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Twelve-month treatment of heroin addicts with methadone or buprenorphine normalized plasma cortisol levels, and controlled withdrawal symptoms as well as craving. During treatment, the time course of plasma cortisol levels and craving was not strictly correlated: heroin craving was more elevated at 12 than at 3 months. The results suggest a correlation between hypercortisolism, withdrawal symptoms and heroin use and suppose a more complex role for craving and its components in drug-taking behaviour. The main goal of the pharmacological treatment of opioid-dependence should be addressed at the normalization of hypothalamic-pituitary-adrenocortical (HPA) axis more than at the control of craving. [ABSTRACT FROM AUTHOR]

Abstract Background Transcriptionally quiescent spermatozoa have been established to be a repository of mRNA coding for several functionally essential cellular proteins. This entourage of mRNA is envisaged to be involved in post-fertilization and early embryogenesis. Minisatellites tagged with mRNA transcripts have been implicated with gene organization, regulation and function. However, the organization and expression of the minisatellite tagged transcript diversity, particularly in spermatozoa, remains unclear. Results In the present study, we identified and characterized 12 mRNA transcripts from the spermatozoa of water buffalo Bubalus bubalis employing minisatellite associated sequence amplification (MASA) and a consensus sequence of 33.15 repeat loci. Of these 33.15 tagged transcripts, only one was found to be homologous to Bovine steroid 21-hydroxylase (P-450-c21) gene. Other ten transcripts showed significant similarity with various mRNAs or chromosomal contigs across the species. The remaining one construed to be novel since this was unreported in the database (NCBI GenBank). All these uncharacterized and known transcripts showed highest expression in testis and spermatozoa compared to that in somatic tissues and ovary. Of these 12 mRNA transcripts, 4 showed differential expression in the forebrain and hindbrain of buffalo. Moreover, genes corresponding to all the 33.15 tagged spermatozoal transcripts were found to be conserved across 13 other species analyzed. Conclusion Our results show MASA as an important tool to capture mRNA transcript diversity tagged with minisatellites in the spermatozoa. Comprehensive characterization of these transcripts is envisaged to augment our understanding on the genes involved in testicular functions and sustenance of a viable paternal genome during pre- and post- fertilization events and early stages of development. Prospects of this approach in genome analysis in general and comparative genomics in particular are highlighted.

Abstract Background Simple sequence repeats (SSRs) of GACA/GATA have been implicated with differentiation of sex-chromosomes and speciation. However, the organization of these repeats within genomes and transcriptomes, even in the best characterized organisms including human, remains unclear. The main objective of this study was to explore the buffalo transcriptome for its association with GACA/GATA repeats, and study the structural organization and differential expression of the GACA/GATA repeat tagged transcripts. Moreover, the distribution of GACA and GATA repeats in the prokaryotic and eukaryotic genomes was studied to highlight their significance in genome evolution. Results We explored several genomes and transcriptomes, and observed total absence of these repeats in the prokaryotes, with their gradual accumulation in higher eukaryotes. Further, employing novel microsatellite associated sequence amplification (MASA) approach using varying length oligos based on GACA and GATA repeats; we identified and characterized 44 types of known and novel mRNA transcripts tagged with these repeats from different somatic tissues, gonads and spermatozoa of water buffalo Bubalus bubalis. GACA was found to be associated with higher number of transcripts compared to that with GATA. Exclusive presence of several GACA-tagged transcripts in a tissue or spermatozoa, and absence of the GATA-tagged ones in lung/heart highlights their tissue-specific significance. Of all the GACA/GATA tagged transcripts, ~30% demonstrated inter-tissue and/or tissue-spermatozoal sequence polymorphisms. Significantly, ~60% of the GACA-tagged and all the GATA-tagged transcripts showed highest or unique expression in the testis and/or spermatozoa. Moreover, ~75% GACA-tagged and all the GATA-tagged transcripts were found to be conserved across the species. Conclusion Present study is a pioneer attempt exploring GACA/GATA tagged transcriptome in any mammalian species highlighting their tissue, stage and species-specific expression profiles. Comparative analysis suggests the gradual accumulation of these repeats in the higher eukaryotes, and establishes the GACA richness of the buffalo transcriptome. This is envisaged to establish the roles of integral simple sequence repeats and tagged transcripts in gene expression or regulation.

