Uncoating of clathrin-coated vesicles in presynaptic terminals: roles for Hsc70 and auxilin.

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.     

Three-dimensional structure of baculovirus-expressed Norwalk virus capsids.

The three-dimensional structure of the baculovirus-expressed Norwalk virus capsid has been determined to a resolution of 2.2 nm using electron cryomicroscopy and computer image processing techniques. The empty capsid, 38.0 nm in diameter, exhibits T = 3 icosahedral symmetry and is composed of 90 dimers of the capsid protein. The striking features of the capsid structure are arch-like capsomeres, at the local and strict 2-fold axes, formed by dimers of the capsid protein and large hollows at the icosahedral 5- and 3-fold axes. Despite its distinctive architecture, the Norwalk virus capsid has several similarities with the structures of T = 3 single-stranded RNA (ssRNA) viruses. The structure of the protein subunit appears to be modular with three distinct domains: the distal globular domain (P2) that appears bilobed, a central stem domain (P1), and a lower shell domain (S). The distal domains of the 2-fold related subunits interact with each other to form the top of the arch. The lower domains of the adjacent subunits associate tightly to form a continuous shell between the radii of 11.0 and 15.0 nm. No significant mass density is observed below the radius of 11.0 mm. It is suspected that the hinge peptide in the adjoining region between the central domain and the shell domain may facilitate the subunits adapting to various quasi-equivalent environments. Architectural similarities between the Norwalk virus capsid and the other ssRNA viruses have suggested a possible domain organization along the primary sequence of the Norwalk virus capsid protein. It is suggested that the N-terminal 250 residues constitute the lower shell domain (S) with an eight-strand beta-barrel structure and that the C-terminal residues beyond 250 constitute the protruding (P1+P2) domains. A lack of an N-terminal basic region and the ability of the Norwalk virus capsid protein to form empty T = 3 shells suggest that the assembly pathway and the RNA packing mechanisms may be different from those proposed for tomato bushy stunt virus and southern bean mosaic virus but similar to that in tymoviruses and comoviruses.     

Differential modulation of dopaminergic systems in the rat brain by dietary protein

Rats that consume a diet 50% rich in protein exhibit hyperactivity and hyperresponsiveness to nociceptive stimuli, in which facilitation of dopaminergic activity has been implicated. We studied the regional changes in the concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of rats that were maintained on high-protein (HP, 50% casein), normal-protein (NP, 20% casein), and low-protein (LP, 8% casein) diets for 36 weeks. Brain nuclei that represented different DAergic systems were punchdissected and analyzed using HPLC. In the substantia nigra, the striatum, and the dentate gyrus, DA concentrations decreased and increased, respectively, with a decrease and increase in dietary protein (p<0.05 compared to the NP diet). Similar trends in the effect of the HP diet were observed in the ventral tegmental area, amygdala, frontal cortex, subiculum, centromedial nucleus (CM) of the thalamus, and inferior colliculi (IC), although the differences in DA concentrations were not statistically significant. These brain areas also showed a pattern of decreased DA concentration in association with the LP diet, and the differences were statistically significant (p<0.05) in the CM and IC. DA concentrations in most regions of the midbrain and brainstem were not different between the diet groups, nor were consistent trends observed in those regions. Also, there were no consistent relationships between DOPAC/DA and HVA/DA ratios and dietary protein level. These data suggest that only discrete dopaminergic neuronal circuits in the rat forebrain were sensitive to changes in dietary protein level.     

GABA Requires Nitric Oxide for Alleviating Arsenate Stress in Tomato and Brinjal Seedlings

In recent years, gamma-aminobutyric acid (GABA) and nitric oxide (NO) have drawn much attention in reducing/alleviating metal toxicity in plants. Arsenic is a potentially toxic metal that imposes severe toxic effects on crop plants. Therefore, the present study was an attempt to explore the role of GABA and NO in the amelioration of arsenate As(V) toxicity in tomato and brinjal seedlings. Arsenate toxicity reduced fresh mass about by 17% in tomato and 25% in brinjal seedlings, increased the level of reactive oxygen species, antioxidant enzymes (except glutathione-S-transferase), and altered photosynthesis. However, exogenously applied GABA and sodium nitroprusside (SNP, a NO donor) improved the growth of both vegetables and reduced As(V) toxicity. Interestingly, As(V) toxicity was further increased on applying biosynthetic inhibitor of NO even in the presence of GABA, and under similar conditions addition of NO donor rescued the negative effect of biosynthetic inhibitor (L-NAME) of NO. This indicates that NO is crucial in GABA-mediated alleviation of As(V) toxicity in tomato and brinjal seedlings. Besides this, results revealed that c-PTIO (a NO scavenger) addition reversed the alleviatory effect of SNP also suggesting that NO is important in curtailing As(V) toxicity. Overall, the results indicated that NO is necessary for mitigation of As(V) stress by GABA in two studied vegetable crops but NO itself does not require GABA for the same.

