Biophysical properties of normal and diseased renal glomeruli

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Suitability of Solanum lycopersicum L. ‘Microtom’ for growth in Bioregenerative Life Support Systems: exploring the effect of high‐LET ionising radiation on photosynthesis,leaf structure and fruit traits

• The realisation of manned space exploration requires the development of Bioregenerative Life Support Systems (BLSS). In such self‐sufficient closed habitats, higher plants have a fundamental role in air regeneration, water recovery, food production and waste recycling. In the space environment, ionising radiation represents one of the main constraints to plant growth.
• In this study, we explore whether low doses of heavy ions, namely Ca 25 Gy, delivered at the seed stage, may induce positive outcomes on growth and functional traits in plants of Solanum lycopersicum L. ‘Microtom’. After irradiation of seed, plant growth was monitored during the whole plant life cycle, from germination to fruit ripening. Morphological parameters, photosynthetic efficiency, leaf anatomical functional traits and antioxidant production in leaves and fruits were analysed.
• Our data demonstrate that irradiation of seeds with 25 Gy Ca ions does not prevent achievement of the seed‐to‐seed cycle in ‘Microtom’, and induces a more compact plant size compared to the control. Plants germinated from irradiated seeds show better photochemical efficiency than controls, likely due to the higher amount of D1 protein and photosynthetic pigment content. Leaves of these plants also had smaller cells with a lower number of chloroplasts. The dose of 25 Gy Ca ions is also responsible for positive outcomes in fruits: although developing a lower number of berries, plants germinated from irradiated seeds produce larger berries, richer in carotenoids, ascorbic acid and anthocyanins than controls.
• These specific traits may be useful for ‘Microtom’ cultivation in BLSS in space, in so far as the crew members could benefit from fresh food richer in functional compounds that can be directly produced on board.

     

Cadmium-Resistance Plasmid Affected Cd+2 Uptake More Than Cd+2 Adsorption in Klebsiella oxytoca

A bacterial strain was isolated from Petra City Wastewater Treatment Plant. This isolate was identified as Klebsiella oxytoca based on 16S rDNA analysis. A single plasmid (> 23 kb) was detected in this strain and transformed into Esherichia coli JM83. The transformed E. coli cells exhibited elevated resistance to cadmium as compared to parental plasmid-free cells. The sodium dodecyl sulfate (SDS)-treated cells showed higher efficiency in plasmid curing than the ethidium bromide–treated cells. The ethidium bromide–cured cells grew only in a 10 μ g/ml Cd^{+ 2} minimal tolerable concentration, whereas the SDS-treated cells had no growth in any of the Cd concentrations tested (2, 5, 10, 20, 30, 40, and 50 ppm). Contrary to the Freundlich model, the Langmuir model gave a good fit to the Cd biosorption data by K. oxytoca cells. Plasmid curing caused 80%, 82%, and 70% inhibition in the Cd biosorption, adsorption, and uptake, respectively. Furthermore, the absence of lysine decarboxylase (LDC) activity in the cured strain strongly implies that the structural gene-encoding LDC in this bacterium is plasmid encoded. After curing of the plasmid, 100% of the antibiotic-resistant loci were observed as chromosomal encoded. All of the results shown above indicated that the Cd resistance is plasmid mediated.     

Is the oxidative stress theory of aging dead?

Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. While data from studies in invertebrates (e.g., C. elegans and Drosophila) and rodents show a correlation between increased lifespan and resistance to oxidative stress (and in some cases reduced oxidative damage to macromolecules), direct evidence showing that alterations in oxidative damage/stress play a role in aging are limited to a few studies with transgenic Drosophila that overexpress antioxidant enzymes. Over the past eight years, our laboratory has conducted an exhaustive study on the effect of under- or overexpressing a large number and wide variety of genes coding for antioxidant enzymes. In this review, we present the survival data from these studies together. Because only one (the deletion of the Sod1 gene) of the 18 genetic manipulations we studied had an effect on lifespan, our data calls into serious question the hypothesis that alterations in oxidative damage/stress play a role in the longevity of mice.     

Interaction of nitroaromatic radiosensitizers with irradiated polyadenylic acid as measured by an indirect immunochemical assay with specificity for the 8,5'-cycloadenosine moiety

The relative reactivity of a series of nitroaromatic radiosensitizers toward the C(5') radical intermediate leading to 8,5'-cycloadenosine formation in deoxygenated solutions of irradiated polyadenylic acid (poly A) was assessed using standard competition kinetic analysis. Formation of 8,5'-cycloadenosine was assayed by an indirect, competitive, enzyme-linked immunosorbent assay (ELISA) described in an earlier report. In the absence of oxygen, the nitroaromatics inhibit 8,5'-cyclonucleoside formation in a way which generally increases with radiosensitizer electron affinity. Although hydroxyl radical scavenging by the nitroaromatics may account for a relatively small decrease in 8,5'-cyclonucleoside formation, the data suggest that oxidation of the C(5') radical intermediate is the more plausible explanation for the decreased yield of the 8,5'-cyclonucleoside with increasing nitroaromatic electron affinity.     

3H-SCH 23390 binding sites in the rat substantia nigra: evidence for a presynaptic localization and innervation by dopamine

Chronic treatment with SCH 23390, a selective D-1 dopamine receptor antagonist, elicited a 32% increase in the density of 3H-SCH 23390 binding sites in nigral membrane preparations but failed to change the apparent KD of the ligand for its binding sites. Haloperidol, a D-2 dopamine receptor antagonist which blocks the dopamine-sensitive adenylate cyclase and (-) sulpiride, a selective D-2 dopamine receptor blocker, which does not block the dopamine-sensitive adenylate cyclase, failed to change both the Bmax and KD of 3H-SCH 23390 binding. Finally, the intrastriatal injection of kainic acid produced a marked decrease of both GAD activity and GABA content and 3H-SCH 23390 binding sites (65%) in the homolateral substantia nigra. The results show that in the rat substantia nigra most of the 3H-SCH 23390 binding sites have a presynaptic localization on the striato-nigral GABAergic afferent terminals and suggest that dopamine released from nigral dendrites exerts a tonic influence on these presynaptic D-1 dopamine receptors.     

The cytosol as site of phosphorylation of the cyclic AMP-dependent protein kinase in adrenal steroidogenesis

The mitochondria, the microsomes and the cystosol have been described as possible sites of cAMP-dependent phosphorylation. However, there has been no direct demonstration of a cAMP-dependent kinase associated with the activation of the side-chain cleavage of cholesterol. We have investigated the site of action of the cAMP-dependent kinase using a sensitive cell-free assay. Cytosol derived from cells stimulated with ACTH or cAMP was capable of increasing progesterone synthesis in isolated mitochondria when combined with the microsomal fraction. Cytosol derived from cyclase or kinase of negative mutant cells did not. Cyclic AMP and cAMP-dependent protein kinase stimulated in vitro a cytosol derived from unstimulated adrenal cells. This cytosol was capable of stimulating progesterone synthesis in isolated mitochondria. Inhibitor of cAMP-dependent protein kinase abolished the effect of the cAMP. ACTH stimulation of cytosol factors is a rapid process observable with a half maximal stimulation at about 3 pM ACTH. The effect was also abolished by inhibitor of arachidonic acid release. The function of cytosolic phosphorylation is still unclear. The effect of inhibitors of arachidonic acid release, and the necessity for the microsomal compartment in order to stimulate mitochondrial steroidogenesis, suggest that the factor in the cytosol may play a role in arachidonic acid release.     

The rhGM-CSF-EPO hybrid protein MEN 11300 induces anti-EPO antibodies and severe anaemia in rhesus monkeys

A recombinant human GM-CSF-EPO hybrid protein named MEN 11300 was administered biweekly for a total of 6 weeks to rhesus monkeys in order to evaluate its pharmacokinetic behaviour, tolerability and immunogenicity. In this primate species a strong antibody response was induced which neutralized the in vitro biological activity of human EPO while no antibody response could be detected against human GM-CSF. A severe drop in reticulocyte counts at approximately 2 weeks after initiation of treatment was followed by a dramatic decrease in the number of erythrocytes. No effects were observed on GM-CSF-dependent hematopoietic lineages and the clinical chemistry analyses did not reveal signs of general toxicity. Reticulocyte and erythrocyte counts started to recover 3-4 weeks after discontinuation of treatment in concert with a decline in anti-EPO antibody titres. Nevertheless, cell numbers remained below basal levels up to 50 days after the last MEN 11300 administration. Haematological impairment indicates that the administration to non-human primate of human EPO fused to human GM-CSF, induces neutralizing autoantibodies to the self EPO. Present data do not allow prediction of the immunogenic potential of the fusion protein in humans and a dose-escalating phase I study should be addressed to investigate the safety of the product.     

Overproduction of soluble, extracellular cytotoxin α-sarcin inEscherichia coli

The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin α-sarcin to be used for immunoconjugate production. α-Sarcin cDNA was isolated fromAspergillus giganteus strain MDH 18894 and its expression inEscherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant α-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernant. A variant form of α-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.