Abstract Background Secreted modular calcium binding protein-1 (Smoc-1) belongs to the BM-40 family which has been implicated with tissue remodeling, angiogenesis and bone mineralization. Besides its anticipated role in embryogenesis, Smoc-1 has been characterized only in a few mammalian species. We made use of the consensus sequence (5' CACCTCTCCACCTGCC 3') of 33.15 repeat loci to explore the buffalo transcriptome and uncovered the Smoc-1 transcript tagged with this repeat. The main objective of this study was to gain an insight into its structural and functional organization, and expressional status of Smoc-1 in water buffalo, Bubalus bubalis. Results We cloned and characterized the buffalo Smoc-1, including its copy number status, in-vitro protein expression, tissue & age specific transcription/translation, chromosomal mapping and localization to the basement membrane zone. Buffalo Smoc-1 was found to encode a secreted matricellular glycoprotein containing two EF-hand calcium binding motifs homologous to that of BM-40/SPARC family. In buffalo, this single copy gene consisted of 12 exons and was mapped onto the acrocentric chromosome 11. Though this gene was found to be evolutionarily conserved, the buffalo Smoc-1 showed conspicuous nucleotide/amino acid changes altering its secondary structure compared to that in other mammals. In silico analysis of the Smoc-1 proposed its glycoprotein nature with a calcium dependent conformation. Further, we unveiled two transcript variants of this gene, varying in their 3'UTR lengths but both coding for identical protein(s). Smoc-1 evinced highest expression of both the variants in liver and modest to negligible in other tissues. The relative expression of variant-02 was markedly higher compared to that of variant-01 in all the tissues examined. Moreover, expression of Smoc-1, though modest during the early ages, was conspicuously enhanced after 1 year and remained consistently higher during the entire life span of buffalo with gradual increment in expression of variant-02. Immunohistochemically, Smoc-1 was localized in the basement membrane zones and extracellular matrices of various tissues. Conclusion These data added to our understandings about the tissue, age and species specific functions of the Smoc-1. It also enabled us to demonstrate varying expression of the two transcript variants of Smoc-1 amongst different somatic tissues/gonads and ages, in spite of their identical coding frames. Pursuance of these variants for their roles in various disease phenotypes such as hepatocellular carcinoma and angiogenesis is envisaged to establish broader biological significance of this gene.

In DNA, electron excitation allows adjacent pyrimidine bases to dimerize by [2 + 2] cycloaddition, creating chemically stable but lethal and mutagenic cyclobutane pyrimidine dimers (CPDs). The usual cause is ultraviolet radiation. Alternatively, CPDs can be made in the dark (dCPDs) via chemically mediated electron excitation of the skin pigment melanin, after it is oxidized by peroxynitrite formed from the stress-induced radicals superoxide and nitric oxide. We now show that the dark process is not limited to the unusual structural molecule melanin: signaling biomolecules such as indolamine and catecholamine neurotransmitters and hormones can also be chemiexcited to energy levels high enough to form dCPDs. Oxidation of serotonin, dopamine, melatonin, and related biogenic amines by peroxynitrite created triplet-excited species, evidenced by chemiluminescence, energy transfer to a triplet-state reporter, or transfer to O 2 resulting in singlet molecular oxygen. For a subset of these signaling molecules, triplet states created by peroxynitrite or peroxidase generated dCPDs at levels comparable to ultraviolet (UV). Neurotransmitter catabolism by monoamine oxidase also generated dCPDs. These results reveal a large class of signaling molecules as electronically excitable by biochemical reactions and thus potential players in deviant mammalian metabolism in the absence of light.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO - ), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO - scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa , Xpc , and Mitf . AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Some early reports demonstrate that levels of cyclobutane pyrimidine dimers (CPD) may increase after UVR exposure had ended, although these observations were treated as artifacts. More recently, it has been shown unequivocally that CPD formation does occur post-irradiation, with maximal levels occurring after about 2-3 h. These lesions have been termed "dark CPD" (dCPD). Subsequent studies have confirmed their presence in vitro, in mouse models and in human skin in vivo. Melanin carbonyls have a role in the formation of dCPD, but they have also been observed in amelanotic systems, indicating other, unknown process(es) exist. In both cases, the formation of dCPD can be prevented by the presence of certain antioxidants. We lack data on the spectral dependence of dCPD, but it is unlikely to be the same as for incident CPD (iCPD), which are formed only during irradiation. There is evidence that iCPD and dCPD may have different repair kinetics, although this remains to be elucidated. It is also unknown whether iCPD and dCPD have different biological properties. The formation of dCPD in human skin in vivo has implications for post solar exposure photoprotection, and skin carcinogenesis, with a need for this to be investigated further., (© 2021 American Society for Photobiology.)

