Sex without sex chromosomes: genetic architecture of multiple loci independently segregating to determine sex ratios in the copepod Tigriopus californicus.

Sex-determining systems are remarkably diverse and may evolve rapidly. Polygenic sex-determination systems are predicted to be transient and evolutionarily unstable, yet examples have been reported across a range of taxa. Here, we provide the first direct evidence of polygenic sex determination in Tigriopus californicus, a harpacticoid copepod with no heteromorphic sex chromosomes. Using genetically distinct inbred lines selected for male- and female-biased clutches, we generated a genetic map with 39 SNPs across 12 chromosomes. Quantitative trait locus mapping of sex ratio phenotype (the proportion of male offspring produced by an F2 female) in four F2 families revealed six independently segregating quantitative trait loci on five separate chromosomes, explaining 19% of the variation in sex ratios. The sex ratio phenotype varied among loci across chromosomes in both direction and magnitude, with the strongest phenotypic effects on chromosome 10 moderated to some degree by loci on four other chromosomes. For a given locus, sex ratio phenotype varied in magnitude for individuals derived from different dam lines. These data, together with the environmental factors known to contribute to sex determination, characterize the underlying complexity and potential lability of sex determination, and confirm the polygenic architecture of sex determination in T. californicus.

Physico-chemical confinement of helical nanofilaments.

Helical nanofilaments (HNFs) have attracted much interest because of their unique optical properties, but there have been many hurdles to overcome in using them for the practical applications due to their structural complexity. Here we demonstrate that the molecular configuration and layer conformation of a modulated HNF (HNFs(mod)) can be studied using a physicochemical confinement system. The layer directions affected by the chemical affinity between the mesogen and surface were drastically controlled in surface-modified nanochannels. Furthermore, an in situ experiment using grazing-incidence X-ray diffraction (GIXD) was carried out to investigate in detail the structural evolution through thermal transitions. The results demonstrate that the HNF(mod) structure can be perfectly controlled for functional HNF device applications, and a combined system with chemical and physical confinement effects will be helpful to better understand the fundamentals of soft matter.

Multigenerational response to artificial selection for biased clutch sex ratios in Tigriopus californicus populations.

Polygenic sex determination (PSD) is relatively rare and theoretically evolutionary unstable, yet has been reported across a range of taxa. Evidence for multilocus PSD is provided by (i) large between-family variance in sex ratio, (ii) paternal and maternal effects on family sex ratio and (iii) response to selection for family sex ratio. This study tests the polygenic hypothesis of sex determination in the harpacticoid copepod Tigriopus californicus using the criterion of response to selection. We report the first multigenerational quantitative evidence that clutch sex ratio responds to artificial selection in both directions (selection for male- and female-biased families) and in multiple populations of T. californicus. In the five of six lines that showed a response to selection, realized heritability estimated by multigenerational analysis ranged from 0.24 to 0.58. Divergence of clutch sex ratio between selection lines is rapid, with response to selection detectable within the first four generations of selection.

Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study.

Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of (R)- or (S)-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2'-bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2'-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel-Crafts reaction with SCP_Q111BpyAla achieving the best selectivity with an enantioselectivity of 72% ee (S). Interestingly, despite using the same protein scaffold, different attachment strategies for Bpy at the same residue (Q111) led to a switch in the enantiopreference of the ArM. X-ray crystal structures of SCP_Q111CBpy and SCP_Q111BpyAla ArMs with bound Cu(ii) ions unveiled crucial differences in the orientation of the catalytic centre. Combining structural information, alanine scanning studies, and computational analysis shed light on the distinct active sites of the ArMs, clarifying that these active sites stabilise the nucleophilic substrate on different sides of the electrophile leading to the observed switch in enantioselectivity. This work underscores the importance of integrating structural studies with catalytic screening to unravel the intricacies of ArM behaviour and facilitate their development for targeted applications in biocatalysis.

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe-AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.

Reduction in protein tyrosine phosphorylation during differentiation of human leukemia cell line K-562.

Human chronic myelogenous leukemia cell line K-562 expresses the bcr/c-abl fusion protein which is an active protein tyrosine kinase. Multiple tyrosine-phosphorylated proteins were detected in K-562 cells by immunoblotting with a high-affinity anti-phosphotyrosine antibody. When K-562 cells were induced with hemin to progress through the erythroid differentiation pathway, reduction in the levels of these tyrosine-phosphorylated proteins was observed. This reduction in tyrosine-phosphorylated proteins was not found in another chronic myelogenous leukemia cell line which could not be induced to differentiate by hemin. This and other observations established that the reduction in protein tyrosine phosphorylation is a specific differentiation response. The bcr/c-abl protein synthesis was reduced in hemin-treated K-562 cells. Thus, erythroid differentiation of K-562 cells reduces the level of the bcr/c-abl tyrosine kinase and the phosphotyrosine content of its substrate proteins.

