A large-scale screen in S. pombe identifies seven novel genes required for critical meiotic events

Meiosis is a specialized form of cell division by which sexually reproducing diploid organisms generate haploid gametes. During a long prophase, telomeres cluster into the bouquet configuration to aid chromosome pairing, and DNA replication is followed by high levels of recombination between homologous chromosomes (homologs). This recombination is important for the reductional segregation of homologs at the first meiotic division; without further replication, a second meiotic division yields haploid nuclei. In the fission yeast Schizosaccharomyces pombe, we have deleted 175 meiotically upregulated genes and found seven genes not previously reported to be critical for meiotic events. Three mutants (rec24, rec25, and rec27) had strongly reduced meiosis-specific DNA double-strand breakage and recombination. One mutant (tht2) was deficient in karyogamy, and two (bqt1 and bqt2) were deficient in telomere clustering, explaining their defects in recombination and segregation. The moa1 mutant was delayed in premeiotic S phase progression and nuclear divisions. Further analysis of these mutants will help elucidate the complex machinery governing the special behavior of meiotic chromosomes.     

Chemoanatomical separation of vibrissal trigeminal primary afferents in the rat: a special central representation of supraorbital vibrissae

A choleratoxin B subunit transganglionic labelling technique and NPY immunohistochemistry were applied in the rat to achieve the chemoanatomical separation of myelinated vibrissal primary afferents, previously considered to be morphologically indistinguishable. Further, a special central representation pattern of supraorbital vibrissae was observed in the trigeminal brainstem nuclear complex: (1) Choleratoxin-labelled supraorbital vibrissal primary afferents terminated densely in their appropriate barrelettes in the trigeminal principal sensory nucleus, in the spinal oral subnucleus, in the caudal part of the spinal interpolar subnucleus, and in lamina IV of the caudal part of the spinal caudal subnucleus. (2) A second population of choleratoxin-labelled vibrissal afferents was also observed, terminating only in lamina III of the caudal subnucleus. (3) After peripheral nerve transection, NPY-immunoreactive supraorbital vibrissal primary afferent fibres appeared in their appropriate barrelettes in the principal sensory nucleus and the caudal part of the interpolar subnucleus, while in the caudal part of the caudal subnucleus NPY-immunoreactive vibrissal primary afferent terminals were found exclusively in the inner part of lamina II, extending over the outer part of lamina III. NPY-immunoreactive supraorbital vibrissal primary afferents were never found in the oral subnucleus. In contrast with the rules of the central representation of the mystacial (infraorbital) vibrissae, the multiple representation of the supraorbital vibrissae in the caudal subnucleus and the dense, barrelette-like terminal arborization of the choleratoxin-labelled supraorbital vibrissal primary afferents in the oral subnucleus apparently indicate an enhanced role of supraorbital vibrissae in reflexes that protect the eyes and the head from damage.     

Impact of cytokines on replication in the thymus of primary human immunodeficiency virus type 1 isolates from infants

Early infection of the thymus with the human immunodeficiency virus (HIV) may explain the more rapid disease progression among children infected in utero than in children infected intrapartum. Therefore, we analyzed infection of thymocytes in vitro by HIV type 1 primary isolates, obtained at or near birth, from 10 children with different disease outcomes. HIV isolates able to replicate in the thymus and impact thymopoiesis were present in all infants, regardless of the timing of viral transmission and the rate of disease progression. Isolates from newborns utilized CCR5, CXCR4, or both chemokine receptors to enter thymocytes. Viral expression was observed in discrete thymocyte subsets postinfection with HIV isolates using CXCR4 (X4) and isolates using CCR5 (R5), despite the wider distribution of CXCR4 in the thymus. In contrast to previous findings, the X4 primary isolates were not more cytopathic for thymocytes than were the R5 isolates. The cytokines interleukin-2 (IL-2), IL-4, and IL-7 increased HIV replication in the thymus by inducing differentiation and expansion of mature CD27(+) thymocytes expressing CXCR4 or CCR5. IL-2 and IL-4 together increased expression of CXCR4 and CCR5 in this population, whereas IL-4 and IL-7 increased CXCR4 but not CCR5 expression. IL-2 plus IL-4 increased the viral production of all pediatric isolates, but IL-4 and IL-7 had a significantly higher impact on the replication of X4 isolates compared to R5 isolates. Our studies suggest that coreceptor use by HIV primary isolates is important but is not the sole determinant of HIV pathogenesis in the thymus.     