UV-like DNA damage is created in the dark by chemiexcitation, in which UV-activated enzymes generate reactive oxygen and nitrogen species that create a dioxetane on melanin. Thermal cleavage creates an electronically excited triplet-state carbonyl whose high energy transfers to DNA. Screening natural compounds for the ability to quench this energy identified polyenes, polyphenols, mycosporine-like amino acids, and related compounds better known as antioxidants. To eliminate false positives such as ROS and RNS scavengers, we then used the generator of triplet-state acetone, tetramethyl-1,2-dioxetane (TMD), to excite the triplet-energy reporter 9,10-dibromoanthracene-2-sulfonate (DBAS). Quenching measured as reduction in DBAS luminescence revealed three clusters of 50% inhibitory concentration, ~50 μM, 200-500 μM, and >600 μM, with the former including sorbate, ferulic acid, and resveratrol. Representative triplet-state quenchers prevented chemiexcitation-induced "dark" cyclobutane pyrimidine dimers (dCPD) in DNA and in UVA-irradiated melanocytes. We conclude that (i) the delocalized pi electron cloud that stabilizes the electron-donating activity of many common antioxidants allows the same molecule to prevent an electronically excited species from transferring its triplet-state energy to targets such as DNA and (ii) the most effective class of triplet-state quenchers appear to operate by energy diversion instead of electron donation and dissipate that energy by isomerization.

Exposures from the external and internal environments lead to the modification of genomic DNA, which is implicated in the cause of numerous diseases, including cancer, cardiovascular, pulmonary and neurodegenerative diseases, together with ageing. However, the precise mechanism(s) linking the presence of damage, to impact upon cellular function and pathogenesis, is far from clear. Genomic location of specific forms of damage is likely to be highly informative in understanding this process, as the impact of downstream events (e.g. mutation, microsatellite instability, altered methylation and gene expression) on cellular function will be positional-events at key locations will have the greatest impact. However, until recently, methods for assessing DNA damage determined the totality of damage in the genomic location, with no positional information. The technique of "mapping DNA adductomics" describes the molecular approaches that map a variety of forms of DNA damage, to specific locations across the nuclear and mitochondrial genomes. We propose that integrated comparison of this information with other genome-wide data, such as mutational hotspots for specific genotoxins, tumour-specific mutation patterns and chromatin organisation and transcriptional activity in non-cancerous lesions (such as nevi), pre-cancerous conditions (such as polyps) and tumours, will improve our understanding of how environmental toxins lead to cancer. Adopting an analogous approach for non-cancer diseases, including the development of genome-wide assays for other cellular outcomes of DNA damage, will improve our understanding of the role of DNA damage in pathogenesis more generally., (© 2021. The Author(s).)