Primary structure determinants of the pH- and temperature-dependent aggregation of thioredoxin.

Thioredoxins are small proteins found in all living organisms. We have previously reported that Chlamydomonas reinhardtii thioredoxin h exhibited differences both in its absorption spectrum and its aggregation properties compared to thioredoxin m. In this paper, we demonstrate, by site-directed mutagenesis, that the particularity of the absorption spectrum is linked to the presence of an additional tryptophan residue in the h isoform. The pH and temperature dependence of the aggregation of both thioredoxins has been investigated. Our results indicate that the aggregation of TRX is highly dependent on pH and that the differences between the two TRX isoforms are linked to distinct pH dependencies. We have also analyzed the pH and temperature dependence of 12 distinct variants of TRX engineered by site-directed mutagenesis. The results obtained indicate that the differences in the hydrophobic core of the two TRX isoforms do not account for the differences of aggregation. On the other hand, we show the importance of His-109 as well as the second active site cysteine, Cys-39 in the aggregation mechanism.

Structure of the glycosylphosphatidylinositol membrane anchor glycan of a class-2 variant surface glycoprotein from Trypanosoma brucei.

The neutral glycan fraction of the glycosylphosphatidylinositol (GPI) membrane anchor of a class-2 variant surface glycoprotein (VSG) from Trypanosoma brucei was isolated following aqueous hydrogen fluoride dephosphorylation and nitrous acid deamination of the purified glycoprotein. The neutral glycans were fractionated by high-pH anion exchange chromatography and gel-filtration and six major glycan structures were solved by a combination of one and two-dimensional NMR, composition analysis, methylation linkage analysis and electrospray-mass spectrometry. The glycans were similar to those previously described for class-1 VSGs, in that they contained the linear trimannosyl sequence Manalpha1-2Manalpha1-6Man and a complex alpha-galactose branch of up to Galalpha1-2Galalpha1-6(Galalpha1-2)Gal, but most also contained an additional galactose residue attached alpha1-2 to the non-reducing terminal mannose residue and about one-third contained an additional galactose residue attached beta1-3 to the middle mannose residue. The additional complexity of the class-2 VSG GPI glycans is discussed in terms of a biosynthetic model that explains the full range of mature GPI structures that can be expressed on different VSG classes by the same trypanosome clone.

Ventricular preexcitation. Practical considerations.

Ventricular preexcitation syndromes are important because they are associated with paroxysmal tachycardias that can result in serious cardiovascular complications and sudden death. These syndromes are also important because the ECG findings, if unrecognized, are frequently misdiagnosed as something else. With the use of "classic" syndrome names, for example, the Wolff-Parkinson-White (WPW) syndrome, physicians tend to think of ventricular preexcitation in a rigidly defined sense (short PR interval, delta wave, and abnormal QRS). A broader view of ventricular preexcitation allows one to think of it as an entity with endless variations. Failure to recognize the wide ECG variations in which ventricular preexcitation occurs may have substantial clinical consequences relative to misdiagnosis and treatment.

Cloning, overexpression, purification, and spectroscopic characterization of human S100P.

The calcium-binding protein S100P has been found to be associated with human prostate cancer. We have overexpressed S100P in Escherichia coli using a T7 expression system. A rapid two-step procedure for the isolation of overexpressed S100P leads to a preparation of >95% pure protein with a yield of approximately 150 mg per liter of culture. The structural integrity of recombinant S100P was analyzed using CD and fluorescence spectroscopic techniques. The far-UV CD shows that secondary structure of recombinant S100P consists predominantly of a-helical structure. Both near-UV CD and tyrosine fluorescence spectra show that aromatic residues are involved in the formation of a specific, well packed structure, indicating that the recombinant S100P protein adopts a compact folded conformation. Ca2+ has a profound effect on S100P structure. Near-UV CD and fluorescence intensity of both internal (tyrosine) and external (ANS) probes suggest significant structural rearrangements in the tertiary structure of the molecule. The similarity of far-UV CD spectrum of S100P in the presence and in the absence of Ca2+ suggests that Ca2+ binding has only minor effects on secondary structure.

Anion binding to the ubiquitin molecule.