     

Marker-trait association analysis for gall midge (Orseolia oryzae) resistance in a diverse rice population

Damage caused by insect herbivores, notably Asian rice gall midge, Orseolia oryzae is more prevalent in the rice-growing belts of India's southern and north-eastern states. As a prelude to resistant cultivar development, the identification of genomic regions for resistance in the source population is crucial. In the present investigation, 202 rice genotypes were phenotyped and assayed with genomic markers reported for gall midge resistance. Positive skewness and platykurtic distribution of response scores suggested the inheritance of gall midge resistance in the study population. The marker gm3del3 contributed the most genetic variation, followed by RM28574 and marker RM22709 explained minimal variation. A marker-trait association analysis with a single marker-trait linear regression approach was performed to discover gall midge resistant genomic region/genes. The marker RM17480 on chromosome 4 reported to be linked with gm3 gene was found significantly associated with the gall midge resistance genomic region with allelic effects in a negative direction favouring resistance reaction. The allelic effects of significantly associated markers were correlated significantly with the phenotypic variation of gall midge damage scores. Genes identified in the vicinity of this marker contribute to stress response reactions in rice plants. The 200 bp allele of the marker was associated with susceptibility, while the 250 bp allele was associated with resistance expression. This allelic association with trait variation suggests the importance of associated marker for utilisation in marker-assisted selection programmes to incorporate resistance alleles into elite rice genotypes.     

Synthesis,characterization and absolute configurations of methyl ladderanoates

Methyl esters of [5]-ladderanoic acid and [3]-ladderanoic acid were prepared by esterification of the acids isolated from biomass at a wastewater treatment plant. Optical rotations at six different wavelengths (633, 589, 546, 436, 405 and 365 nm) and vibrational circular dichroism (VCD) spectra in the 1800–900 cm⁻¹ region were measured in CDCl₃ solvent and compared with quantum chemical (QC) predictions using B3LYP functional and 6-311++G(2d,2p) basis set with polarizing continuum model representing the solvent. QC predictions gave negative optical rotations at all six wavelengths for (R)-methyl [5]-ladderanoate and positive optical rotations for (R)-methyl [3]-ladderanoate, the same signs as previously reported for the corresponding acids. The crystal structure of (−)-methyl [5]-ladderanoate independently confirmed (R) configuration. The QC-predicted VCD spectra using Boltzmann population weighted spectra of individual conformers did not provide satisfactory quantitative agreement with the experimental VCD spectra. An improved quantitative agreement for VCD spectra could be obtained when conformer populations were optimized to maximize the similarity between experimental and predicted VCD spectra, but more improvements in VCD predictions are needed.     

Influence of salinity on axillary bud cultures of six lowland tropical varieties of potato (Solanum tuberosum)

Axillary bud cultures of six low-land tropical varieties of potato Solanum tuberosum (L. T. 2, 5, 6, 7, 8 and 9) were studied for their response to salinity stress using crude sea salt. Concentrations of 0.8% and above severely inhibited the growth, while decreased growth was observed between 0.4% and 0.6% salt in all the varieties, with occasional shoot tip necrosis and compound leaf formation. A lower concentration of salt (0.2%) was beneficial and increased shoot weight in most varieties. Protein content showed a positive correlation with proline levels, indicating that proline accumulation was due to new synthesis rather than breakdown of existing proteins. Soluble carbohydrates increased in the stem with increasing salt. Roots showed an increase in sodium content while shoots showed increased potassium levels, with increasing salt concentrations. There was a positive correlation between dry weight yields and K/Na ratio. It is apparent that the synthesis of proline, accumulation of soluble carbohydrates as well as increase in potassium and sodium content were all used to adjust the physiology in response to salt stress by the different varieties of potato, with varying degrees, in the present study.     

The diagenetic fate of collagen as revealed by analytical pyrolysis of fossil fish scales from deep time

The mechanism of protein degradation has remained a topic of debate (specifically concerning their preservation in deep time), which has recently been invigorated due to multiple published reports of preservation ranging from Miocene to the Triassic that potentially challenge the convention that protein preservation beyond the Cenozoic is extremely uncommon or is expected to be absent altogether, and thus have attracted skepticism. In this paper, we analyze fossil fish scales from the Cretaceous, Jurassic, and Triassic using comprehensive pyrolysis gas chromatography coupled with time-of-flight mass spectrometry and compare the pyrolytic products so obtained with a well-preserved fish scale from Late Pliocene, in an attempt to better understand the effects of diagenesis on protein degradation at the molecular level through deep time. We find that the Pliocene fish scale displays a large number of N-bearing pyrolytic products, including abundant substituted cyclic 2,5-diketopiperazines (2,5-DKPs) which are diagnostic products of peptide and amino acid pyrolysis. We identify N-bearing compounds in the Mesozoic fish scales—however, among the 2,5-DKPs that were identified in the Pliocene scale, only diketodipyrrole (or cyclo (Pyr-Pyr)) is present in the Mesozoic scales. We discuss the implications of N-bearing pyrolytic products with emphasis on 2,5-DKPs in geological samples and conclude that the discrepancy in abundance and variety of N-bearing products between Pliocene and Mesozoic scales indicates that the protein component in the latter has been extensively diagenetically altered, while a suite of DKPs such as in the former would imply stronger evidence to indicate preservation of protein. We conclude that analytical pyrolysis is an effective tool for detecting preservation of intact proteins, as well as for providing insights into their degradation mechanisms, and can potentially be utilized to assign proteinaceous origin to a fossil sample of unknown affinity.