Melanoma is the deadliest type of skin cancer. Human melanomas often show hyperactivity of nitric oxide synthase (NOS) and NADPH oxidase (NOX), which, respectively, generate nitric oxide (NO · ) and superoxide (O 2 ·- ). The NO · and O 2 ) which is the driver of melanin chemiexcitation. Melanoma precursors, the melanocytes, are specialized skin cells that synthesize melanin, a potent shield against sunlight's ultraviolet (UV) radiation. However, melanin chemiexcitation paradoxically demonstrates the melanomagenic properties of melanin. In a loop, the NOS activity regulates melanin synthesis, and melanin is utilized by the chemiexcitation pathway to generate carcinogenic melanin-carbonyls in an excited triplet state. These carbonyl compounds induce UV-specific DNA damage without UV. Additionally, the carbonyl compounds are highly reactive and can make melanomagenic adducts with proteins, DNA and other biomolecules. Here we review the role of the melanin chemiexcitation pathway in melanoma initiation, progression, and drug resistance. We conclude by hypothesizing a non-classical, positive loop in melanoma where melanin chemiexcitation generates carcinogenic reactive carbonyl species (RCS) and DNA damage in normal melanocytes. In parallel, NOS and NOX regulate melanin synthesis generating raw material for chemiexcitation, and the resulting RCS and reactive nitrogen species (RNS) regulate cellular proteome and transcriptome in favor of melanoma progression, metastasis, and resistance against targeted therapies.- react instantly with each other to generate peroxynitrite (ONOO - ) which is the driver of melanin chemiexcitation. Melanoma precursors, the melanocytes, are specialized skin cells that synthesize melanin, a potent shield against sunlight's ultraviolet (UV) radiation. However, melanin chemiexcitation paradoxically demonstrates the melanomagenic properties of melanin. In a loop, the NOS activity regulates melanin synthesis, and melanin is utilized by the chemiexcitation pathway to generate carcinogenic melanin-carbonyls in an excited triplet state. These carbonyl compounds induce UV-specific DNA damage without UV. Additionally, the carbonyl compounds are highly reactive and can make melanomagenic adducts with proteins, DNA and other biomolecules. Here we review the role of the melanin chemiexcitation pathway in melanoma initiation, progression, and drug resistance. We conclude by hypothesizing a non-classical, positive loop in melanoma where melanin chemiexcitation generates carcinogenic reactive carbonyl species (RCS) and DNA damage in normal melanocytes. In parallel, NOS and NOX regulate melanin synthesis generating raw material for chemiexcitation, and the resulting RCS and reactive nitrogen species (RNS) regulate cellular proteome and transcriptome in favor of melanoma progression, metastasis, and resistance against targeted therapies., (Copyright © 2020 Premi.)

If the genome contains outlier sequences extraordinarily sensitive to environmental agents, these would be sentinels for monitoring personal carcinogen exposure and might drive direct changes in cell physiology rather than acting through rare mutations. New methods, adductSeq and freqSeq, provided statistical resolution to quantify rare lesions at single-base resolution across the genome. Primary human melanocytes, but not fibroblasts, carried spontaneous apurinic sites and TG sequence lesions more frequent than ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs). UV exposure revealed hyperhotspots acquiring CPDs up to 170-fold more frequently than the genomic average; these sites were more prevalent in melanocytes. Hyperhotspots were disproportionately located near genes, particularly for RNA-binding proteins, with the most-recurrent hyperhotspots at a fixed position within 2 motifs. One motif occurs at ETS family transcription factor binding sites, known to be UV targets and now shown to be among the most sensitive in the genome, and at sites of mTOR/5' terminal oligopyrimidine-tract translation regulation. The second occurs at A 2-15 TTCTY, which developed "dark CPDs" long after UV exposure, repaired CPDs slowly, and had accumulated CPDs prior to the experiment. Motif locations active as hyperhotspots differed between cell types. Melanocyte CPD hyperhotspots aligned precisely with recurrent UV signature mutations in individual gene promoters of melanomas and with known cancer drivers. At sunburn levels of UV exposure, every cell would have a hyperhotspot CPD in each of the ∼20 targeted cell pathways, letting hyperhotspots act as epigenetic marks that create phenome instability; high prevalence favors cooccurring mutations, which would allow tumor evolution to use weak drivers., Competing Interests: The authors declare no competing interest.

