Selective, Ultra-Sensitive, and Rapid Detection of Serotonin by Optimized ZnO Nanorod FET Biosensor.

BACKGROUND: Fluctuation in serotonin (5-HT) level is an essential manifestation of several neurological disorders. In view of such importance, it is necessary to monitor the levels of 5-HT with good sensitivity, selectivity, affordability and low response time. Zinc oxide (ZnO) based field effect transistors (FET) with attributes like minimized noise levels and large on-off ratio are regarded as emerging high performance biosensor platforms. However, their response is significantly non-linear and there has been no appreciable endeavor for improving the non-linearity. METHOD: In this paper, we have introduced embedded gate electrode encompassing the channel of the FET which improves the uniformity in electric field line distribution through the electrolyte and proportionately enhances the capture of target biomolecule at ultra-low concentrations, thereby increasing the linearity. Further, we have incorporated the optimized parameters of ZnO nanorods reported previously, for rapid and selective detection of 5-HT. RESULTS: It has been observed that the fabricated ZnO FET biosensor lowers the detection limit down to 0.1fM which is at least one order of magnitude lower than the existing reports. The sensor also has wide linear range from 0.1fM to 1nM with a detection time of about 20 minutes. CONCLUSION: The proposed zinc oxide nanorod-based sensor can be used as an excellent tool for future diagnosis of neurological disorders.

Expression profile, transcriptional and post-transcriptional regulation of genes involved in hydrogen sulphide metabolism connecting the balance between development and stress adaptation in plants: a data-mining bioinformatics approach.

Recent research focused on novel aspects of sulphur and sulphur-containing molecules in fundamental plant processes has highlighted the importance of these compounds. Currently, the focus has shifted to the efficacy of hydrogen sulphide (H2 S) as signalling compounds that regulate different development and stress mitigation in plants. Accordingly, we used an in silico approach to study the differential expression patterns of H2 S metabolic genes at different growth/development stages and their tissue-specific expression patterns under a range of abiotic stresses. Moreover, to understand the multilevel regulation of genes involved in H2 S metabolism, we performed computation-based promoter analysis, alternative splice variant analysis, prediction of putative miRNA targets and co-expression network analysis. Gene expression analysis suggests that H2 S biosynthesis is highly influenced by developmental and stress stimuli. The functional annotation of promoter structures reveales a wide range of plant hormone and stress responsive cis-regulatory elements (CREs) that regulate H2 S metabolism. Co-expression analysis suggested that genes involved in H2 S metabolism are also associated with different metabolic processes. In this data-mining study, the primary focus was to understand the genetic architecture governing pathways of H2 S metabolism in different cell compartments under various developmental and stress signalling cascades. The present study will help to understand the genetic architecture of H2 S metabolism via cysteine metabolism and the functional roles of these genes in development and stress tolerance mechanisms.

The histopathologic and molecular basis for the diagnosis of histiocytic sarcoma and histiocyte-associated lymphoma of mice.

Histiocytic sarcoma (HS) and histiocyte-associated lymphoma (HAL) of mice are difficult to distinguish histologically. Studies of multiple cases initially diagnosed as HS or HAL allowed us to define HS as round, fusiform, or mixed cell types that were F4/80+, Mac-2+, and PAX5-; that lacked markers for other sarcomas; and that had immune receptor genes in germline configuration. Two other subsets had clonal populations of lymphocytes. The first, HAL, featured malignant lymphocytes admixed with large populations of normal-appearing histiocytes. The second appeared to be composites of lymphoma and HS. Several cases suggestive of B myeloid-lineage plasticity were also observed.

Erythrocyte abnormality in human myopathy.

Erythrocyte ghosts isolated from myopathic patients responded to 10(-4) molar ouabain with a dramatic increase in adenosine triphosphatase activity, while identical preparations from normal donors were inhibited by the same drug. These results have been interpreted in terms of a disease-related change in membrane integrity bearing upon function of the transport enzyme.

Molecular basis for increased synthesis of albumin in rat liver after thioacetamide administration.

