SRY sequence in maternal plasma: Implications for non-invasive prenatal diagnosis: First report from India

AIM:

The presence of circulatory cell-free fetal DNA in maternal plasma has found new applications in non-invasive risk-free prenatal diagnosis.

MATERIALS AND METHODS:

We made use of a size separation approach along with real time polymerase chain reaction (PCR) to evaluate the use of fetal DNA in the detection of the sex of the fetus. Cell-free fetal DNA was isolated from the plasma of 30 women (10–20 weeks gestation) using a size separation approach. We made use of Taq Man Chemistry and real time PCR using primers and probes for GAPDH and SRY.

RESULTS:

Only 24 cases could be studied as there was no amplification in six cases. Fetal sex was accurately determined in all of the 24 cases wherein 19 women were carrying male fetuses and five women were carrying female fetuses. An increase in the amount of fetal DNA was observed with an increase in the gestational age.

CONCLUSIONS:

Real time PCR analysis is a highly sensitive and accurate tool for non-invasive prenatal diagnosis, allowing detection of the sex of the fetus as early as 10 weeks of gestation. Non-invasive prenatal diagnosis eliminates the risk of fetal loss associated with the invasive procedure.     

The Amino Terminus of Herpes Simplex Virus Type 1 Glycoprotein K (gK) Modulates gB-Mediated Virus-Induced Cell Fusion and Virion Egress

Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gKΔ31-47, gKΔ31-68, and gKΔ31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gKΔ31-47 and gKΔ31-117 exhibited a gK-null (ΔgK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gKΔ31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gKΔ31-47 and gKΔ31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gBΔ28syn) caused extensive virus-induced cell fusion. However, the gBΔ28syn mutation was unable to cause virus-induced cell fusion in the presence of the gKΔ31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gKΔ31-68, ΔgK, or UL20-null virus. The gKa peptide complemented the gKΔ31-47 and gKΔ31-68 mutant viruses for infectious-virus production and for gKΔ31-68/gBΔ28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5, 42, 44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7, 15, 35, 53, 54, 57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown that gD interacts with both gB and gH/gL (1, 2). Binding of gD to its cognate receptors, including Nectin-1, HVEM, and others (12, 29, 48, 59, 60, 62, 63), is thought to trigger conformation changes in gH/gL and gB that cause fusion of the viral envelope with cellular membranes during virus entry and virus-induced cell fusion (32, 34). Transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (49, 68). However, this phenomenon does not accurately model viral fusion, because other viral glycoproteins and membrane proteins known to be important for virus-induced cell fusion are not required (6, 14, 31). Specifically, gK and UL20 were shown to be absolutely required for virus-induced cell fusion (21, 46). Moreover, syncytial mutations within gK (7, 15, 35, 53, 54, 57) or UL20 (5, 42, 44) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than wild-type virus into susceptible cells (25). Furthermore, transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while coexpression of the wild-type gK with gB, gD, and gH/gL inhibited cell fusion (3).Glycoproteins gB and gH are highly conserved across all subfamilies of herpesviruses. gB forms a homotrimeric type I integral membrane protein, which is N glycosylated at multiple sites within the polypeptide. An unusual feature of gB is that syncytial mutations that enhance virus-induced cell fusion are located exclusively in the carboxyl terminus of gB, which is predicted to be located intracellularly (51). Single-amino-acid substitutions within two regions of the intracellular cytoplasmic domain of gB were shown to cause syncytium formation and were designated region I (amino acid [aa] positions 816 and 817) and region II (aa positions 853, 854, and 857) (9, 10, 28, 69). Furthermore, deletion of 28 aa from the carboxyl terminus of gB, disrupting the small predicted alpha-helical domain H17b, causes extensive virus-induced cell fusion as well as extensive glycoprotein-mediated cell fusion in the gB, gD, and gH/gL transient-coexpression system (22, 49, 68). The X-ray structure of the ectodomain of gB has been determined and is predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB. Therefore, perturbation of the carboxyl terminus of gB may alter the conformation of the amino terminus of gB, thus favoring one of the two predicted conformational structures that causes membrane fusion (34).The UL53 (gK) and UL20 genes encode multipass transmembrane proteins of 338 and 222 aa, respectively, which are conserved in all alphaherpesviruses (15, 42, 55). Both proteins have multiple sites where posttranslational modification can occur; however, only gK is posttranslationally modified by N-linked carbohydrate addition (15, 35, 55). The specific membrane topologies of both gK and UL20 protein (UL20p) have been predicted and experimentally confirmed using epitope tags inserted within predicted intracellular and extracellular domains (18, 21, 44). Syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (18), while syncytial mutations of UL20 are located within the amino terminus of UL20p, shown to be located intracellularly (44). A series of recent studies have shown that HSV-1 gK and UL20 functionally and physically interact and that these interactions are necessary for their coordinate intracellular transport and cell surface expression (16, 18, 21, 26, 45). Specifically, direct protein-protein interactions between the amino terminus of HSV-1 UL20 and gK domain III, both of which are localized intracellularly, were recently demonstrated by two-way coimmunoprecipitation experiments (19).According to the most prevalent model for herpesvirus intracellular morphogenesis, capsids initially assemble within the nuclei and acquire a primary envelope by budding into the perinuclear spaces. Subsequently, these virions lose their envelope through fusion with the outer nuclear lamellae. Within the cytoplasm, tegument proteins associate with the viral nucleocapsid and final envelopment occurs by budding of cytoplasmic capsids into specific trans-Golgi network (TGN)-associated membranes (8, 30, 47, 70). Mature virions traffic to cell surfaces, presumably following the cellular secretory pathway (33, 47, 61). In addition to their significant roles in virus-induced cell fusion, gK and UL20 are required for cytoplasmic virion envelopment. Viruses with deletions in either the gK or the UL20 gene are unable to translocate from the cytoplasm to extracellular spaces and accumulated as unenveloped virions in the cytoplasm (5, 15, 20, 21, 26, 35, 36, 38, 44, 55). Current evidence suggests that the functions of gK and UL20 in cytoplasmic virion envelopment and virus-induced cell fusion are carried out by different, genetically separable domains of UL20p. Specifically, UL20 mutations within the amino and carboxyl termini of UL20p allowed cotransport of gK and UL20p to cell surfaces, virus-induced cell fusion, and TGN localization, while effectively inhibiting cytoplasmic virion envelopment (44, 45).In this paper, we demonstrate that the amino terminus of gK expressed as a free peptide of 82 aa (gKa) is transported to infected cell surfaces by viral proteins other than gK or UL20p and facilitates virus-induced cell fusion caused by syncytial mutations in the carboxyl terminus of gB. Thus, functional domains of gK can be genetically separated, as we have shown previously (44, 45), as well as physically separated into different peptide portions that retain functional activities of gK. These results are consistent with the hypothesis that the amino terminus of gK directly or indirectly interacts with and modulates the fusogenic properties of gB.     

Effect of chronic nNOS inhibition on blood pressure, vasoactivity, and arterial wall structure in Wistar rats