The function of α-synuclein (α-syn) has been long debated, and two seemingly divergent views have emerged. In one, α-syn binds to VAMP2, acting as a SNARE chaperone-but with no effect on neurotransmission-while another posits that α-syn attenuates neurotransmitter release by restricting synaptic vesicle mobilization and recycling. Here, we show that α-syn-VAMP2 interactions are necessary for α-syn-induced synaptic attenuation. Our data connect divergent views and suggest a unified model of α-syn function., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Sunlight's ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO) and superoxide (O2(-)); these combine to form the powerful oxidant peroxynitrite (ONOO(-)). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed "chemiexcitation", is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ultraviolet radiation (UVR) instantaneously generates cyclobutane pyrimidine dimers (CPDs). Paradoxically, we recently observed that UV enables the protective pigment melanin to create CPDs in the dark long after the exposure ends. UV-induced reactive oxygen species (ROS) oxidize melanin to create melanin carbonyls in a high-energy quantum state. These energetic melanin carbonyls transfer their energy to DNA in the dark, creating CPDs in the absence of UVR.

Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts that are typically created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. We found that in melanocytes, CPDs are generated for >3 hours after exposure to UVA, a major component of the radiation in sunlight and in tanning beds. These "dark CPDs" constitute the majority of CPDs and include the cytosine-containing CPDs that initiate UV-signature C→T mutations. Dark CPDs arise when UV-induced reactive oxygen and nitrogen species combine to excite an electron in fragments of the pigment melanin. This creates a quantum triplet state that has the energy of a UV photon but induces CPDs by energy transfer to DNA in a radiation-independent manner. Melanin may thus be carcinogenic as well as protective against cancer. These findings also validate the long-standing suggestion that chemically generated excited electronic states are relevant to mammalian biology., (Copyright © 2015, American Association for the Advancement of Science.)

Despite recent advancements in therapy, melanoma remains a highly lethal skin cancer. A better understanding of the genetic and epigenetic changes responsible for melanoma formation and progression could result in the development of more effective treatments. Advanced melanomas are known to exhibit widespread promoter region CpG island methylation leading to the inactivation of key tumor suppressor genes. Meta-analyses of relevant microarray data sets revealed the hematopoietic stem cell regulator gene latexin (LXN) to be commonly downregulated in approximately 50% of melanomas. The CpG island in the promoter region of LXN was almost universally hypermethylated in melanoma cell lines and tumors, and treatment of the cell lines with the demethylating drug 5-aza-2'-deoxycytidine resulted in increased LXN expression. In this paper, we demonstrate that the exogenous expression of LXN in melanoma cell lines results in a significant inhibition of tumor cell proliferation. In addition, we show that the increased expression of LXN in these lines correlates with reduction in the expression levels of stem cell transcription factors OCT4, NANOG, SOX2, KLF4, and MYCN, indicating that LXN may exert its tumor-suppressive function by altering the stem cell-like properties of melanoma cells.

Owing to clonal inheritance, haploid status and lack of recombination, structural polymorphism in the human Y chromosome is more prevalent than that in the remaining parts of the genome. We studied structural organization of the AZFc region, assessed microdeletions therein and studied copy number variation (CNV) of several candidate genes in 750 Indian males. FISH mapping of 13 Y-specific BAC/cosmid clones uncovered a hitherto unreported AZFc configuration showing inter-DAZ gene sequence onto the Yp instead of Yq region. Such inter-DAZ gene arrangements were also detected in five German males (European Y). In 40-50% males, partial u3 and one of the green amplicons, g1, g2 or g3 was present on the Yp in addition to Yq, suggesting an alteration in the IR3 region. Among other AZFc candidates, complete TTY3 and partial CDY1 BAC sequences were detected on the proximal 5p and distal 15q regions, respectively, in both the sexes. However, primers deduced from these clones showed male specific amplification of TTY3 and CDY1 exons suggesting (re)organization of their flanking sequences between Y and autosomes. Importantly, approximately 5% males showed CNV of various Y-linked genes, and approximately 3%, random microdeletions across the AZF region. Present study demonstrates hitherto unreported singular structural organization with respect to DAZ, TTY3 and CDY1 genes highlighting organizational complexities of the human Y chromosome in the global context.

