both the 70° head-up tilt and 24-hour electrocardiographic holter monitoring. Spectrum analysis of R-R interval variability provided the indices of sympathetic (low frequency [LF_RR]) and vagal (high frequency [HF_RR]) modulation of the sinoatrial node. Homeostasis model assessment of insulin resistance (HOMA-IR) was used to classify obese children/adolescents (n=72) as insulin resistant (n=37) and non-insulin resistant (n=35).

Results

In obese subjects: a) left ventricular mass was significantly (p<0.05) increased, whereas both the e/a ratio and the e''/a'' ratio were decreased; b) at rest, HF_RR was lower, and the LF_RR/HF_RR ratio was higher; c) during tilting, magnitude of tilt-induced inhibition of HF_RR was lower; d) during 24-hour electrocardiographic holter monitoring, LF_RR and the LF_RR/HF_RR ratio were higher, whereas HF_RR was lower; e) HOMA-IR inversely correlated with both the e''/a'' ratio (r=-0.655; p<0.001) and the tilt-induced LF_RR/HF_RR ratio (r=-0.933; p<0.001); and, f) the e''/a'' ratio correlated with the tilt-induced LF_RR/HF_RR ratio (r=0.501; p<0.001). Moreover, HF_RR at rest, magnitude of tilt-induced HF_RR reduction, and the e''/a'' ratio in insulin resistant obese children/adolescents were markedly lower when compared with the remaining subjects.

Conclusions

Subclinical abnormalities of myocardial function and of cardiac autonomic regulation were closely associated in obese children/adolescents and both correlated with the degree of insulin resistance.     

Discontinuous Occurrence of the hsp70 (dnaK) Gene among Archaea and Sequence Features of HSP70 Suggest a Novel Outlook on Phylogenies Inferred from This Protein

Occurrence of the hsp70 (dnaK) gene was investigated in various members of the domain Archaea comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria Aquifex pyrophilus and Thermotoga maritima representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in A. pyrophilus and T. maritima, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A. pyrophilus and T. maritima form a thermophilic cluster with that from the green nonsulfur bacterium Thermomicrobium roseum and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the T. maritima HSP70 clusters with the homologues from the archaea Methanobacterium thermoautotrophicum and Thermoplasma acidophilum, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from T. maritima lacks the insert, although T. maritima is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of hsp70 (dnaK) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two hsp70 (dnaK) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of hsp70 (dnaK), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous hsp70 (dnaK) gene.     

Heterologous protein production in Zygosaccharomyces bailii: physiological effects and fermentative strategies

The optimisation and scale-up of a specific protein production process have to take into account cultivation conditions as well as cell physiology of growth and the influence of foreign protein expression on host cell metabolism. The ability of Zygosaccharomyces bailii to tolerate high sugar concentrations as well as high temperatures and acidic environments renders this "non-conventional" yeast suitable for the development of biotechnological processes like heterologous protein production. This work addresses the production of human interleukin-1beta by a recombinant Z. bailii strain. We found that the heterologous protein production causes some modifications of the Z. bailii carbon metabolism, leading to a reduced biomass yield. The other important factor is the dependence of the recombinant IL-1beta production/secretion on the growth rate. Among the cultivation strategies studied, the most appropriate in terms of production and productivity was the fed-batch mode.     

A basal level of DNA damage and telomere deprotection increases the sensitivity of cancer cells to G-quadruplex interactive compounds

Here, with the aim of obtaining insight into the intriguing selectivity of G-quadruplex (G4) ligands toward cancer compared to normal cells, a genetically controlled system of progressive transformation in human BJ fibroblasts was analyzed. Among the different comparative evaluations, we found a progressive increase of DNA damage response (DDR) markers throughout the genome from normal toward immortalized and transformed cells. More interestingly, sensitivity to G4 ligands strongly correlated with the presence of a basal level of DNA damage, including at the telomeres, where the chromosome ends were exposed to the DDR without concurrent induction of DNA repair activity, as revealed by the lack of 53BP1 recruitment and telomere aberrations. The link between telomere uncapping and the response to G4 stabilization was directly assessed by showing that a partial TRF2 depletion, causing a basal level of telomere localized DDR, rendered telomerized fibroblasts prone to G4-induced telomere damage and anti-proliferative defects. Taken together these data strongly indicate that the presence of a basal level of telomere-associated DDR is a determinant of susceptibility to G4 stabilization.