Differences in muscle contractile characteristics among bodybuilders, endurance trainers and control subjects

The purpose of this investigation was to compare the myosin heavy chain (MHC) isoform expression of the triceps brachii muscle and isoinertial, isometric and isokinetic strength indices in competitive bodybuilders (CB, n = 5), recreational resistance trainers (RT, n = 5), endurance-trained rowers (ER, n = 5) and control (C, n = 5) subjects. Muscle tissue samples were analysed for MHC isoform content using 6% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The CB possessed significantly smaller (P < 0.05) percentage of MHC type IIb proteins [12.92 (SD 7.08)%] than RT [30.08 (SD 6.58)%] ER [31.20 (SD 2.74)%] and C [38.22 (SD 2.95)%] groups (i.e. CB < RT ≈ ER < C). While the content of MHC type IIa isoforms did not differ significantly between the two resistance-trained groups [CB = 55.76 (SD 5.38)%; RT = 45.72 (SD 7.8)%], CB presented significantly more type IIa MHC isoforms than ER [42.84 (SD 2.98)%] and C [34.72 (SD 1.57)%] subjects (i.e. CB ≈ RT > ER ≈ C). The MHC type I protein content did not differ significantly among RT [24.20 (SD 4.89)%] ER [25.38 (SD 1.67)%] and C [27.06 (SD 1.81)%] groups. The CB [31.32 (SD 2.67)%] presented significantly more type I MHC isoforms only in comparison with RT. However, when changes in the percentage of MHC type I isoforms were converted to effect sizes (ES), it appeared that low statistical power rather than the absence of an effect accounted for the nonsignificant differences between CB and other groups (i.e. CB > RT ≈ ER ≈ C). Significant differences existed in isoinertial strength among the trained athletes (i.e. CB > RT > ER ≈ C), while isometric and isokinetic strength were not significantly different among any of the trained groups. However, the ES transformation of data demonstrated that large differences existed between resistance-trained groups and ER for isometric and isokinetic strength (i.e. CB ≈ RT > ER ≈ C). A statistically significant negative correlation (P < 0.001) was found between MHC type IIb isoforms and isoinertial strength index (r = − 0.68). The MHC type IIa proteins were positively related to all the strength measures considered (r = 0.51 – 0.61; P < 0.001). These data demonstrated different patterns of MHC isoform expression among the different groups of athletes and it is suggested that these differences on occasion may affect the expression of strength. Accepted: 24 September 1996     

Some mechanical factors in the dynamics of the thin-walled,spherical viscus

Purely mechanical factors involved in the dynamics of the thin-walled, spherical viscus may be elucidated by studying the behavior of an artificial viscus. An equation is developed on theoretical grounds which expresses the pressure-distention characteristics of such a model and apparently corrects the errors contained in similar equations developed by other investigators. A precise definition for wall “tone” is offered. On this basis the mathematics of “tone”-distention relationships have been developed. Experimental investigation of the artificial viscus (spherical rubber balloon) permits the differences in behavior between the ideal and the actual model to be composed, and confirms the validity of our theoretical developments. This investigation was financially supported by USPHS grant RG 101. The material presented here is part of a thesis for qualification for the degree of M.S. (Pharmacology).     

A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome

While macroalgal microbiomes are the focus of many recent studies, there is little information about microbial spatial diversity across the thallus. Reliance on field material makes it difficult to discern whether recovered microbiomes belong to the host or its epiphytes, and technical comparisons of macroalgal samples for microbial studies are needed. Here, we use a common garden approach that avoids the problem of epiphytes, particularly at holdfasts, to examine the microbiome of Porphyra umbilicalis (strain Pum1). We used the V6 hypervariable region of the 16S rDNA with Illumina HiSeq sequencing and developed PNA clamps to block recovery of organelle V6 sequences. The common garden approach allowed us to determine differences in the microbiome at the holdfast versus blade margin. We found a notable increase in the relative abundance of Planctomycetes and Alphaproteobacteria at the holdfast, particularly of the possible symbiont Sulfitobacter sp. Nonadjacent 1.5 cm² samples of blade margin had microbiomes that were not statistically different. The most abundant phylum in the overall microbiome was Proteobacteria, followed by Bacteroidetes. Because phycologists often work in remote sites, we compared three stabilization and preparation techniques and found silica gel desiccation/bead‐beating and flash‐freezing/lyophilization/bead‐beating to be interchangeable. Core taxa (≥0.1% of sequences) across treatments were similar and accounted for ≥95% of all sequences. Finally, statistical conclusions for all comparisons were the same, regardless of which microbial community analysis tool was used: mothur or minimum entropy decomposition.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Characterizing clearance of helper adenovirus by a clinical rAAV1 manufacturing process.

