Blockade of M1 muscarinic acetylcholine receptors modulates the methamphetamine-induced psychomotor stimulant effect.

Muscarinic acetylcholine receptors (M1-M5) regulate many key functions of the CNS and peripheral nervous system. In the present study, the role of M1 muscarinic receptors (M1R) in the psychomotor stimulant and sensitizing properties of methamphetamine (METH) is investigated using molecular, neurochemical, and behavioral approaches. Acute and repeated treatment with METH increased M1R mRNA expression in the frontal cortex and the CA2 region of the hippocampus. Repeated treatment with METH also increased M1R mRNA expression in the dentate gyrus. Dicyclomine, an M1R antagonist, did not affect the psychomotor effect of METH, but it attenuated METH-induced increases in the dopamine (DA) efflux in the nucleus accumbens (NAc). Dicyclomine enhanced the psychomotor effect of METH after repeated treatment with METH and 8.0 mg/kg of dicyclomine, and also augmented the increase in the NAc DA overflow evoked by repeated METH treatment. These results suggest that M1R plays a role in the METH-induced psychomotor stimulant effect by changing the release of DA in the NAc of mice.

Cardiac sympathetic denervation is correlated with Parkinsonian midline motor symptoms.

BACKGROUND: In patients with Parkinson's disease (PD), myocardial (123)I-metaiodobenzylguanidine (MIBG) uptake is significantly reduced even without apparent autonomic abnormalities. Several studies have suggested that the disease duration, severity and specific phenotype influence MIBG uptake in PD. The objective of this study was to investigate prospectively the relationship between the myocardial MIBG uptake and the Parkinsonian motor handicap in patients with minimal to severe disability. METHODS: Sixty-nine patients with PD who underwent MIBG scintigraphy and clinical assessments off medication were included. MIBG uptake was assessed using the ratio of the heart to the upper mediastinum (H/M) according to planar scintigraphic data and correlated with the age, disease duration and severity as measured by the modified Hoehn and Yahr (H & Y) stage and Unified Parkinson's Disease Rating Scale (UPDRS). RESULTS: There was a significant negative correlation between the H/M ratio and midline symptoms such as speech, posture and gait. However, neither the severity measured by H & Y stage and UPDRS motor scores nor the non-midline symptoms were related to the degree of cardiac sympathetic denervation. CONCLUSION: The results of this study suggest that the severity of midline motor symptoms is closely related to myocardial sympathetic dysfunction, although the implications of these findings require further study.

Aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faecium.

Escherichia coli SCH92111602 expresses an aminoglycoside resistance profile similar to that conferred by the aac(6')-Ie-aph(2")-Ia gene found in gram-positive cocci and was found to contain the aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im (only 44 nucleotides apart). aph(2")-Ib had been reported previously in Enterococcus faecium SF11770. aac(6')-Im had not been detected previously in enterococci and was found to be present also 44 nucleotides downstream from aph(2")-Ib in E. faecium SF11770. aph(2")-Ib and aac(6')-Im are separate open reading frames, each with its own putative ribosome binding site, whereas aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes with just one start and one stop codon. The deduced AAC(6')-Im protein exhibits 56% identity and 80% similarity to the AAC(6')-Ie domain of the bifunctional enzyme AAC(6')-APH(2"). Our results document the existence of a member of the aph(2") family of genes in gram-negative bacteria and provide evidence suggesting the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria.

Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium.

A new high-level gentamicin resistance gene, designated aph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib.

In-vitro synergistic activity of the combination of ampicillin and arbekacin against vancomycin-and high-level gentamicin-resistant Enterococcus faecium with the aph(2")-Id gene.

The combination of ampicillin plus arbekacin produced synergistic killing against 8 of 13 vancomycin-, ampicillin-, gentamicin-, and streptomycin-resistant Enterococcus faecium isolates that possess the high-level gentamicin resistance gene, aph(2")-Id. This combination may prove useful in treating infections caused by multiresistant enterococci.

In-vitro activity of arbekacin alone and in combination with vancomycin against gentamicin- and methicillin-resistant Staphylococcus aureus.

In-vitro susceptibility studies were performed on 99 clinical Staphylococcus aureus isolates. A total of 68 of 73 methicillin-resistant S. aureus and 2 of 26 methicillin-susceptible S. aureus were gentamicin-resistant (gentamicin MIC range 16 to 1,024 microg/mL). All 70 gentamicin-resistant isolates contained the aac(6')-Ie-aph(2'')-Ia aminoglycoside resistance gene, and none possessed the aph(2'')-Ic or aph(2'')-Id aminoglycoside resistance genes. The arbekacin MIC for the 70 gentamicin-resistant isolates ranged from 0.25 to 4 microg/mL. The combination of arbekacin plus vancomycin produced synergistic killing against 12 of 13 gentamicin-resistant MRSA isolates. The combination of gentamicin plus vancomycin produced synergistic killing against 7 of the same 13 isolates. Arbekacin may prove useful when used in combination with vancomycin in treating infections caused by gentamicin-resistant MRSA.

