Use of Vertical Slab Isoelectric Focusing and Immunoblotting to Evaluate Steady-State Phosphorylation of eIF2α in Cultured Cells

The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the α-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2α phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts is rarely as high as that found in extracts produced from reticulocytes, and in part this reflects the fact that some component(s) of cell extracts interferes with the entry of eIF2α into the isoelectric focusing gel. To overcome these difficulties, we have modified the earlier method to include immunoprecipitation of eIF2 from cell extracts prior to isoelectric focusing, as well as a low sodium dodecyl sulfate concentration in the isoelectric focusing sample buffer. Since the PKR activation state and therefore the eIF2α phosphorylation state change with cell density and nutritional status, we routinely set up consistent feeding schedules and recommend the collection of data over a range of cell densities.     

Characterization and Functional Properties of Lactobacilli Isolated from Kefir Grains

Applied Biochemistry and Microbiology - As a natural, symbiotic consortium of bacteria and yeasts, kefir grains provide a unique source of new probiotic microorganisms. In this work, three new...     

European checkerspots (Melitaeini: Lepidoptera,Nymphalidae) are not aposematic – the point of view of great tits (Parus major)

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The mouse anterior chamber angle and trabecular meshwork develop without cell death

Background

The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice.

Results

The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation.

Conclusions

These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.     

Diagnostic and epidemiological significance of F-donor activity of Enterobacteria strains

The study of the circulating strains of enteropathogenic Escherichia coli (EPEC), shigellae and salmonellae for the presence of F-factor (the conjunction factor) was carried out. The study revealed that 85% of Shigella flexneri strains 2a had conjugative (F)-factor. The proportion of such strains was 63% among EPEC of different serovars and 15% among Salmonella enteritidis. As 63% EPEC strains had hemolytic activity and were found to carry F-conjugative plasmid it should be taken into consideration in the identification of microorganisms. The presence of conjugation plasmids is an important epidemiological marker.     

Synthesis and Antiviral Activity of Hydrazides and Substituted Benzalhydrazides of Betulinic Acid and Its Derivatives

New nitrogen-containing derivatives of betulinic and betulonic acids, hydrazides and N"-benzalhydrazides, were synthesized. Their antiviral activities toward viruses of influenza A virus, herpes simplex type I virus, enterovirus ECHO6, and HIV-1 were studied in vitro. Betulinic acid 3-oxime was found to have the highest activity against the influenza virus. Betulonic acid, betulinic acid 4-chlorobenzalhydrazide, betulonic acid 3-oxime benzalhydrazide, and betulinic acid hydrazide inhibited the replication of herpes simplex type I virus. Betulinic acid hydrazide also showed antiviral activity toward HIV-1. All the derivatives of betulinic acid under study displayed a low antiviral activity toward enterovirus ECHO6.     

Preparation of androsta-1,4-diene-3,17-dione from sterols using <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis> VKPM Ac-1656 strain

A product of microbiological cleavage of the sterols side chain, androsta-1,4-diene-3,17-dione, is toxic for bacteria, in particular, actinobacteria of the genera Mycobacterium and Arthrobacter. Sterols were transformed into androsta-1,4-diene-3,17-dione by culturing the M. neoaurum VKPM Ac-1656 strain in a high yield, provided that a sorbent was used for elimination of contact between the bacterial cells and the product. Unlike the cholesterol side chain, the more branched chains of phytosterols were cleaved in the presence of M. neoaurum at a high rate only under turbulent stirring of the culture medium, which intensified the formation of hydrocarbonate ion from NaNI₃ in situ.     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner

The NF-kappaB p50 is the N-terminal processed product of the precursor, p105. It has been suggested that p50 is generated not from full-length p105 but cotranslationally from incompletely synthesized molecules by the proteasome. We show that the 20S proteasome endoproteolytically cleaves the fully synthesized p105 and selectively degrades the C-terminus of p105, leading to p50 generation in a ubiquitin-independent manner. As small as 25 residues C-terminus to the site of processing are sufficient to promote processing in vivo. However, any p105 mutant that lacks complete ankyrin repeat domain (ARD) is processed aberrantly, suggesting that native processing must occur from a precursor, which extends beyond the ARD. Remarkably, the mutant p105 that lacks the internal region including the glycine-rich region (GRR) is completely degraded by 20S proteasome in vitro. This suggests that the GRR impedes the complete degradation of the p105 precursor, thus contributing to p50 generation.