Changes in osmotic and ionic concentrations in the hemolymph of Macrobrachium rosenbergii exposed to varying salinities and correlation to ionic and crystalline composition of the cuticle

Osmotic and ionic regulatory ability were examined in the giant freshwater prawn, Macrobrachium rosenbergii in response to varying salinities. In freshwater, and under conditions of low salinity, hemolymph osmolality was maintained around 450 mOsm. Under high salinity, osmolality values increased in a time-wise manner until reaching levels of the surrounding rearing water. Changes in sodium concentration generally paralleled osmotic change, and potassium and magnesium concentrations increased upon exposure to extremely high salinity. In contrast, total calcium concentration was maintained at high levels regardless of salinity treatment. Examination of crystalline structure and ionic composition of the cuticle revealed that it was comprised principally of an α-chitin-like material, and calcite (calcium carbonate). Calcite accounted for 25% of total bulk weight in freshwater, while sodium, potassium and magnesium constituents combined comprised less than 2.5% of this total. Although sodium, potassium and magnesium contents increased nearly 2-fold in response to changing salinity, calcium levels remained relatively constant.     

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.     

Evidence for archaic Asian ancestry on the human X chromosome

The human RRM2P4 pseudogene has a pattern of nucleotide polymorphism that is unlike any locus published to date. A gene tree constructed from a 2.4-kb fragment of the RRM2P4 locus sequenced in a sample of 41 worldwide humans clearly roots in East Asia and has a most-recent common ancestor approximately 2 Myr before present. The presence of this basal lineage exclusively in Asia results in higher nucleotide diversity among non-Africans than among Africans. A global survey of a single-nucleotide polymorphism that is diagnostic for the basal, Asian lineage in 570 individuals shows that it occurs at frequencies up to 53% in south China, whereas only one of 177 surveyed Africans carries this archaic lineage. We suggest that this ancient lineage is a remnant of introgressive hybridization between expanding anatomically modern humans emerging from Africa and archaic populations in Eurasia.     

Heterogeneous patterns of variation among multiple human x-linked Loci: the possible role of diversity-reducing selection in non-africans

Studies of human DNA sequence polymorphism reveal a range of diversity patterns throughout the genome. This variation among loci may be due to natural selection, demographic influences, and/or different sampling strategies. Here we build on a continuing study of noncoding regions on the X chromosome in a panel of 41 globally sampled humans representing African and non-African populations by examining patterns of DNA sequence variation at four loci (APXL, AMELX, TNFSF5, and RRM2P4) and comparing these patterns with those previously reported at six loci in the same panel of 41 individuals. We also include comparisons with patterns of noncoding variation seen at five additional X-linked loci that were sequenced in similar global panels. We find that, while almost all loci show a reduction in non-African diversity, the magnitude of the reduction varies substantially across loci. The large observed variance in non-African levels of diversity results in the rejection of a neutral model of molecular evolution with a multi-locus HKA test under both a constant size and a bottleneck model. In non-Africans, some loci harbor an excess of rare mutations over neutral equilibrium predictions, while other loci show no such deviation in the distribution of mutation frequencies. We also observe a positive relationship between recombination rate and frequency spectra in our non-African, but not in our African, sample. These results indicate that a simple out-of-Africa bottleneck model is not sufficient to explain the observed patterns of sequence variation and that diversity-reducing selection acting at a subset of loci and/or a more complex neutral model must be invoked.     

Map of seven polymorphic markers on rat Chromosome 14: linkage conservation with human Chromosome 4

Seven polymorphic markers identified by polymerase chain reaction (PCR) amplification, including markers for six genes—DRD1L (dopamine receptor, D1-like-2), GLUKA (glucokinase), PF4 (platelet factor 4), ALB (albumin), AFP (-fetoprotein), and BSP (bone sialoprotein)—and one anonymous locus (D14N52), were mapped to a single 67-cM linkage group with F₂ intercross progeny of F344/N and LEW/N inbred rat strains. Two of these markers, ALB and AFP, have previously been assigned to rat Chromosome (Chr) 14, allowing assignment of this entire linkage group. Five of the markers—DRD1L, PF4, ALB, AFP, and PBSP—have been physically mapped to a large region of human Chr 4 encompassing the p arm and the q arm to band q28. Homologs of two of the markers, ALB and AFP, have been mapped to Chr 5 in the mouse. Comparison of human Chr 4 with the homologous regions on Chr 14 of the rat and Chr 5 of the mouse indicated that linkage conservation with human Chr 4 extends over a greater region in the rat than in the mouse. The markers described here were found to be highly polymorphic in twelve inbred strains (F344/N, LEW/N, ACI/N, BUF/N, BN/SsN, LOU/MN, MNR/N, MR/N, SHR/N, WBB1/N, WBB2/N, and WKY/N). These polymorphic markers should be useful in genetic linkage studies of important phenotypes in rats.     

Homologies of the adductor mandibulae muscles in Tetraodontiformes as indicated by nerve branching patterns

Homologies of the adductor mandibulae muscles in eight families of Tetraodontiformes were hypothesized from the branching patterns of ramus mandibularis trigeminus. Insertions of the muscles to the upper or lower jaw were weak indicators of homology, migrations of the sites occurring frequently in A1, A2, A2, and A3. In monacanthids, tetraodontids, and diodontids, A1 tended to be split into numerous subsections, whereas in aracanids and ostraciids, A3 was highly developed, comprising three or four subsections. In tetraodontids, A2 was found to be a composite of A1 subsection and A2. The methods of and limits to applying nerve branching patterns to muscle homology are discussed. A new naming system that reflects both muscle homologies and insertions is proposed.     

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.     

Contributions of juvenile and adult diet to the lifetime reproductive success and lifespan of a spider

Food availability can vary widely for animals in nature and can have large effects on growth, reproduction and survival. While the consequences of food limitation for animals have been extensively studied, significant questions still remain including how ontogenetic variation in food availability contributes to lifetime reproductive success. We tested the effects of juvenile and adult food limitation on the lifetime reproductive success and lifespan of bridge spiders, Larinioides sclopetarius. Food availability was manipulated (low or high) over the entire juvenile and adult stage in a full‐factorial design and reproductive output and lifespan were measured. Juvenile and adult food limitation both reduced lifetime egg and hatchling production with effect sizes that were not significantly different from each other. Unlike some other arthropods, where juvenile food limitation reduces fecundity by reducing adult body size, body size was not affected by juvenile diet in bridge spiders. Clutch size was also significantly reduced by both juvenile and adult food limitation. The effect of adult diet on clutch size was stronger than that of juvenile diet. Juvenile and adult food limitation both extended total lifespan, and adult food limitation extended adult longevity (i.e. time from maturation to death). However, juvenile food limitation decreased adult longevity, in contrast to what would be predicted by dietary or caloric restriction. Compensatory feeding and growth are widely recognized mechanisms through which animals can ameliorate some of the negative effects of periods of food limitation. Yet our results combined with studies of a range of other species suggest that there may be lasting consequences of juvenile food limitation on lifetime reproductive success that cannot be compensated for by adult feeding in some species.