Administration of thioacetamide to rats for 4 days has been shown previously to increase the activity of liver messenger RNA (mRNA) and to disproportionately increase the activity of albumin mRNA in the wheat germ translation system. We have explored the possibility that administration of thioacetamide quantitatively alters the transcription of rat liver unique DNA. Nucleic acid hybridization between the radioactive rat liver unique DNA and RNA from untreated as well as 4-day thioacetamide-treated rat liver indicates that the normal extent of transcription is not altered by the drug treatment. We also investigated the possibility that thioacetamide treatment increases the level of mRNA in general or of albumin mRNA in particular. Albumin mRNA, which is th most abundantly represented population in rat liver cytoplasmic mRNA, was further enriched by sucrose gradient fractionation of the liver mRNA. Synthesis of complementary DNA (cDNA) and then use of the strategy of limited hybridization yielded the cDNA corresponding to albumin mRNA. Hybridization of the fractionated cDNA with mRNA from untreated livers as well as from livers with increasing days of treatment shows no change in the level of albumin mRNA. The results indicate that drug treatment induces the augmented synthesis of albumin by disproportionately increasing the translational activity of albumin mRNA. Quantitation of gene dosage by comparison of hybridization kinetics of fractionated cDNA and unique [3H]DNA with total rat cellular DNA indicates that there are not more than two copies of the albumin gene present in the rat genome.

Correlation of the induction of transcription of the AKR mouse genome 5-lododeoxyuridine with the activation of an endogenous murine leukemia virus.

5-iododeoxyuridine (IdUrd) is highly effective in inducing the production of endogenous viruses (e.g., RNA-containing murine leukemia virus) from a variety of cell lines that normally do not release such viruses. For the activation of murine leukemia virus by IdUrd, its incorporation into cellular DNA is necessary. We have explored the possibility that incorporated IdUrd qualitatively alters the transcription of cell DNA. Nucleic acid hybridization between radioactive mouse unique DNA and RNA from the highly activatable mouse AKR-2B cell line indicates that the normal extent of transcription in AKR-2B cells is considerably lower than that observed in other lines of mouse cells studied. Treatment of AKR-2B cells with IdUrd increases the extent of transcription of unique DNA by 60%, which corresponds to an induction of approximately 2.5 X 10(4) gene equivalents. Included among this new set of RNA's are sequences that are transcribed from the DNA genome of the endogenous AKR-type murine leukemia virus present in AKR-2B cells. IdUrd treatment also markedly increases the synthesis and/or the accumulation of those RNA transcripts which are normally expressed in untreated cells. These results suggest that IdUrd stimulates the overall transcription activity of AKR-2B cells. It is possible that IdUrd-induced activation of endogenous murine leukemia virus is a consequence of this stimulation.

Regulation of viral transcription in cells infected with iododeoxyuridine-substituted simian virus 40 as a model for the activation by iododeoxyuridine of latent viral genomes.

5-Iododeoxyuridine (IdUrd) induces the expression of viruses in a variety of cell lines that harbor latent viral genomes. Moreover, IdUrd stimulates cellular as well as viral RNA synthesis in certain cells. In order to understand better the action of IdUrd on RNA metabolism, we have examined viral RNA synthesis in monkeys cell infected with IdUrd-substituted simian virus 40 (SV40). Extensively substituted SV40, in which 18 to 35% of the thymidine residues were substituted by IdUrd, was 100-fold less viable (by plaque analysis) than was unsubstituted SV40, although the substituted virus induced 30 to 50% as much viral-specific RNA as did the unsubstituted virus. In contrast, SV40, containing only 10 to 15% IdUrd, substitution was almost as viable as unsubstituted virus, and the substituted SV40 induced 5-fold more viral-specific RNA, as well as longer viral messenger RNA transcripts, than did the unsubstituted virus. These results suggest that the lightly substituted, mutagenized SV40 genome may produce defective proteins which fall to regulate their own transcription. Cellular DNA into which halogenated pyrimidines have been incorporated may also induce the synthesis of defective regulatory proteins, including cellular repressors of transcription which normally maintain the latent state of integrated viral genomes.

Examination of Epstein-Barr virus and C-type proviral sequences in American and African lymphomas and derivative cell lines.

While Epstein-Barr viral (EBV) DNA is nearly always detectable in African Burkitt's lymphoma (BL), the relatively low frequency of BL in EBV-positive African children and the infrequent finding of EBV in American BL suggest that other cofactors may contribute to the malignant transformation. Among these possible cofactors are type C oncornaviruses. To evaluate this possibility, we screened the cellular DNA from 16 lymphomas (2 African, 14 American) and the DNA from 20 lymphoma-derived cell lines (4 African, 16 American) with a radiolabeled viral DNA probe from EBV and two oncornaviral probes (murine amphotropic 1504A virus and simian sarcoma virus). The radiolabeled EBV DNA probe hybridized with 18 of 36 tumor or cell line DNA's. Only 2 of 11 American BL tumors contained detectable EBV sequences. However, the cell lines derived from three EBV-negative tumors converted in vitro to EBV positivity, suggesting that some of the tumor cells could be infected with EBV. In contrast, none of the tumors or the cell lines derived therefrom hybridized with either the 1504A or the simian sarcoma virus probes, decreasing the likelihood that type C viruses are cofactors with EBV.

Microcalorimetric determination of binding sites of acetylcholinesterase.

Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7), phosphorylated with dichlorvos, showed relatively more reactivity toward the substrate indophenyl acetate than the enzyme that was carbamylated with carbaryl. When the anionic subsite of the phosphorylated or carbamylated enzyme was alkylated with an aziridinium ion, the reaction velocity toward indophenyl acetate increased in the phosphorylated enzyme but not in the carbamylated enzyme. The organophosphate binding site--which appears to be different from that of carbamate or indophenyl acetate, but probably the same as that of acetylcholine -- is apparently alkylated in such a way that the modified (phosphorylated) enzyme is better-fit for the binding (and hydrolysis) of indophenyl acetate. The modified conformation presumably results in the release of the phosphoryl group from the esteratic subsite.

Cholesterol oxidase: thermochemical studies and the influence of hydroorganic solvents on enzyme activity.

Thermal and binary cosolvent studies of the cholesterol oxidase (cholesterol: oxygen oxidoreductase, EC 1.1.3.6) reaction have been carried out using batch microcalorimetry and ultraviolet spectrophotometry respectively. Heat conduction measurements are shown to provide the basis for a serum cholesterol assay yielding results comparable to conventional automated clinical assay. The enthalpy of the reaction for cholesterol oxidation, measured with different sources of the enzyme in the presence and absence of catalase is -113 +/- 7.2 mJ/mumol. The value is agreement with calculated estimates based on bond energies, enthalpies of formation and trigonal additivity contribution calculations. From this heat of reaction the deltaHf0 of cholestenone (c) is calculated to be -490 kJ . mol-1. No evidence for the reverse reaction could be adduced. Enzyme activation with detergent (Surfal) is attributed to the formation of mixed micelles of cholesterol with detergent molecules. The detergent concentration at which the enzyme is half activated corresponds to the critical micelle concentration of Surfal. The enhanced enzyme activity found when ethanol, acetonitrile and dioxane were examined as binary cosolvents with water is ascribed to a conformational change in the enzyme mediated through the altered structuredness of water. This cosolvent effect is abolished in the presence of 0.18% Surfal due to the formation of inverted mixed micelles of detergent with cholesterol.

Charlotte Friend Memorial Lecture: murine leukemia virus (MuLV) tumorigenesis.

Recent analysis of over 500 lymphomas occurring in NFS.V mice, congenic for Akv-type ecotropic MuLV structural genes, has revealed that about 90% are of B cell lineage as determined by demonstration of clonal rearrangements of Ig heavy chain genes, phenotyping by immunocytochemistry or cytofluorometric analysis, and by site and morphology of tumor. At least 40% of the B cell lymphomas were found to have their origin in the splenic marginal zone, a site only once before described for mouse lymphomas. Clonal somatic integrations of ecotropic MuLV occurred in 85% of tumors.

Immunologic havoc induced by the 60 kD Gag protein of the MAIDS defective virus.

The mechanisms responsible for development of profound immunodeficiency and extensive lymphoproliferation that characterize infection of different species with retroviruses are only partially understood. In mice, it has been shown the activities of an unusual Gag protein are necessary and sufficient to induce these abnormalities in a syndrome designated mouse AIDS (MAIDS). Current studies suggest that complex, antigen-driven interactions between T cells and B cells result in polyclonal activation of both types of lymphocytes, aberrant cytokine production and late lymphomas.

In vitro transformation by bovine papillomavirus.

We have studied tumorigenic transformation of mouse tissue culture cells by bovine papillomavirus (BPV) as a model of papillomavirus-induced cell proliferation. When BPV or its 7.9-kb full-length viral DNA genome induces focal transformation of mouse calls, the viral DNA is maintained in the transformed cells as multiple extrachromosomal copies. The transforming capacity was initially localized to a 69% subgenomic fragment of the viral DNA genome. We have further characterized the BPV DNA sequences that can encode the transforming function by generating and analyzing the transforming activity of a series of BPV DNA deletion mutants. The results indicated that two discontinuous segments of the viral DNA are required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA. A retroviral control element (the long terminal repeat DNA. A retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) will activate the 2.3-kb segment at the 3' end of the 69% fragment to transform the mouse cells.

Central nervous system infection in a murine retrovirus-induced immunodeficiency syndrome.

