SAIVGeM: spreadsheet analysis of immunoglobulin VH gene mutations

The analysis of mutations in immunoglobulin heavy chain variable (IGHV) region genes is a tedious process when performed by hand on multiple sequences. This report describes a set of linked Microsoft Excel files that perform several common analyses on large numbers of IGHV sequences. The spreadsheet analysis of immunoglobulin VH gene mutations (SAIVGeM) package determines the distribution of mutations among each nucleotide, the nature of the mutation at both the nucleotide and amino acid level, the frequency of mutation in the A/G G C/T A/T (RGYW) hotspot motifs of both strand polarity, and the distribution of replacement and silent mutations among the complementarity determining regions (CDRs) and the framework regions (FRs) of the immunoglobulin gene as defined by either the Kabat or IMGT conventions. These parameters are summarized and graphically presented where appropriate. In addition, the SAIVGeM package analyzes those mutations that occur in third positions of redundant codons. Because any nucleotide change in these positions is inherently silent, these positions can be used to study the mutational spectra without biases from the selection of protein structure.     

High mobility group box protein 1: an endogenous signal for dendritic cell maturation and Th1 polarization

High mobility group box protein 1 (HMGB1), a DNA binding nuclear and cytosolic protein, is a proinflammatory cytokine released by monocytes and macrophages. This study addressed the hypothesis that HMGB1 is an immunostimulatory signal that induces dendritic cell (DC) maturation. We show that HMGB1, via its B box domain, induced phenotypic maturation of DCs, as evidenced by increased CD83, CD54, CD80, CD40, CD58, and MHC class II expression and decreased CD206 expression. The B box caused increased secretion of the proinflammatory cytokines IL-12, IL-6, IL-1alpha, IL-8, TNF-alpha, and RANTES. B box up-regulated CD83 expression as well as IL-6 secretion via a p38 MAPK-dependent pathway. In the MLR, B box-activated DCs acted as potent stimulators of allogeneic T cells, and the magnitude of the response was equivalent to DCs activated by exposure to LPS, nonmethylated CpG oligonucleotides, or CD40L. Furthermore, B box induced secretion of IL-12 from DCs as well as IL-2 and IFN-gamma secretion from allogeneic T cells, suggesting a Th1 bias. HMGB1 released by necrotic cells may be a signal of tissue or cellular injury that, when sensed by DCs, induces and/or enhances an immune reaction.     

G209A mutant alpha synuclein expression specifically enhances dopamine induced oxidative damage

Alpha synuclein protein may play an important role in familial and sporadic Parkinson's disease pathology. We have induced G209A mutant or wild-type alpha-synuclein expression in stable HEK293 cell models to determine if this influences markers of oxidative stress and damage under normal conditions or in the presence of dopamine or paraquat. Induced wild-type or mutant alpha-synuclein expression alone had no effect upon levels of oxidative stress or damage, as measured by glutathione levels or aconitase activity. Both wild-type and mutant alpha-synuclein expression decreased the oxidative damage induced by paraquat, although the protection was less marked with mutant alpha-synuclein expression. This suggests that alpha-synuclein expression may either have anti-oxidant properties or may upregulate cellular antioxidant levels, a function that was diminished by the G209A mutation. However, mutant but not wild-type alpha-synuclein expression specifically enhanced dopamine associated oxidative damage. Non-expressing cells treated with reserpine to inhibit the vesicular monoamine compartmentalisation produced similar results. However, consistent with the hypothesis that mutant alpha-synuclein disrupts vesicular dopamine compartmentalization, this effect was diminished in cells expressing mutant alpha-synuclein. This may result in increased dopamine metabolism and cause selective oxidative damage to dopaminergic cells.     

CONCORDANCE BETWEEN GENETIC AND SPECIES DIVERSITY IN CORAL REEF FISHES ACROSS THE PACIFIC OCEAN BIODIVERSITY GRADIENT

The relationship between genetic diversity and species diversity provides insights into biogeography and historic patterns of evolution and is critical for developing contemporary strategies for biodiversity conservation. Although concordant large‐scale clines in genetic and species diversity have been described for terrestrial organisms, whether these parameters co‐vary in marine species remains largely unknown. We examined patterns of genetic diversity for 11 coral reef fish species sampled at three locations across the Pacific Ocean species diversity gradient (Australia: ～1600 species; New Caledonia: ～1400 species; French Polynesia: ～800 species). Combined genetic diversity for all 11 species paralleled the decline in species diversity from West to East, with French Polynesia exhibiting lowest total haplotype and nucleotide diversities. Haplotype diversity consistently declined toward French Polynesia in all and nucleotide diversity in the majority of species. The French Polynesian population of most species also exhibited significant genetic differentiation from populations in the West Pacific. A number of factors may have contributed to the general positive correlation between genetic and species diversity, including location and time of species origin, vicariance events, reduced gene flow with increasing isolation, and decreasing habitat area from West to East. However, isolation and habitat area, resulting in reduced population size, are likely to be the most influential.     