Recombinant adeno-associated viral vectors (rAAV) are being developed as gene therapy delivery vehicles and as genetic vaccines, and some of the most scaleable manufacturing methods for rAAV use live adenovirus to induce production. One aspect of establishing safety of rAAV products is therefore demonstrating adequate and reliable clearance of this helper virus by the vector purification process. The ICH Q5A regulatory guidance on viral safety provides recommendations for process design and characterization of viral clearance for recombinant proteins, and these principles were adapted to a rAAV serotype 1 purification process for clinical vectors. Specific objectives were to achieve overall adenovirus clearance factors significantly greater than input levels by using orthogonal separation and inactivation methods, and to segregate adenovirus from downstream operations by positioning a robust clearance step early in the process. Analytical tools for process development and characterization addressed problematic in-process samples, and a viral clearance validation study was performed using adenovirus and two non-specific model viruses. Overall clearance factors determined were >23 LRV for adenovirus, 11 LRV for BVDV, and >23 LRV for AMuLV.     

Sex-biased natal dispersal and inbreeding avoidance in American black bears as revealed by spatial genetic analyses

We tested the hypothesis that sex-biased natal dispersal reduces close inbreeding in American black bears, a solitary species that exhibits nearly complete male dispersal and female philopatry. Using microsatellite DNA and spatial data from reproductively mature bears (>or= 4 years old), we examined the spatial genetic structure of two distinct populations in New Mexico from 1993 to 2000. As predicted, relatedness (r) and the frequency of close relationships (parent-offspring or full siblings) decreased with distance among female dyads, but little change was observed among male or opposite-sex dyads. Neighbouring females were more closely related than neighbouring males. The potential for inbreeding was low. Most opposite-sex pairs that lived sufficiently close to facilitate mating were unrelated, and few were close relatives. We found no evidence that bears actively avoided inbreeding in their selection of mates from this nearby pool, as mean r and relationship frequencies did not differ between potential and actual mating pairs (determined by parentage analysis). These basic patterns were apparent in both study areas despite a nearly two-fold difference in density. However, the sex bias in dispersal was less pronounced in the lower-density area, based on proportions of bears with male and female relatives residing nearby. This result suggests that male bears may respond to reduced competition by decreasing their rate or distance of dispersal. Evidence supports the hypothesis that inbreeding avoidance is achieved by means of male-biased dispersal but also indicates that competition (for mates or resources) modifies dispersal patterns.     

A pentapeptide motif related to a pigment binding site in the major light-harvesting protein of photosystem II, LHCII, governs substrate-dependent plastid import of NADPH:protochlorophyllide oxidoreductase A

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only known example thus far of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex. Here we demonstrate that a pentapeptide motif, threonine-threonine-serine-proline-glycine (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate dependent. An additional motif was identified in the NH(2)-terminal part of transA that was needed for binding of the precursor to the Pchlide-dependent translocon complex. Point mutations of the TTSPG motif identified glycine as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of glycine may bind directly or via a water molecule to the central Mg atom of the pigment.     

Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Hypoxic (low oxygen) and reperfusion (post‐hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent‐free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro‐octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non‐reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO‐solubilized occludin revealed that occludin oligomeric assemblies co‐localizing with ‘TJ‐associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO‐solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non‐covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide‐bonded inner core, and dispersal of these non‐covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.     

Non-peptide calcitonin gene-related peptide receptor antagonists from a benzodiazepinone lead

High-throughput screening of the Merck sample collection identified benzodiazepinone tetralin-spirohydantoin 1 as a CGRP receptor antagonist with micromolar activity. Comparing the structure of 1 with those of earlier peptide-based antagonists such as BIBN 4096 BS, a key hydrogen bond donor-acceptor pharmacophore was hypothesized. Subsequent structure activity studies supported this hypothesis and led to benzodiazepinone piperidinyldihydroquinazolinone 7, CGRP receptor K(i)=44nM and IC(50)=38nM. Compound 7 was orally bioavailabile in rats and is a lead in the development of orally bioavailable CGRP antagonists for the treatment of migraine.