Certification of the methylmercury content in SRM 2977 mussel tissue (organic contaminants and trace elements) and SRM 1566b oyster tissue.

The methylmercury content in two new marine bivalve mollusk tissue Standard Reference Materials (SRMs) has been certified using results of analyses from the National Institute of Standards and Technology (NIST) and two other laboratories. The certified concentrations of methylmercury were established based on the results from four and six different (independent) analytical methods, respectively, for SRM 1566b Oyster Tissue (13.2 +/- 0.7 microg/kg) and SRM 2977 Mussel Tissue (organic contaminants and trace elements) (36.2 +/- 1.7 microg/kg). The certified concentration of methylmercury in SRM 1566b is among the lowest in any certified reference material (CRM).

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues.

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.

Isolation of cDNA for an NADP-malic enzyme from Aloe arborescens.

NADP-malic enzyme catalyzes the reaction of decarboxylation from malate. In CAM plants, functions of this enzyme diverged to include both photosynthetic and non-photosynthetic roles. A full length cDNA for an NADP-malic enzyme was isolated from an 'obligate' CAM plant aloe (Aloe arborescens). The cDNA contains an ORF encoding 592 amino acid residues, whose sequence is highly homologous to the known plant NADP-malic enzymes. This gene is constitutively expressed in all organs in a low level. The amount of the transcript exhibited no diurnal variation, suggesting that this gene is not involved in photosynthetic functions.

Function and modulation of the cloned glycine receptor channels expressed in Xenopus oocytes.

Complementary DNAs encoding glycine receptor subunits alpha 1 and alpha 2 were isolated from rat cDNA libraries. The alpha 2 protein had 71% homology to the alpha 1 protein. The alpha 1 mRNA is abundant in the spinal cord and brain stem of mature rats whereas the alpha 2 mRNA was expressed in the tissues during the restricted period from late embryonic (E18) to early postnatal stage (P10). Homomeric glycine receptor channels consisting of alpha 1 or alpha 2 subunit expressed in Xenopus oocytes had an ability to generate Cl- currents, and the currents were suppressed by strychnine, a selective antagonist. Single channel kinetics between the homomeric channels differed considerably (alpha 1 << alpha 2). The currents generated through homomeric alpha 1 receptor channels were augmented in the presence of Zn2+ (100 nM to 10 microM), and depressed by 4 beta-phorbol 12-myristate 13-acetate (10 nM), an activator of protein kinase C.

The hair-organ relationship in mercury concentration in contemporary Japanese.

The hair-organ relationship of mercury concentration was investigated in 46 autopsy samples in Tokyo, Japan. Hair mercury levels were highly significantly correlated with organ Hg levels in the cerebrum, cerebellum, heart, spleen, liver, kidney cortex, and kidney medulla, when the total mercury or methyl mercury value in the organ was compared with the hair total mercury or organic mercury, respectively. When the inorganic mercury value was tested, significant correlations remained, with weaker coefficients in all the organs but the spleen. Stepwise multiple regression analysis evidenced that the hair organic mercury value was the major explanatory variable for the organ total mercury or organ methyl mercury value in all the organs. To explain the organ inorganic mercury value, the hair organic mercury value was the major variable for the cerebrum and kidney (both cortex and medulla), the hair inorganic mercury value was the major variable for the cerebellum and heart, and the hair phosphorous and hair organic mercury were the major variables for the liver; no explanatory variable existed for the spleen. Auxiliary explanatory variables accounted for the organ total mercury and inorganic mercury levels, among which the hair selenium value was conspicuous with negative regression coefficients.

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development.

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR.

Interrelationship between the concentrations of some elements in the organs of Japanese with special reference to selenium-heavy metal relationships.

Multi-element analyses were conducted on internal organs (cerebrum, cerebellum, heart, spleen, liver and kidney) of Japanese obtained from 45 forensic medical autopsy cadavers. Elements analyzed and analytical methods were as follows: Ca, Cd, Cu, Fe, K, Mg, Na, P and Zn by inductively coupled plasma atomic emission spectrometry; total Hg (T-Hg) and inorganic Hg (I-Hg) by cold vapour atomic absorption spectrometry; methyl Hg (MeHg) by gas chromatography; and Se by fluorometry. A significantly positive correlation between Se and T-Hg was observed in kidney, liver, heart and spleen. The T-Hg/Se molar ratio was less than 1 in all the organs examined. The correlation between Zn and Cd was significant in liver and kidney. Multiple regression analyses using Se as a dependent and I-Hg, MeHg, Zn, Cd, Cu as independent variables were conducted on each organ. Inorganic Hg was a significant independent variable in kidney, whereas in liver and spleen it was MeHg. Cadmium was significant in explaining the variations in Se in liver and kidney medulla, indicating Cd-Se co-accumulation in humans.

Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors.