Cloning of the <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene and its heterologous expression in <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

In this study, the gene hmgR encoding the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) was cloned and characterized in the zygomycete fungus Rhizomucor miehei. The hmgR gene comprises a total of 3,585 bp including the coding sequence of a 1,058 amino acids length putative protein and five introns (137, 83, 59, 60 and 69 bp in length) dispersed in the whole coding region. Southern hybridization analysis revealed that the gene is present only in one copy in the R. miehei genome. The isolated Rhizomucor gene was expressed in the related fungus, Mucor circinelloides. Transformants harbouring the Rhizomucor hmgR gene in an autoreplicative plasmid proved to be more tolerant to statins (e.g. lovastatin, simvastatin, and fluvastatin), the competitive inhibitors of the HMG-CoA reductase, than the original M. circinelloides strain. At the same time, heterologous expression of the Rhizomucor hmgR did not affect the carotenoid production of M. circinelloides.     

An integrated view of protein evolution

Why do proteins evolve at different rates? Advances in systems biology and genomics have facilitated a move from studying individual proteins to characterizing global cellular factors. Systematic surveys indicate that protein evolution is not determined exclusively by selection on protein structure and function, but is also affected by the genomic position of the encoding genes, their expression patterns, their position in biological networks and possibly their robustness to mistranslation. Recent work has allowed insights into the relative importance of these factors. We discuss the status of a much-needed coherent view that integrates studies on protein evolution with biochemistry and functional and structural genomics.     

Improving the specificity of high-throughput ortholog prediction

Background  

Orthologs (genes that have diverged after a speciation event) tend to have similar function, and so their prediction has become an important component of comparative genomics and genome annotation. The gold standard phylogenetic analysis approach of comparing available organismal phylogeny to gene phylogeny is not easily automated for genome-wide analysis; therefore, ortholog prediction for large genome-scale datasets is typically performed using a reciprocal-best-BLAST-hits (RBH) approach. One problem with RBH is that it will incorrectly predict a paralog as an ortholog when incomplete genome sequences or gene loss is involved. In addition, there is an increasing interest in identifying orthologs most likely to have retained similar function.     

Activation of the poly(ADP-ribose) polymerase pathway in human heart failure

Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of acute and chronic myocardial dysfunction and heart failure. The goal of the present study was to investigate PARP activation in human heart failure, and to correlate PARP activation with various indices of apoptosis and oxidative and nitrosative stress in healthy (donor) and failing (NYHA class III-IV) human heart tissue samples. Higher levels of oxidized protein end-products were found in failing hearts compared with donor heart samples. On the other hand, no differences in tyrosine nitration (a marker of peroxynitrite generation) were detected. Activation of PARP was demonstrated in the failing hearts by an increased abundance of poly-ADP ribosylated proteins. Immunohistochemical analysis revealed that PARP activation was localized to the nucleus of the cardiomyocytes from the failing hearts. The expression of full-length PARP-1 was not significantly different in donor and failing hearts. The expression of caspase-9, in contrast, was significantly higher in the failing than in the donor hearts. Immunohistochemical analysis was used to detect the activation of mitochondrial apoptotic pathways. We found no significant translocation of apoptosis-inducing factor (AIF) into the nucleus. Overall, the current data provide evidence of oxidative stress and PARP activation in human heart failure. Interventional studies with antioxidants or PARP inhibitors are required to define the specific roles of these factors in the pathogenesis of human heart failure.     

Immunotoxicological investigation of subacute combined exposure with low doses of Pb, Hg and Cd in rats

Detectable interactions between NOEL (No Observed Effect Level) doses of Pb, Hg and Cd in general toxicological, hematological, and immune function parameters were investigated. The metals (Pb-acetate, 20 mg/kg; HgCl2, 0.40 mg/kg; CdCl2, 1.61 mg/kg) were combined. First, the rats received the combination Pb + Hg + Cd for 4 weeks per os. Significant difference vs. control was found only in the weight of lung and popliteal lymph node (PLN). The Pb + Hg and Pb + Cd combinations significantly decreased the PLN to 100 g body weight and PLN to brain weight ratio, and Pb+Hg also decreased the relative adrenal weight. After 12 weeks treatment with the same doses, effects on the thymus, kidney, and adrenal weights in the Pb + Hg, and thymus weight in the Pb + Cd, combination were seen. Pb + Cd also affected the white and red blood cell count and hematocrit. Combined with Hg or Cd, NOEL dose Pb showed toxicity, indicating that exposure limits may be inefficient in combined exposure situations.     

Correlation between first polar body morphology and further embryo development

First polar body (PB) morphology of human oocytes can indicate further embryo development and viability. However, controversial data have been published in this topic. Our retrospective study analyses the fertilization and further development of oocytes in relation to different morphological features of the first PB. The morphology of 3387 MII oocytes from 522 in vitro fertilization (IVF) treatments were assessed before intracytoplasmic sperm injection (ICSI). Oocytes were classified according to their first PB morphology. Assessment of fertilization and embryonic development (cell number, embryo grade, amount of anuclear fragmentation and presence of multinucleated blastomeres) was performed 16-20 and 42-48 hours after ICSI. Our results show that fertilization rate and embryo quality is influenced by PB morphology, while speed of development is not affected by the morphology of the first PB. Contrary to previous findings, our results suggest that oocytes with a fragmented PB had a higher developmental ability than those with an intact PB. However, we observed a lower viability of oocytes with a large PB. Since there are contradictions in this and previous observations, an extensive study is needed with standard hormonal stimulation protocol and oocyte evaluation criteria.     

Autophagy is required for zebrafish caudal fin regeneration

Regeneration is the ability of multicellular organisms to replace damaged tissues and regrow lost body parts. This process relies on cell fate transformation that involves changes in gene expression as well as in the composition of the cytoplasmic compartment, and exhibits a characteristic age-related decline. Here, we present evidence that genetic and pharmacological inhibition of autophagy – a lysosome-mediated self-degradation process of eukaryotic cells, which has been implicated in extensive cellular remodelling and aging – impairs the regeneration of amputated caudal fins in the zebrafish (Danio rerio). Thus, autophagy is required for injury-induced tissue renewal. We further show that upregulation of autophagy in the regeneration zone occurs downstream of mitogen-activated protein kinase/extracellular signal-regulated kinase signalling to protect cells from undergoing apoptosis and enable cytosolic restructuring underlying terminal cell fate determination. This novel cellular function of the autophagic process in regeneration implies that the role of cellular self-digestion in differentiation and tissue patterning is more fundamental than previously thought.     

Electrodichroism of Purple Membrane: Ionic Strength Dependence

The dichroism of purple membrane suspension was measured in dc and ac electric fields. From these measurements three parameters can be obtained: the permanent dipole moment, μ, the electrical polarizability, α, and the retinal angle, δ, (relative to the membrane normal). The functional dependence of the dichroism on the electric field is analyzed. There is a small decrease (~2°) in retinal angle going from dark adapted to the light adapted form. No measurable difference in μ, α, and δ was found under the photocycle. The dichroism was measured in two different salt solutions (KCl and CaCl₂) in the range 0-10 mM. The retinal angle increases from 64° to 68° with increasing ionic strength going through a minimum. This is attributed to the changing (decreasing) inner electric field in the membrane. The polarizability, α, consists of two parts. One component is related to the polarization of the purple membrane and the second component to the ionic cloud. The second component decreases with ion concentration approximately as κ^-3 (κ is the Debye parameter) in agreement with a model calculation for the polarization of the ionic cloud. The origin of the slightly ionic strength dependent permanent dipole moment is not well understood.