While the unequivocal pattern of endothelial nitric oxide (NO) synthase (eNOS) inhibition in cardiovascular control has been recognised, the role of NO produced by neuronal NOS (nNOS) remains unclear. The purpose of the present study was to describe the cardiovascular effects of NO production interference by inhibition of nNOS with 7-nitroindazole (7-NI). Wistar rats (10 weeks old) were used: control and experimental rats were administered 7-NI 10 mg/kg b.w./day in drinking water for 6 weeks. Systolic blood pressure (BP) was measured by the tail-cuff plethysmographic method. Isolated thoracic aortas (TAs) were used to study vasomotor activity of the conduit artery in vitro. The BP response of anaesthetised animals was used to follow the cardiovascular-integrated response in vivo. Geometry of the TA was measured after perfusion fixation (120 mm Hg) by light microscopy. Expression of eNOS was measured in the TA by immunoblot analysis. Although 6 weeks of nNOS inhibition did not alter systolic BP, the heart/body weight ratio was decreased. Relaxation of the TA in response to acetylcholine (10⁻⁹–10⁻⁵ mol/L) was moderately inhibited. However, no difference in the BP hypotensive response after acetylcholine (0.1, 1, 10 μg) was observed. The contraction of TA in response to noradrenaline (10⁻¹⁰–10⁻⁵ mol/L), and the BP pressor response to noradrenaline (0.1, 1 μg) was attenuated. The inner diameter of the TA was increased, and the wall thickness, wall cross-sectional area, and wall thickness/inner diameter ratio were decreased. The expression of eNOS in the TA was increased. In summary, cardiac and TA wall hypotrophy, underlined by decreased contractile efficiency, were observed. The results suggested that two constitutive forms of NOS (nNOS, eNOS) likely participate in regulation of cardiovascular tone by different mechanisms.     

CDD: a curated Entrez database of conserved domain alignments

The Conserved Domain Database (CDD) is now indexed as a separate database within the Entrez system and linked to other Entrez databases such as MEDLINE(R). This allows users to search for domain types by name, for example, or to view the domain architecture of any protein in Entrez's sequence database. CDD can be accessed on the WorldWideWeb at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=cdd. Users may also employ the CD-Search service to identify conserved domains in new sequences, at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. CD-Search results, and pre-computed links from Entrez's protein database, are calculated using the RPS-BLAST algorithm and Position Specific Score Matrices (PSSMs) derived from CDD alignments. CD-Searches are also run by default for protein-protein queries submitted to BLAST(R) at http://www.ncbi.nlm.nih.gov/BLAST. CDD mirrors the publicly available domain alignment collections SMART and PFAM, and now also contains alignment models curated at NCBI. Structure information is used to identify the core substructure likely to be present in all family members, and to produce sequence alignments consistent with structure conservation. This alignment model allows NCBI curators to annotate 'columns' corresponding to functional sites conserved among family members.     

Induced cell death of preimplantation mouse embryos cultured in vitro evaluated by comet assay

The occurrence of apoptosis in mouse preimplantation embryos was analyzed using DNA staining (Hoechst 33342, PI) for the visualization of nuclear changes and by the comet assay, a single-cell gel electrophoresis assay, modified for the analysis of blastocysts. Mouse preimplantation embryos isolated 56 h after superovulation were cultured in vitro for 64 h. Apoptosis was induced by treatment with camptothecin and actinomycin D during the first 15 h of culture. After culture in vitro, a number of embryos were stained and analyzed using morphological criteria. The remaining embryos were examined using the comet assay for the detection of DNA fragmentation. The proportion of damaged embryos in experimental groups, in comparison to controls, was dependent on the dose of apoptosis inductor. At high doses (camptothecin, microg/ml and actinomycin D, 0.05 microg/ml) over 90% (chi-square test, P<0.001) of embryos had apoptotic comets, at medium doses (camptothecin, 0.01 microg/ml and actinomycin D, 0.005 microg/ml) comets appeared only in 30-70% of embryos (camptothecin, P<0.01 and actinomycin D, P<0.001). At low doses (camptothecin, 0.001 microg/ml and actinomycin D, 0.0005 microg/ml) the increase in damaged embryos was not statistically significant. Hoechst/PI staining showed a higher percentage of damaged blastomeres at high doses. Morphological changes correlated with the outcome of the comet assay. Our results show that comet assay is an appropriate method for studying apoptosis in preimplantation embryos, and it appears to be more sensitive than the classically used morphological analyses.     

Effect of thyroid hormones on the gene expression of calcium transport systems in rat muscles

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.     