Effects of different salts (NaCl, MgCl2, CaCl2, GdmCl, NaBr, NaClO4, NaH2PO4, Na2SO4) on the stability of the ubiquitin molecule at pH 2.0 have been studied by differential scanning calorimetry, circular dichroism, and Tyr fluorescence spectroscopies. It is shown that all of the salts studied significantly increase the thermostability of the ubiquitin molecule, and that this stabilization can be interpreted in terms of anion binding. Estimated thermodynamic parameters of binding for Cl- show that this binding is relatively weak (Kd = 0.15 M) and is characterized by a negative enthalpy of -15 kJ/mol per site. Particularly surprising was the observed stabilizing effect of GdmCl through the entire concentration range studied (0.01-2 M), however, to a lesser extent than stabilization by NaCl. This stabilizing effect of GdmCl appears to arise from the binding of Cl- ions. Analysis of the observed changes in the stability of the ubiquitin molecule in the presence of GdmCl can be adequately described by combining the thermodynamic model of denaturant binding with Cl- binding effects.

Regulation of polymeric immunoglobulin A receptor messenger ribonucleic acid expression in rodent uteri: effect of sex hormones.

Previously we have shown that estradiol stimulates the production of secretory component, the external domain of the polymeric immunoglobulin A (IgA) receptor (pIgR) responsible for transporting IgA from tissues into secretions. In the present study, levels of pIgR messenger RNA (mRNA) in uterine tissues of rats were correlated with pIgR expression in epithelial cells and secretory component in uterine secretions. Analysis of uterine pIgR mRNA and pIgR expression in epithelial cells during the estrous cycle indicated that levels were high at proestrus and estrus and low at diestrus. When ovariectomized rats were treated with estradiol for 3 days, and pIgR mRNA was measured 4 and 12 h after the last injection, levels of uterine pIgR mRNA were significantly greater than those in saline-treated controls. High levels of pIgR were also detected in uterine epithelial cells and uterine secretions. When estradiol and progesterone were given in combination, progesterone partially reversed the effect of estradiol on pIgR mRNA levels and expression of pIgR in epithelial cells. These studies demonstrate that changes in uterine pIgR mRNA levels correlate with pIgR expression during the estrous cycle and in response to estradiol and progesterone. These findings suggest that mucosal immune responses in the reproductive tract are regulated in part by the actions of estradiol and progesterone on pIgR mRNA expression.

Leptin is a four-helix bundle: secondary structure by NMR.

Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

The relative roles of adaptation and phylogeny in determination of larval traits in diversifying anuran lineages.

I measured phenotypic traits important to the fitness of larval anurans to assess the relative roles of ancestral trait value and selective regime in determining present-day phenotypes. The positions of 14 species from three taxonomic families and three different habitats in a phenotypic space defined by 19 traits provided measures of taxonomic and ecological similarity. The distribution of phenotypic distances among species revealed that neither taxonomy nor habitat overwhelmingly determined phenotype. There appear to be multiple ways in which anurans can exploit pond types. However, the direction of phenotypic movement was not random from one species to the next. Independent contrasts revealed significant correlations in the evolution of traits that were consistent among lineages. These correlations reflected well-known trade-offs that result from functional relationships among the constituent traits. Although there is no simple pattern in the distribution of mean phenotypes across environments and lineages, the pattern of the evolutionary trajectories that created that distribution is consistent with a predictive theory of multivariate evolution.

The glycosylation of the variant surface glycoproteins and procyclic acidic repetitive proteins of Trypanosoma brucei.

Trypanosoma brucei, in common with the other African trypanosomes, exhibits unusual cell-surface molecular architecture. The bloodstream form of the parasite is coated with a continuous layer of approximately five million variant surface glycoprotein (VSG) dimers that provide the parasite with a macromolecular diffusion barrier to guard against lysis by the alternative complement pathway. The procyclic form of the parasite has a more diffuse cell-surface coat made up of approximately 2.5 million copies of procyclic acidic repetitive protein (PARP). Within the VSG and PARP coats exist lower-abundance surface glycoproteins such as receptors and nutrient transporters. Both the VSG molecules and the PARP molecules are attached to the membrane via glycosylphosphatidylinositol (GPI) membrane anchors and the VSGs and one form of PARP are N-glycosylated. In this article, the structures of the N-glycans and the GPI anchors of T. brucei VSGs and PARPs are reviewed and simple models of the surfaces of bloodstream and procyclic trypomastigotes are presented.

Uterine stromal cell suppression of pIgR production by uterine epithelial cells in vitro: a mechanism for regulation of pIgR production.