The effects of biotin and fatty acids on SV3T3 cell growth in the presence of normal calf serum

The growth of SV3T3 cells in medium containing a low concentration (0.20% v/v) of normal calf serum is enhanced by the addition of biotin or certain unsaturated fatty acids. The biotin effect on the final viable cell density is 5- to 10-fold over the control and is extremely potent, exerting a saturating response at a a concentration of approximately 200 pg/ml. The optimal growth response observed with fatty acids in 5-fold over the control and requires the combination of nervonic acid, palmitoleic acid, and arachidonic acid. The fatty acids are probably not replacing the function of biotin since these two substances are additive in their growth effects.     

The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414–530) and CC2 (aa 530–630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.     

Increasing ocean temperatures reduce activity patterns of a large commercially important coral reef fish

Large‐bodied fish are critical for sustaining coral reef fisheries, but little is known about the vulnerability of these fish to global warming. This study examined the effects of elevated temperatures on the movement and activity patterns of the common coral trout Plectropomus leopardus (Serranidae), which is an important fishery species in tropical Australia and throughout the Indo West‐Pacific. Adult fish were collected from two locations on Australia's Great Barrier Reef (23°S and 14°S) and maintained at one of four temperatures (24, 27, 30, 33 °C). Following >4 weeks acclimation, the spontaneous swimming speeds and activity patterns of individuals were recorded over a period of 12 days. At 24–27 °C, spontaneous swimming speeds of common coral trout were 0.43–0.45 body lengths per second (bls^?1), but dropped sharply to 0.29 bls^?1 at 30 °C and 0.25 bls^?1 at 33 °C. Concurrently, individuals spent 9.3–10.6% of their time resting motionless on the bottom at 24–27 °C, but this behaviour increased to 14.0% at 30 °C and 20.0% of the time at 33 °C (mean ± SE). The impact of temperature was greatest for smaller individuals (<45 cm TL), showing significant changes to swimming speeds across every temperature tested, while medium (45–55 cm TL) and large individuals (>55 cm TL) were first affected by 30 °C and 33 °C, respectively. Importantly, there was some indication that populations can adapt to elevated temperature if presented with adequate time, as the high‐latitude population decreased significantly in swimming speeds at both 30 °C and 33 °C, while the low‐latitude population only showed significant reductions at 33 °C. Given that movement and activity patterns of large mobile species are directly related to prey encounter rates, ability to capture prey and avoid predators, any reductions in activity patterns are likely to reduce overall foraging and energy intake, limit the energy available for growth and reproduction, and affect the fitness and survival of individuals and populations.     

Modulation of aggregate size- and shape-distributions of the amyloid-β peptide by a designed β-sheet breaker

A peptide with 42 amino acid residues (Aβ42) plays a key role in the pathogenesis of the Alzheimer’s disease. It is highly prone to self aggregation leading to the formation of fibrils which are deposited in amyloid plaques in the brain of diseased individuals. In our study we established a method to analyze the aggregation behavior of the Aβ peptide with a combination of sedimentation velocity centrifugation and enhanced data evaluation software as implemented in the software package UltraScan. Important information which becomes accessible by this methodology is the s-value distribution and concomitantly also the shape-distribution of the Aβ peptide aggregates generated by self-association. With this method we characterized the aggregation modifying effect of a designed β-sheet breaker molecule. This compound is built from three head-to-tail connected aminopyrazole moieties and represents a derivative of the already described Tripyrazole. By addition of this compound to a solution of the Aβ42 peptide the maximum of the s-value distribution was clearly shifted to smaller s-values as compared to solutions where only the vehicle DMSO was added. This shift to smaller s-values was stable for at least 7 days. The information about size- and shape-distributions present in aggregated Aβ42 solutions was confirmed by transmission electron microscopy and by measurement of amyloid formation by thioflavin T fluorescence.     

Medium-chain fatty acids undergo elongation before beta-oxidation in fibroblasts

Although mitochondrial fatty acid beta-oxidation (FAO) is considered to be well understood, further elucidation of the pathway continues through evaluation of patients with FAO defects. The FAO pathway can be examined by measuring the 3-hydroxy-fatty acid (3-OHFA) intermediates. We present a unique finding in the study of this pathway: the addition of medium-chain fatty acids to the culture media of fibroblasts results in generation of 3-OHFAs which are two carbons longer than the precursor substrate. Cultured skin fibroblasts from normal and LCHAD-deficient individuals were grown in media supplemented with various chain-length fatty acids. The cell-free medium was analyzed for 3-OHFAs by stable-isotope dilution gas-chromatography/mass-spectrometry. Our finding suggests that a novel carbon chain-length elongation process precedes the oxidation of medium-chain fatty acids. This previously undescribed metabolic step may have important implications for the metabolism of medium-chain triglycerides, components in the dietary treatment of a number of disorders.