Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin

Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37 °C. LCE, on the other hand, was although optimally active at 37 °C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics.     

Comparison of Liver Fat Indices for the Diagnosis of Hepatic Steatosis and Insulin Resistance

Context

Hepatic steatosis, defined as increased hepatocellular lipid content (HCL), associates with visceral obesity and glucose intolerance. As exact HCL quantification by ¹H-magnetic resonance spectroscopy (¹H-MRS) is not generally available, various clinical indices are increasingly used to predict steatosis.

Objective

The purpose of this study was to test the accuracy of NAFLD liver fat score (NAFLD-LFS), hepatic steatosis index (HSI) and fatty liver index (FLI) against ¹H-MRS and their relationships with insulin sensitivity and secretion.

Design, Setting and Participants

Ninety-two non-diabetic, predominantly non-obese humans underwent clinical examination, ¹H-MRS and an oral glucose tolerance test (OGTT) to calculate insulin sensitivity and β-cell function. Accuracy of indices was assessed from the area under the receiver operating characteristic curve (AROC).

Results

Median HCL was 2.49% (0.62;4.23) and correlated with parameters of glycemia across all subjects. NAFLD-LFS, FLI and HSI yielded AROCs of 0.70, 0.72, and 0.79, respectively, and related positively to HCL, insulin resistance, fasting and post-load β-cell function normalized for insulin resistance. Upon adjustment for age, sex and HCL, regression analysis revealed that NAFLD-LFS, FLI and HSI still independently associated with both insulin sensitivity and β-cell function.

Conclusion

The tested indices offer modest efficacy to detect steatosis and cannot substitute for fat quantification by ¹H-MRS. However, all indices might serve as surrogate parameters for liver fat content and also as rough clinical estimates of abnormal insulin sensitivity and secretion. Further validation in larger collectives such as epidemiological studies is needed.     

Genetic systems of six species ofPlantago (Plantaginaceae)

Investigation of the genetic system of six species ofPlantago has revealed striking differences in their breeding and meiotic systems.Plantago patagonica is an inbreeder on account of preanthesis cleistogamy, whereasP. lanceolata is an obligate outbreeder, as it is self-incompatible.Plantago drummondii, P. lagopus, P. ovata, andP. major show mixed mating but in varying proportions. In terms of their energy budgets, outbreeding species invest more in floral advertisement and male function, while inbreeders invest more in female function. The contribution of the meiotic system to genetic variability, as revealed through recombination index, is more important in the inbreeding species.     

UV and chemical mutagenesis in rev7 mutants of yeast

Summary We have examined induced mutagenesis in rev7-1 mutants of Baker's yeast' Saccharomyces cerevisiae, using a variety of contrasting test systems and several different mutagens. UV-induced reversion frequencies of the ochre allele arg4-17, the putative missense allele ilv1-92 and the frameshift allele his4-38 were 10 to 200 fold lower in haploid and diploid rev7-1 mutants compared with wild type strains, but UV-induced reversion frequencies of the frameshift allele leu2-3 and the proline missense allele cyc1-115 were reduced only a few fold. Ilv1-92 reversion frequencies induced by methyl methane sulfonate or by N-methyl-N-nitro-N-nitrosoguanidine were 10 to 20 times lower in rev7-1 mutants, but normal frequencies of these revertants were induced with ethyl methane sulfonate, even though rev7-1 strains are slightly sensitive to this mutagen as well as to the others tested. We conclude that the rev7 mutants, like the rev3 mutants they closely resemble, have a substantial but not total deficiency concerning induced mutagenesis.     

Akumal's reefs: stony coral communities along the developing Mexican Caribbean coastline

Fringing coral reefs along coastlines experiencing rapid development and human population growth have declined worldwide because of human activity and of natural causes. The "Mayan Riviera" in Quintana Roo, México, attracts large numbers of tourists in part because it still retains some of the natural diversity and it is important to obtain baseline information to monitor changes over time in the area. In this paper, the condition of the stony corals in the developing coastline of the Akumal-area fore reefs is characterized at the start of the new millennium at two depths, and along an inferred sedimentation gradient. Transect surveys were conducted in five fringing reefs starting at haphazardly chosen points, with respect to species composition, live cover, colony density, relative exposure to TAS mats and, for one species (Diploria strigosa, Dana, 1848), tissue regression rates in the presence of TAS mats. Fish population density and herbivory rates are also assessed. Data from line intercept transects (n=74) show that live stony coral cover, density and relative peripheral exposure of colonies to turf algal/sediment (TAS) mats were inversely related to an inferred sediment stress gradient at 13m. In 2000, live stony coral cover had decreased by 40-50% at two sites studied in 1990 by Mu?oz-Chagin and de la Cruz-Agüero (1993). About half of this loss apparently occurred between 1998 and 2000 during an outbreak of white plague disease that mostly affected Montastraea faveolata, and M. annularis. At a 13 m site, where inferred sedimentation rates are relatively high, time series photography of tagged Diploria strigosa, (n=38) showed an average loss of 70 cm2 of live tissue/coral/year to encroachment by TAS mats during the same period. Whereas densities of carnivorous fishes and herbivores (echinoids, scarids, acanthurids and Microspathodon chrysurus) in 2000 were low in belt transects at 10-19 m (n=106), turf-algal gardening pomacentrids were relatively common on these reefs.     

Modeling of Noisy Spindle Dynamics Reveals Separable Contributions to Achieving Correct Orientation

The mechanisms by which the mammalian mitotic spindle is guided to a predefined orientation through microtubule-cortex interactions have recently received considerable interest, but there has been no dynamic model that describes spindle movements toward the preferred axis in human cells. Here, we develop a dynamic model based on stochastic activity of cues anisotropically positioned around the cortex of the mitotic cell and we show that the mitotic spindle does not reach equilibrium before chromosome segregation. Our model successfully captures the characteristic experimental behavior of noisy spindle rotation dynamics in human epithelial cells, including a weak underlying bias in the direction of rotation, suppression of motion close to the alignment axis, and the effect of the aspect ratio of the interphase cell shape in defining the final alignment axis. We predict that the force exerted per cue has a value that minimizes the deviation of the spindle from the predefined axis. The model has allowed us to systematically explore the parameter space around experimentally relevant configurations, and predict the mechanistic function of a number of established regulators of spindle orientation, highlighting how physical modeling of a noisy system can lead to functional biological understanding. We provide key insights into measurable parameters in live cells that can help distinguish between mechanisms of microtubule and cortical-cue interactions that jointly control the final orientation of the spindle.     

Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes

After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.