DNA lesions and cytogenetic changes induced by N-nitrosomorpholine in HepG2, V79 and VH10 cells: the protective effects of Vitamins A, C and E

INTRODUCTION: N-Nitrosomorpholine (NMOR), present in the workplace of tyre chemical factories, is a known hepatocarcinogen. This compound belongs to the group of N-nitrosamines, which are indirect-acting and require metabolic activation. However, the mechanism of its carcinogenic effect is not completely clear. AIMS: The objective of this study was (i) to compare the DNA-damaging and clastogenic effects of NMOR in three cell lines (HepG2, V79 and VH10) with different levels of metabolizing enzymes and (ii) to determine the protective effects of Vitamins A, C and E against deleterious effects of NMOR. METHODS: The exponentially growing cells were pre-treated with Vitamins A, C and E and treated with NMOR. Genotoxic effects of NMOR were evaluated by single-cell gel electrophoresis (SCGE, comet assay), while the chromosomal aberration assay was used for the study of clastogenic effects. KEY RESULTS: NMOR-induced a significant dose-dependent increase of DNA damage as analyzed by SCGE, but the extent of DNA migration in the electric field was unequal in the different cell lines. Although the results obtained by SCGE confirmed the genotoxicity of NMOR in all cell lines studied, the number of chromosomal aberrations was significantly increased only in HepG2 and V79 cells, while no changes were observed in VH10 cells. In HepG2 cells pre-treated with Vitamins A, C and E we found a significant decrease of the percentage of tail DNA induced by NMOR. The reduction of the clastogenic effects of NMOR was observed only after pretreatment with Vitamins A and E; Vitamin C did not alter the frequency of NMOR-induced chromosomal aberrations under the experimental conditions of this study. CONCLUSIONS: The fat-soluble Vitamins A and E, which are dietary constituents, reduce the harmful effects of N-nitrosomorpholine in human hepatoma cells HepG2, which are endowed with the maximal capacity for metabolic activation of several drugs.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

Evolution and Divergence of the Genes for Cytoplasmic,Mitochondrial, and Flagellar Creatine Kinases

Creatine kinase (CK) plays a central role in energy homeostasis in cells that display high and variable rates of energy turnover. A number of CK genes exist, each being targeted to particular intracellular compartments. In the vertebrates, two genes code for proteins which form homo- and heterodimers targeted to the cytoplasm, while two additional genes code for primarily octameric proteins targeted to the mitochondrial intermembrane space. Yet another gene is present in certain groups which codes for three fused, complete CK domains and is typically targeted to the flagellar membrane of primitive-type spermatozoa. CK is widely distributed in protochordates and both protostome and deuterostome invertebrate groups. The evolutionary relationships of these CK genes have not been fully elucidated. The present communication reports new cDNA-derived deduced amino acid sequences for four cytoplasmic and three mitochondrial CKs and one flagellar CK from lophotrochozoan, protostome invertebrates as well as a new cytoplasmic CK sequence from a protochordate tunicate. These new sequences, coupled with available sequences in the databases and sequences extracted from genome sequencing projects, provide revealing insights into the evolution and divergence of CK genes. Phylogenetic analyses showed that single cytoplasmic, mitochondrial, and flagellar CK genes were present prior to the divergence of the protostomes and deuterostomes. The flagellar CK gene may have evolved within the cytoplasmic gene clade, although the evidence is somewhat equivocal. The two cytoplasmic genes in the vertebrates, and most likely the two mitochondrial genes, evolved after the divergence of the craniates from the protochordates. Comparison of the structure of the genes for selected cytoplasmic, mitochondrial, and flagellar CKs revealed two identical intron boundaries, further reinforcing the notion of a common evolutionary origin, but also showed patterns of changes in structure consistent with each gene type. These studies show that the cytoplasmic, mitochondrial, and flagellar CK genes are rather ancient and that there has been a systematic pattern of duplication and divergence consistent with changing nature of energy demands and physicochemical environment in the cells where they are expressed.[Reviewing Editor: Martin Kreitman]