A "de novo" mutation of the LDL-receptor gene as the cause of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a common genetic disorder caused by mutations of the LDL-receptor gene and transmitted as a co-dominant trait. However, there are some forms of hypercholesterolemia which have a recessive type of transmission. We have identified a subject with the clinical phenotype of heterozygous FH whose parents had normal plasma lipid values, suggesting a recessive type of transmission. The analysis of the LDL-receptor gene revealed that the patient was heterozygous for a G>C transversion in exon 4, which results in a serine for cysteine substitution at position 88 (C88S) of the receptor protein. Since this novel mutation was not found in the proband's parents and non-paternity was excluded, we concluded that the patient was a carrier of a "de novo" mutation. Haplotype analysis of LDL-receptor locus indicated that this "de novo" mutation occurred in the paternal germ line. The C88S mutation is the likely cause of LDL-receptor defect as it was present in the proband's hypercholesterolemic son and was not found in 200 chromosomes of control subjects.     

Synthesis and structural assignment of two major metabolites of the LTA4H inhibitor DG-051

The same two major CYP mediated metabolites of DG-051 were produced in the presence of rat, dog, monkey and human liver microsomes. Their respective structures were hypothesized based on mass spectrometry data correlated with the parent structure and confirmed by comparison with authentic synthetic samples. The number of regioisomers synthesized as candidates for metabolite M1 was narrowed down using a metabolic study of a selectively deuterated DG-051 analogue.     

Demonstration of a Melanoma-Specific CD44 Alternative Splicing Pattern That Remains Qualitatively Stable,but Shows Quantitative Changes during Tumour Progression

The role of CD44 in the progression of human melanoma has mostly been characterised by qualitative changes in expression of its individual variable exons. These exons however, may be expressed to form a number of molecules, the alternative splice variants of CD44, which may be structurally and functionally different. Using real-time PCR measurements with variable exon specific primers we have determined that all are expressed in human melanoma. To permit comparison between different tumours we identified a stable CD44 variable exon (CD44v) expression pattern, or CD44 ‘fingerprint’. This was found to remain unchanged in melanoma cell lines cultured in different matrix environments. To evaluate evolution of this fingerprint during tumour progression we established a scid mouse model, in which the pure expression pattern of metastatic primary tumours, circulating cells and metastases, non-metastatic primary tumours and lung colonies could be studied. Our analyses demonstrated, that although the melanoma CD44 fingerprint is qualitatively stable, quantitative changes are observed suggesting a possible role in tumour progression.     

Changes in Diversity and Functional Gene Abundances of Microbial Communities Involved in Nitrogen Fixation, Nitrification, and Denitrification in a Tidal Wetland versus Paddy Soils Cultivated for Different Time Periods

In many areas of China, tidal wetlands have been converted into agricultural land for rice cultivation. However, the consequences of land use changes for soil microbial communities are poorly understood. Therefore, we investigated bacterial and archaeal communities involved in inorganic nitrogen turnover (nitrogen fixation, nitrification, and denitrification) based on abundances and relative species richness of the corresponding functional genes along a soil chronosequence ranging between 50 and 2,000 years of paddy soil management compared to findings for a tidal wetland. Changes in abundance and diversity of the functional groups could be observed, reflecting the different chemical and physical properties of the soils, which changed in terms of soil development. The tidal wetland was characterized by a low microbial biomass and relatively high abundances of ammonia-oxidizing microbes. Conversion of the tidal wetlands into paddy soils was followed by a significant increase in microbial biomass. Fifty years of paddy management resulted in a higher abundance of nitrogen-fixing microbes than was found in the tidal wetland, whereas dominant genes of nitrification and denitrification in the paddy soils showed no differences. With ongoing rice cultivation, copy numbers of archaeal ammonia oxidizers did not change, while that of their bacterial counterparts declined. The nirK gene, coding for nitrite reductase, increased with rice cultivation time and dominated its functionally redundant counterpart, nirS, at all sites under investigation. Relative species richness showed significant differences between all soils with the exception of the archaeal ammonia oxidizers in the paddy soils cultivated for 100 and 300 years. In general, changes in diversity patterns were more pronounced than those in functional gene abundances.     