Background: The most frequently observed major consequences of ionizing radiation are chromosomal lesions and cancers, although the entire genome may be affected. Owing to its haploid status and absence of recombination, the human Y chromosome is an ideal candidate to be assessed for possible genetic alterations induced by ionizing radiation. We studied the human Y chromosome in 390 males from the South Indian state of Kerala, where the level of natural background radiation (NBR) is ten-fold higher than the worldwide average, and that from 790 unexposed males as control., Results: We observed random microdeletions in the Azoospermia factor (AZF) a, b and c regions in >90%, and tandem duplication and copy number polymorphism (CNP) of 11 different Y-linked genes in about 80% of males exposed to NBR. The autosomal homologues of Y-linked CDY genes largely remained unaffected. Multiple polymorphic copies of the Y-linked genes showing single Y-specific signals suggested their tandem duplication. Some exposed males showed unilocus duplication of DAZ genes resulting in six copies. Notably, in the AZFa region, approximately 25% of exposed males showed deletion of the DBY gene, whereas flanking genes USP9Y and UTY remained unaffected. All these alterations were detected in blood samples but not in the germline (sperm) samples., Conclusions: Exposure to high levels of NBR correlated with several interstitial polymorphisms of the human Y chromosome. CNPs and enhanced transcription of the SRY gene after duplication are envisaged to compensate for the loss of Y chromosome in some cells. The aforesaid changes, confined to peripheral blood lymphocytes, suggest a possible innate mechanism protecting the germline DNA from the NBR. Genome analysis of a larger population focusing on greater numbers of genes may provide new insights into the mechanisms and risks of the resultant genetic damages. The present work demonstrates unique signatures of NBR on human Y chromosomes from Kerala, India.

Presence of the human Y-chromosome in females with Turner Syndrome (TS) enhances the risk of development of gonadoblastoma besides causing several other phenotypic abnormalities. In the present study, we have analyzed the Y chromosome in 15 clinically diagnosed Turner Syndrome (TS) patients and detected high level of mosaicisms ranging from 45,XO:46,XY = 100:0% in 4; 45,XO:46,XY:46XX = 4:94:2 in 8; and 45,XO:46,XY:46XX = 50:30:20 cells in 3 TS patients, unlike previous reports showing 5-8% cells with Y- material. Also, no ring, marker or di-centric Y was observed in any of the cases. Of the two TS patients having intact Y chromosome in >85% cells, one was exceptionally tall. Both the patients were positive for SRY, DAZ, CDY1, DBY, UTY and AZFa, b and c specific STSs. Real Time PCR and FISH demonstrated tandem duplication/multiplication of the SRY and DAZ genes. At sequence level, the SRY was normal in 8 TS patients while the remaining 7 showed either absence of this gene or known and novel mutations within and outside of the HMG box. SNV/SFV analysis showed normal four copies of the DAZ genes in these 8 patients. All the TS patients showed aplastic uterus with no ovaries and no symptom of gonadoblastoma. Present study demonstrates new types of polymorphisms indicating that no two TS patients have identical genotype-phenotype. Thus, a comprehensive analysis of more number of samples is warranted to uncover consensus on the loci affected, to be able to use them as potential diagnostic markers.