Previous studies have shown that the polymeric Ig receptor (pIgR) is produced by rat uterine epithelial cells both in vivo and vitro. The expression of the pIgR is regulated by sex hormones and/or cytokines at mucosal sites, however the mechanism of regulation in the uterus is not clear. In these studies, co-culture of stromal cells from mature rat uteri with uterine epithelial cells decreased epithelial cell pIgR production. Conditioned supernatants from stromal cells incubated with epithelial cells also decreased pIgR production. Immunohistochemical studies confirmed that expression of pIgR on uterine epithelial cells decreased in the presence of stromal cells. Viability of epithelial cells was sustained during these experiments, as evidenced by the maintenance of high transepithelial resistance. These studies are the first report of stromal cell regulation of pIgR production by epithelial cells at any site in the body and suggest that stromal cells can provide a signal that leads to the regulation of pIgR production.

Decreased axial and peripheral bone density in patients taking long-term warfarin.

Impaired vitamin K metabolism is associated with under-carboxylation of the non-collagenous bone-matrix protein osteocalcin, which is required in its fully carboxylated state for normal bone formation. Post-menopausal women have under-carboxylation of osteocalcin which increases with age and is marked in the elderly. A similarly marked degree of impaired carboxylation occurs during coumarin therapy, and a key question is whether this may lead to accelerated loss of bone mass which is clinically important. We measured axial and peripheral bone mineral density (BMD) in 40 male patients on warfarin and 40 controls individually matched for age, disease and other drug therapy. A consistent trend for reduced BMD at all sites was observed in the warfarin-treated patients. This was particularly marked in the cancellous bone at the distal radius (9% reduction, p = 0.023) and at the cancellous rich lumbar spine site (10.4% reduction, p < 0.004). No significant relationship was observed between warfarin dose, International Normalized Ratio (INR) or duration of therapy and bone density. Because of the biochemical similarity, this study provides a new lead on post-menopausal osteoporosis, and supports the hypothesis that impaired carboxylation of osteocalcin plays a role in the pathogenesis of bone loss in the elderly through deficiency in vitamin K metabolism.

Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium.

In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (vanA phenotype) accumulate UDP-N-acetylmuramyl(UDP-Mur-NAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-D-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.

Synchrony of nutrient supply to the rumen and dietary energy source and their effects on the growth and metabolism of lambs.

The objective of the current series of experiments was to assess the effects of dietary synchrony of OM and N supply to the rumen, achieved by altering the sequence of feeding individual ingredients and in diets with different energy sources, on the metabolism and performance of growing lambs. In Exp. 1, the in situ degradability coefficients of OM and N were determined for five feed ingredients and subsequently was used to formulate two diets, based either on barley or sugar beet pulp, to have a similar predicted nutrient content. Within each diet, specific ingredients were shifted between the 0900 and 1600 feeding to provide either a synchronous, intermediate, or asynchronous supply of OM and N to the rumen. In Exp. 2, these diets were fed at a restricted level to 48 growing lambs with an initial live weight of 25.1 +/- 4.22 kg and a slaughter weight of 41.4 +/- 1.94 kg. There was no significant effect of dietary treatment on live weight gain or feed conversion efficiency. Lambs fed the synchronous diets deposited more kidney knob and channel fat than lambs on the asynchronous or intermediate diets (P < 0.05), whereas lambs fed the barley-based diets deposited more carcass (P < 0.05) and noncarcass (P < 0.001) fat than lambs on the sugar beet-based diets. Lambs fed the asynchronous diets retained less energy over the course of the experiment than lambs on the intermediate or synchronous diets (P < 0.05), and had a lower energy efficiency (0.079, 0.097, and 0.093 MJ retained/ MJ of intake, respectively, P < 0.05). Lambs fed the barley-based diets retained more energy than lambs on the sugar beet-based (P < 0.001) and had a higher energy balance (0.095 vs. 0.084 MJ retained/MJ intake, respectively; P < 0.01). Plasma ammonia concentrations mirrored ruminal ammonia concentrations on the barley-based diets, but not sugar beet-based diets. In Exp. 3, lambs fed the sugar beet-based diets had a higher digestibility of OM and NDF (P < 0.001). By contrast, lambs on the barley-based diets had a higher level of purine derivative excretion and microbial N production (P < 0.001). The results indicate that neither dietary synchrony nor energy source significantly influenced growth rate. However, both the asynchronous and sugar beet pulp-based diets resulted in a lower efficiency of dietary energy use, and the avoidance of asynchronous patterns of nutrient release within the rumen can improve energy efficiency in growing lambs.