Hypohydration does not impair skeletal muscle glycogen resynthesis after exercise

The purpose of this investigation was to examine the effects of moderate hypohydration (HY) on skeletal muscle glycogen resynthesis after exhaustive exercise. On two occasions, eight males completed 2 h of intermittent cycle ergometer exercise (4 bouts of 17 min at 60% and 3 min at 80% of maximal O2 consumption/10 min rest) to reduce muscle glycogen concentrations (control values 711 +/- 41 mumol/g dry wt). During one trial, cycle exercise was followed by several hours of light upper body exercise in the heat without fluid replacement to induce HY (-5% body wt); in the second trial, sufficient water was ingested during the upper body exercise and heat exposure to maintain euhydration (EU). In both trials, 400 g of carbohydrate were ingested at the completion of exercise and followed by 15 h of rest while the desired hydration level was maintained. Muscle biopsy samples were obtained from the vastus lateralis immediately after intermittent cycle exercise (T1) and after 15 h of rest (T2). During the HY trial, the muscle water content was lower (P less than 0.05) at T1 and T2 (288 +/- 9 and 265 +/- 5 ml/100 g dry wt, respectively; NS) than during EU (313 +/- 8 and 301 +/- 4 ml/100 g dry wt, respectively; NS). Muscle glycogen concentration was not significantly different during EU and HY at T1 (200 +/- 35 vs. 251 +/- 50 mumol/g dry wt) or T2 (452 +/- 34 vs. 491 +/- 35 mumol/g dry wt). These data indicate that, despite reduced water content during the first 15 h after heavy exercise, skeletal muscle glycogen resynthesis is not impaired.     

5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation

Most eukaryotic mRNAs are characterized by the presence of a 5'-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNAs in the presence of cap analogues (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNAs. One way of explaining the importance of the 5'-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure, and thus allow the 40S ribosomal subunit to bind to the mRNA [Sonenberg, N., Guertin, D., Cleveland, D., & Trachsel, H. (1981) Cell (Cambridge, Mass.) 27, 563-572]. Our data and those of others indicate that the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogues or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5'-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases has been performed, and the experimental data have been compared with computer-generated models of AMV-4 secondary structure. In addition, the in vitro translatability of AMV-4 has been examined as a function of increasing potassium concentration, conditions that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5'-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5'-terminus can form weak base pairs with internal regions of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator.

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.     

Worldwide patterns of mitochondrial DNA differentiation in the harbor seal (Phoca vitulina)

The harbor seal (Phoca vitulina) has one of the broadest geographic distributions of any pinniped, stretching from the east Baltic, west across the Atlantic and Pacific Oceans to southern Japan. Although individuals may travel several hundred kilometers on annual feeding migrations, harbor seals are generally believed to be philopatric, returning to the same areas each year to breed. Consequently, seals from different areas are likely to be genetically differentiated, with levels of genetic divergence increasing with distance. Differentiation may also be caused by long-standing topographic barriers such as the polar sea ice. We analyzed samples of 227 harbor seals from 24 localities and defined 34 genotypes based on 435 bp of control region sequence. Phylogenetic analysis and analysis of molecular variance showed that populations in the Atlantic and Pacific Oceans and east and west coast populations of these oceans are significantly differentiated. Within these four regions, populations that are geographically farthest apart generally are the most differentiated and often do not share genotypes or differ in genotype frequency. The average corrected sequence divergence between populations in the Atlantic and Pacific Oceans is 3.28% +/- 0.38% and those among populations within each of these oceans are 0.75% +/- 0.69% and 1.19% +/- 0.65%, respectively. Our results suggest that harbor seals are regionally philopatric, on the scale of several hundred kilometers. However, genetic discontinuities may exist, even between neighboring populations such as those on the Scottish and east English coasts or the east and west Baltic. The mitochondrial data are consistent with an ancient isolation of populations in both oceans, due to the development of polar sea ice. In the Atlantic and Pacific, populations appear to have been colonized from west to east with the European populations showing the most recent common ancestry. We suggest the recent ancestry of European seal populations may reflect recolonization from Ice Age refugia after the last glaciation.      

Extent of stimulation controls the formation of memory CD8 T cells

Only a small fraction of effector CD8 T cells survives to become long-lived memory cells, whereas the majority of them die after an acute infection. What controls the formation of memory CD8 T cells remains mostly unknown. In this study, we showed CD8 T cells primed earlier during vaccinia viral infection received stronger stimulation, divided more extensively, and survived better than those primed later, leading to generation of a larger memory pool. Despite differentiation into effectors, the late-primed CD8 T cells lacked full cell division, displayed increased apoptosis, and failed to develop into memory cells, suggesting that the extent of stimulation influences the survival of effector CD8 T cells. We further demonstrated that the extent of stimulation, which included both the duration and the levels of antigenic stimulation/costimulation, during priming determined the formation of memory CD8 T cells via controlling the extent of Akt activation, and functional suppression of Akt led to defective CD8 memory formation in vivo. Collectively, our data suggest that the extent of stimulation controls CD8 memory formation via activation of Akt and may provide important insights into the design of effective vaccines.     

Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo

Tight junctions (TJs) are major components of the blood–brain barrier (BBB) that physically obstruct the interendothelial space and restrict paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS. TJs are dynamic structures whose intricate arrangement of oligomeric transmembrane and accessory proteins rapidly alters in response to external stressors to produce changes in BBB permeability. In this study, we investigate the constitutive trafficking of the TJ transmembrane proteins occludin and claudin-5 that are essential for forming the TJ seal between microvascular endothelial cells that inhibits paracellular diffusion. Using a novel, detergent-free OptiPrep density-gradient method to fractionate rat cerebral microvessels, we identify a plasma membrane lipid raft domain that contains oligomeric occludin and claudin-5. Our data suggest that oligomerization of occludin involves disulfide bond formation within transmembrane regions, and that assembly of the TJ oligomeric protein complex is facilitated by an oligomeric caveolin scaffold. This is the first time that distribution of oligomeric TJ transmembrane proteins within plasma membrane lipid rafts at the BBB has been examined in vivo. The findings reported in this study are critical to understand the mechanism of assembly of the TJ multiprotein complex that is essential for maintaining BBB integrity.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Nonpeptide oxytocin antagonists: analogs of L-371,257 with improved potency.

Structure-activity studies on the oxytocin antagonist 1 (L-371,257; Ki = 9.3 nM) have led to the identification of a related series of compounds containing an ortho-trifluoroethoxyphenylacetyl core which are orally bioavailable and have significantly improved potency in vitro and in vivo, e.g., compound 8 (L-374,943; Ki = 1.4 nM).