Ionizing radiations are known to induce tumors, chromosomal lesions and minisatellite length variations, yet no correlation has been demonstrated between radiation exposure and indels or copy number polymorphism (CNP) of the genes. We studied the impact of natural background radiation (NBR) on the human Y chromosome owing to its haploid status and clonal inheritance. We analyzed the AZFc region using the DNA from blood and semen of 100 males living near the coastal peninsula in Kerala (India), exposed to NBR along with other 50 normal fertile males. STS mapping of AZFc region showed random microdeletions without conclusive gr/gr or b1/b3 phenotypes. Using a highly specific novel Taqman assay based on sY587 sequence, we detected four copies of the DAZ genes in normal males and 4-16 in those exposed to NBR. Amongst NBR exposed males with multiples copies of the DAZ genes, 75% showed varying FISH signals for DAZ genes with cosmid 18E8 whereas 30% showed mosaicism in terms of presence/absence of the signals in 6-8% cells and unexpected number of signals in 9-12% interphase nuclei. Startlingly, all germline samples studied were found to be free from AZFc microdeletions and CNP of the DAZ genes. Since the DAZ genes are heavily implicated with the germ cell development, the cells with DAZ deletion/duplication are unlikely to survive. Alternatively, an innate mechanism may be operative to protect the germline from the effects of NBR.

In 42 alcoholic inpatients we performed an open randomized study to compare the effects of diazepam and gamma-hydroxybutyrate (GHB) on the suppression of severe alcohol withdrawal syndrome and hypercortisolism. Both diazepam (.5 mg/kg bodyweight, q.i.d.) and GHB (50 mg/kg bodyweight, q.i.d.) were orally administered for three weeks. During all study period, GHB was more able than diazepam in reducing both withdrawal syndrome and hypercortisolism. These effects were evident during the first week of treatment and persisted throughout the study period. The results confirm a strict correlation between high levels of plasma cortisol and alcohol withdrawal symptoms and they show a slight superiority of GHB over diazepam in the suppression of both ethanol withdrawal and hypercortisolism. Taken together, our data suggest that GHB may act as potent anti-withdrawal agent in severe abstinent alcoholics.

An open randomized study was conducted to compare different treatments of alcoholism on ethanol intake, craving, and on biochemical measures of alcohol consumptions. Eighty-six alcoholics were abstinent for a mean of two weeks prior to random assignment to g-hydroxybutyrate (GHB, 50 mg/kg of body weight t.i.d), naltrexone (NTX, 50 mg/day) or disulfiram (DSF, 200 mg/ day) treatment for 12 months. All treatments were equally effective in reducing alcohol intake and in maintaining abstinence. In all patients, the treatments were able to reduce both craving and the altered biological markers of alcohol abuse. The maximum effects were observed in GHB-treated patients. The results of the present study suggest that GHB might act both as anticraving and cellular protector agent.

Protooncogene c-kit receptor is implicated with spermatogenesis, melanogenesis, and hematopoeisis, and undergoes tissue/stage specific alternate splicing. We have isolated 2973-bp full-length cDNA sequence (CDS) of this gene from testis and other tissues of water buffalo Bubalus bubalis. Upon comparison, the c-kit sequences showed tissue specific nucleotide changes resulting in novel truncated peptides. These peptides lacked intracellular and/or transmembrane domains in all the tissues except testis. Other alternately spliced tissue-specific transcripts were also detected, which are the integral parts of the open reading frame and have been reported in other mammals. Phylogenetic analysis of the sequences revealed unique tyrosine kinase domain in buffalo. Copy number calculation and expressional analysis of c-kit using real-time PCR established its single copy status and highest expression (137-177 folds) in testis compared to that (least) in liver. c-kit expression was detected in semen samples although 10 times lesser compared to that in testis. The highest expression of c-kit in testis and the presence of mRNA transcript in sperms substantiate its predominant role in spermatogenesis. This study establishes unequivocal involvement of an autosomal gene c-kit receptor in testicular function.

