Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Endotoxin-induced alterations in hepatic glucose-6-phosphatase activity and gene expression

The mechanisms responsible for the glycemic changes associated with endotoxic shock are not fully understood, but are known to involve the ability of the liver to produce glucose. The purpose of the present study was to determine whether endotoxin (LPS) influences the expression and activity of glucose-6-phosphatase (Glu-6-Pase) during the early hyperglycemic phase and the later hypoglycemic phase. Rats were injected with a relatively large dose of LPS (20 mg/kg) or saline (control), and sacrificed at 1 or 5 h post-injection. Both the plasma glucose concentration and glucose production were elevated 1 h post-LPS (2-fold) and both decreased at 5 h postinjection (50%). Compared to time-matched control values, hepatic glucose-6-phosphate and fructose-6-phosphate levels were significantly decreased at both 1 and 5 h. Hepatic Glu-6-Pase activity and mRNA levels were moderately increased, 1 h after injection of LPS. At 5 h, an 88% decrease in mRNA abundance for Glu-6-Pase was associated with a 30% decrease in activity of this enzyme. Plasma insulin concentrations were not different 1 h after LPS and were elevated 2-fold from control values at 5 h. Circulating levels of glucagon and corticosterone were elevated at both time points following LPS. Our data indicate that the LPS-induced hypoglycemia and reduction in hepatic glucose production were accompanied by a depression in Glu-6-Pase activity and gene expression.     

Melatonin rhythms in the pineal and non-pineal tissues and their physiological implications in subtropical fish

Abstract

Melatonin (N-acetyl-5-methoxy tryptamine), following discovery from the extracts of bovine pineal gland, has been detected in the pineal as well as several extra-pineal tissues/organs of different vertebrates including fish. The unique feature of melatonin in the pineal gland is its rhythmic biosynthesis and release in blood in synchronization with the environmental light-–dark cycle. Accordingly, melatonin produced in the pineal of an animal living in a changing environment is implicated to the regulation of seasonal reproduction by acting as a hormone at one or more levels of hypothalamo-hypophyseal-gonadal axis. Additionally, melatonin is known to act as a potent free-radical scavenger or antioxidant to influence maturation of oocytes. However, possible relationship between extra-pineal melatonin and seasonality of reproduction in any animal remains enigmatic. Perhaps, carp is the only known animal in which temporal patterns of melatonin levels in the serum as well as in the extracts of pineal, retina, ovary, gut, and liver have been studied in relation to the reproductive events in an annual cycle. The purpose of current review is to bring those fascinating, and arguably most important data together to underline their significance in the control of seasonal reproduction in subtropical fish in general and in carp in particular.     

Differential membrane proteomics using 18O-labeling to identify biomarkers for cholangiocarcinoma

Quantitative proteomic methodologies allow profiling of hundreds to thousands of proteins in a high-throughput fashion. This approach is increasingly applied to cancer biomarker discovery to identify proteins that are differentially regulated in cancers. Fractionation of protein samples based on enrichment of cellular subproteomes prior to mass spectrometric analysis can provide increased coverage of certain classes of molecules. We used a membrane protein enrichment strategy coupled with 18O labeling based quantitative proteomics to identify proteins that are highly expressed in cholangiocarcinomas. In addition to identifying several proteins previously known to be overexpressed in cholangiocarcinoma, we discovered a number of molecules that were previously not associated with cholangiocarcinoma. Using immunoblotting and immunohistochemical labeling of tissue microarrays, we validated Golgi membrane protein 1, Annexin IV and Epidermal growth factor receptor pathway substrate 8 (EPS8) as candidate biomarkers for cholangiocarcinomas. Golgi membrane protein 1 was observed to be overexpressed in 89% of cholangiocarcinoma cases analyzed by staining tissue microarrays. In light of recent reports showing that Golgi membrane protein 1 is detectable in serum, further investigation into validation of this protein has the potential to provide a biomarker for early detection of cholangiocarcinomas.     

Library-based numerical reduction of the Hodgkin–Huxley neuron for network simulation

We present an efficient library-based numerical method for simulating the Hodgkin–Huxley (HH) neuronal networks. The key components in our numerical method involve (i) a pre-computed high resolution data library which contains typical neuronal trajectories (i.e., the time-courses of membrane potential and gating variables) during the interval of an action potential (spike), thus allowing us to avoid resolving the spikes in detail and to use large numerical time steps for evolving the HH neuron equations; (ii) an algorithm of spike-spike corrections within the groups of strongly coupled neurons to account for spike-spike interactions in a single large time step. By using the library method, we can evolve the HH networks using time steps one order of magnitude larger than the typical time steps used for resolving the trajectories without the library, while achieving comparable resolution in statistical quantifications of the network activity, such as average firing rate, interspike interval distribution, power spectra of voltage traces. Moreover, our large time steps using the library method can break the stability requirement of standard methods (such as Runge–Kutta (RK) methods) for the original dynamics. We compare our library-based method with RK methods, and find that our method can capture very well phase-locked, synchronous, and chaotic dynamics of HH neuronal networks. It is important to point out that, in essence, our library-based HH neuron solver can be viewed as a numerical reduction of the HH neuron to an integrate-and-fire (I&F) neuronal representation that does not sacrifice the gating dynamics (as normally done in the analytical reduction to an I&F neuron).     

Mycobacterium avium subspecies paratuberculosis diagnosis and geno-typing: genomic insights

Effective control of paratuberculosis and investigations of potential link to Crohn's disease have been hampered by the lack of effective assays for easy and accurate diagnosis of Mycobacterium avium subspecies paratuberculosis (Map). Map is extremely fastidious and depends on iron chelator (Mycobactin). Map strains from humans and sheep are very difficult to isolate and may require years to emerge. Therefore, small numbers of Map isolates have been maintained in available collections. This situation has limited the study of biodiversity of Map. Though, much is known about environmental and host factors that contribute to paratuberculosis disease, but little is known about bacterial genetic mechanism of infection. Diagnostic and strain typing markers still demand improvements. Complete genome sequence of Map K10 strain is available in public domain for comparative genomics with other mycobacteria and clinical isolates of Map. It is anticipated that the genome sequence will help in carrying molecular diagnosis and strain typing with respect to Map forward at rapid pace. This paper reviews the current diagnostic and strain typing markers, which may be useful in typing of clinical isolates in near future.