Astrocyte-enriched primary glial cultures (AGC) from C57BL/6 mice were found to be highly susceptible to infection with the replication competent components of LP-BM5, consisting of the ecotropic and mink cell focus-inducing (MCF) helper murine leukemia viruses (MuLVs). The presence in infected AGC of defective LP-BM5 MuLV genome, a critical component for induction of the disease referred to as murine AIDS, was confirmed by Southern blot hybridization using a probe reactive with the p12 gag sequence of the 4.9 kb defective genome. Electron microscopic studies demonstrated C-type retrovirus particles in both astrocytes and microglial cells. In vivo studies demonstrated that the ecotropic MuLVs and the defective genome could be detected within AGC obtained form either 14-day-old mice following intraperitoneal inoculation or 7-day-old mice following intracranial inoculation. These findings suggest that: (1) the central nervous system (CNS) infection is present at an early stage in murine AIDS, (2) both astrocytes and microglial cells are possible CNS targets in which helper MuLVs replicate, and (3) these cells can harbor the defective genome that is a critical component for disease induction.

Expression of cyclin D1 in mouse B cell lymphomas of different histologic types and differentiation stages.

The G1 cyclin, cyclin D1, has been implicated in the development of human and mouse tumors. Here we describe immunohistochemical analyses of cyclin D1 for a large panel of mouse B cell tumors. In addition, we characterize cyclin D1 expression in a series of cultured cell lines that represent transformed B cells at different stages of development. Immunohistochemical analysis showed that for low-grade lymphomas, cyclin D1 was expressed by 83% of centroblastic centrocytic (CBCC) and 14% of small lymphocytic lymphomas (SLL). For high-grade tumors, 28% of B lymphoblastic and 23% of centroblastic tumors expressed cyclin D1, while all immunoblastic lymphomas were negative. Studies of RNA and protein prepared from cultured B lineage tumors showed that cyclin D1 was expressed by all pre-B and most B cell tumors but not by cell lines representative of late B cell differentiation or by plasma cells. Expression of cyclin D1 in the lymphomas was not associated with alterations in the genomic structure of the Fis-1 (Bcl-1) common proviral integration site or cyclin D1 itself or with cell growth activity as assessed by expression of proliferating cell nuclear antigen (PCNA).

Genomic organisation and expression of BCL6 in murine B-cell lymphomas.

BCL6 encodes a transcription factor deregulated by chromosomal translocations in human diffuse large cell B lymphomas (DLCL). This study was designed to determine whether Bcl6 might also be involved in lymphomas of mice. BCL6 protein was expressed at high levels in 90% or more of DLCL but not in low grade B lymphomas. Southern hybridisation studies demonstrated altered organisation of Bcl6 in three primary DLCL and the WEHI 231 B-cell lymphoma cell line but not in low grade tumours. Chromosomal painting and fluorescence in situ hybridisation (FISH) analyses of the WEHI 231 metaphase spreads revealed a T(5;16) translocation with Bcl6 on Chromosome 16 at the translocation breakpoint. Deregulated expression of BCL6 is thus likely to contribute to the genesis of DLCL of mice as well as of humans.

Combined histologic and molecular features reveal previously unappreciated subsets of lymphoma in AKXD recombinant inbred mice.

Hematopoietic neoplasms developing in AKXD recombinant inbred, NFS.V(+) and ICSBP knockout mice were assessed using morphologic, cytologic and molecular criteria that relate these disorders to human lymphoma and leukemia. Lymphoma types included precursor T-cell and B-cell lymphoblastic, small lymphocytic, splenic marginal zone, follicular, and diffuse large cell (DLCL). In addition to previously defined subtypes of DLCL composed of centroblasts or immunoblasts, two additional subtypes are defined here: lymphoblastic lymphoma like (LL) and lymphoma characterized by a histiocytic reaction (HS). DLCL(HS) were distinguished from true histiocytic lymphomas by the presence of clonal Ig gene rearrangements.

Elimination of 118 Da: a characteristic fragmentation in the tandem mass spectra of 11(15 --> 1)-abeo-taxanes.

The mechanism of the elimination of 118 Da from 11(15-->1)-abeo-taxanes was elucidated with the help of the tandem mass spectra of [M + NH(4)](+) and [M + Li](+) ions and the corresponding D-exchanged species. The fragmentation is triggered by the initial loss of the C-10 substituent. Evidence was also obtained for the stepwise elimination of acetone and acetic acid. Acetone is eliminated from the C-1 hydroxyisopropyl group and acetic acid from either the C-9 or C-7 acetoxy groups. The presence of an additional acetoxy group at C-13 leads to the direct elimination of 118 Da from [M + NH(4)](+) and [M + Li](+) ions involving the C-13 acetoxy group.

A brevifoliol analogue from the Himalyan yew Taxus wallichiana.

The needles of Taxus wallichiana gave a new brevifoliol derivative whose structure was established by spectral data.

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase.

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.