Higher eukaryotes contain a wide variety of repetitive DNA, although their functions often remain unknown. We describe cloning, chromosomal localization, copy number assessment, and transcriptional status of 1378- and 673-bp repeat fractions in the buffalo genome. The pDS5, representing the 1378-bp fragment, showed FISH signals in the centromeric region of acrocentric chromosomes only, whereas pDS4, corresponding to 673 bp, detected signals in the centromeric regions of all the chromosomes. Crosshybridization studies of pDS5 and pDS4 with genomic DNA from different sources showed signals only in buffalo, cattle, goat, and sheep. Real-time PCR analysis uncovered 1234 and 3420 copies of pDS5 and pDS4 fragments per the haploid genome, corresponding to 30 and 68 copies per chromosome, respectively. Analysis of cDNA from different tissues of buffalo with Real-time PCR showed maximum expression of pDS5 and pDS4 in the spleen and liver, respectively. Phylogenetic analysis of these sequences showed a close relationship between buffalo and cattle. The prospect of this approach in comparative genomics is highlighted.

Mutations in the SRY gene encompassing the HMG box have been well characterized in gonadal dysgenesis, male infertility and other types of sex chromosome related anomalies (SCRA). However, no information is available on copy number status of this gene under such abnormal conditions. Employing 'Taqman Probe Assay' specific to the SRY gene, we screened 16 DNA samples from patients with SCRA and 36 samples from males exposed to high levels of natural background radiation (HNBR). Patients with SCRA showed 2-16 copies of the SRY gene of which, one, Oxen (49, XYYYY) had eight copies with sequences different from one another. Of the 36 HNBR samples, 12 had one copy whereas 24 harboured 2-8 copies of the SRY gene. A HNBR male 33F had one normal and one mutated copy of this gene. Analysis of 25 DNA samples from blood and semen of normal males showed only one copy of this gene. Despite multiple copies in affected males, fluorescence in-situ hybridization (FISH) with SRY probe detected a single signal on the Y chromosome in HNBR males suggesting its possible localized tandem duplication. Copy number status of the other Y-linked loci is envisaged to augment DNA diagnostics facilitating genetic counselling to affected patients.

We conducted minisatellite-associated sequence amplification (MASA) with an oligo (5' CACCTCTCCACCTGCC 3') based on consensus of 33.15 repeat loci using cDNA from the testis, ovary, spleen, kidney, heart, liver, and lung of water buffalo Bubalus bubalis and uncovered 25 amplicons of six different sizes (1,263, 846/847, 602, 576, 487, and 324 base pairs). These fragments, cloned and sequenced, were found to represent several functional, regulatory, and structural genes. Blast search of all the 25 amplicons showed homologies with 43 transcribing genes across the species. Of these, the 846/847-bp fragment, having homology with the adenylate kinase gene, showed nucleotide changes at six identical places in the ovary and testis. The 1,263; 324; and 487-bp fragments showed homology with the secreted modular calcium binding protein (SMOC-1), leucine-rich repeat neuronal 6A (LRRN6A) mRNA, and human TTTY5 mRNA, respectively. Real-time PCR showed maximum expression of AKL, LRRN6A, and T-cell receptor gamma (TCR-gamma)-like genes in the testis, SMOC-1 in the liver, and the T-cell receptor-like (TCRL) gene in the spleen compared to those used as endogenous control. We construe that these genes have evolved from a common progenitor and conformed to various biological functions during the course of evolution. MASA approach coupled with real-time PCR has potentials to uncover accurate expression of a large number of genes within and across the species circumventing the screening of cDNA library.

For several decades a causal relation has been hypothesised between drinking water hardness and cardiovascular and other chronic degenerative diseases in humans. Only recently some epidemiological studies also investigated the association between the concentration of the minerals responsible for the hardness of drinking water (calcium and magnesium) and other chronic diseases. Some case-control studies carried out in Taiwan using aggregated data showed a possible protective effect of water hardness toward the risk of dying from various neoplasms, though more research is needed on the issue, possibly based on individual data, to draw definitive conclusions. There is a substantial evidence that consumption of water with high levels of calcium does not increase, and maybe reduces the risk of developing urinary stones of the most common type in developed countries (calcium oxalate), on the contrary, there is no conclusive evidence on the relation between water hardness and foetal malformations, cognitive functions in old men, diabetes and eczema.