Perimicrovillar membrane assembly: the fate of phospholipids synthesised by the midgut of Rhodnius prolixus

In this study, we describe the fate of fatty acids that are incorporated from the lumen by the posterior midgut epithelium of Rhodnius prolixus and the biosynthesis of lipids. We also demonstrate that neutral lipids (NL) are transferred to the haemolymphatic lipophorin (Lp) and that phospholipids remain in the tissue in which they are organised into perimicrovillar membranes (PMMs). 3H-palmitic acid added at the luminal side of isolated midguts of R. prolixus females was readily absorbed and was used to synthesise phospholipids (80%) and NL (20%). The highest incorporation of 3H-palmitic acid was on the first day after a blood meal. The amounts of diacylglycerol (DG) and triacylglycerol synthesised by the tissue decreased in the presence of Lp in the incubation medium. The metabolic fates of 3H-lipids synthesised by the posterior midgut were followed and it was observed that DG was the major lipid released to Lp particles. However, the majority of phospholipids were not transferred to Lp, but remained in the tissue. The phospholipids that were synthesised and accumulated in the posterior midgut were found to be associated with Rhodnius luminal contents as structural components of PMMs.     

Structural and Functional Characterization of the Enantiomers of the Antischistosomal Drug Oxamniquine

Background

For over two decades, a racemic mixture of oxamniquine (OXA) was administered to patients infected by Schistosoma mansoni, but whether one or both enantiomers exert antischistosomal activity was unknown. Recently, a ~30 kDa S. m ansoni sulfotransferase (SmSULT) was identified as the target of OXA action.

Methodology/Principal Findings

Here, we separate the OXA enantiomers using chromatographic methods and assign their optical activities as dextrorotary [(+)-OXA] or levorotary [(-)-OXA]. Crystal structures of the parasite enzyme in complex with optically pure (+)-OXA and (-)-OXA) reveal their absolute configurations as S- and R-, respectively. When tested in vitro, S-OXA demonstrated the bulk of schistosomicidal activity, while R-OXA had antischistosomal effects when present at relatively high concentrations. Crystal structures R-OXA•SmSULT and S-OXA•SmSULT complexes reveal similarities in the modes of OXA binding, but only the S-OXA enantiomer is observed in the structure of the enzyme exposed to racemic OXA.

Conclusions/Significance

Together the data suggest the higher schistosomicidal activity of S-OXA is correlated with its ability to outcompete R-OXA binding the sulfotransferase active site. These findings have important implications for the design, syntheses, and dosing of new OXA-based antischistosomal compounds.     

Multimode sensors as new tools for molecular recognition of testosterone,dihydrotestosterone and estradiol in children's saliva

Increased levels of testosterone (T₂), dihydrotestosterone (DHT) and estradiol (E₂) in children may be responsible for their early/delayed puberty and obesity conditions. Therefore, multimode sensors based on carbon matrices, such as graphite, graphene, fullerene C₆₀ and multiwall carbon nanotubes modified with maltodextrin, were designed to assess reliably T₂, DHT and E₂ in children saliva. The modes used for the assay of hormones were stochastic mode (for qualitative and quantitative determination of hormones) and differential pulse voltammetry mode (for quantitative determination of hormones). The advantage of this type of sensors, for hormone analysis, is their possibility to reach low concentration levels— are placed for children saliva under the detection limit of standard methods (e.g. ELISA used for the determination of these hormones in saliva). This made the multimode sensors an excellent tool for clinical analysis and especially for determination of substances of clinical importance in saliva samples. The proposed method is fast and simple, and no sampling of saliva is required. Copyright © 2014 John Wiley & Sons, Ltd.