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G.

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.

[A study on the effect that rehabilitation education influence on the knowledge attitude and practice of public health nurse].

The home visiting health nurses are important man-power who can serve various and persistent rehabilitation care to disabled person in community. The Community Based Rehabilitation project (CBR) of national rehabilitation center have been carried out from 1995. As a part of that project national health center performed rehabilitation education program for home visiting health nurses. The purpose of this study is to analysis the effect of this education. In the first stage all of those groups were educated for two weeks in national rehabilitation center. But only two group nurses, one is in a urban and the other in a rural community, have been educated continually in the field through discussing rehabilitation care case study. The data in this study were gathered from three group health nurses and analysed by SAS computer program. The results about knowledge, attitude and practice changes of the three group nurses were as follows. 1. In the pre education state the mean point of all nurses' attitude for rehabilitation was 59, but in the post education state that was 90. The difference between pre and post attitude is very significant (t = -14.1, p < 0.0001). 2. In the pre education state the mean point of all nurses' knowledge for rehabilitation was 45, but in the post education state that was 78. The difference between pre and post knowledge is very significant (t = -12.7, p < 0.0001). 3. In the pre education state the mean point of all nurses' practice for rehabilitation care was 37, but in the post education state that was 62. The difference between pre and post practice is very significant (t = -7.3, p < 0.0001). 4. In practice point, the two group nurses who have been educated continuously were superior to the other (t = -3.9, p < 0.001). 5. All points between the urban and rural nurses were no significant differences (p > 0.1). 6. All changes of the attitude, knowledge and practice did not affected by age (F = 0.58, p > 0.1), professional career (F = 0.61, p > 0.1), educational background (F = 0.97, p > 0.1).

Genetic and molecular analysis of a regulatory region of the herbicide 2,4-dichlorophenoxyacetate catabolic plasmid pJP4.

In Alcaligenes eutrophus JMP134, pJP4 carries the genes coding for 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba) degradation plus mercury resistance. The plasmid genes specifying 2,4-D and 3-Cba catabolism are organized in three operons: tfdA, tfdB, and tfdCDEF. Regulation of these operons by two unlinked genes, tfdR and tfdS, has been proposed. Physical and DNA sequence analyses revealed that the tfdR and tfdS genes were identical and were located within a longer inverted repeat of 1592 bp. Similar stem-loop structures were observed among other 2,4-D plasmids. The tfdR gene is 888 bp long and capable of encoding a polypeptide of 32 kDa. The deduced amino acid sequence of tfdR indicates that it is a member of the LysR-type activators. Investigation of the regulation of the catabolic gene clusters through the construction of a pJP4 defined deletion mutant, pYG1010, which lacks a 4.2 kilobase Xbal fragment containing the inverted repeat region carrying the tfdR and tfdS regulatory genes, showed that Pseudomonas cepacia strains containing pYG1010 became 2,4-D negative, but 3-Cba positive. In vivo recombinants of pYG1010 and a cloned tfdS gene rescued the 2,4-D phenotype, indicating that TfdS is a positive regulator of tfdA expression, but not for tfdCDEF expression.

Nucleotide sequence analysis of the Pseudomonas putida PpG7 salicylate hydroxylase gene (nahG) and its 3'-flanking region.

Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.

Purification and characterization of salicylate hydroxylase from Pseudomonas putida PpG7.

The salicylate hydroxylase from P. putida PpG7 was purified and characterized. The enzyme appears to be monomeric, and it showed one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 45 kDa. The sequence of the first 25 amino acids of salicylate hydroxylase (PpG7) was determined. Also, the total amino acid composition of salicylate hydroxylase (PpG7) was obtained and compared with that of the known salicylate hydroxylase from P. putida.

Operon structure and nucleotide homology of the chlorocatechol oxidation genes of plasmids pJP4 and pAC27.

Alcaligenes eutrophus harboring plasmid pJP4 (strain JMP134) is capable of growing on both 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3-Cba), while Pseudomonas putida carrying plasmid pAC27 (strain AC867) can utilize only 3-Cba as the sole carbon source. The tfdCDEF operon of the pJP4 plasmid and the clcABD operon of plasmid pAC27 each encode enzymes for the degradation of chlorocatechols (Clc), key intermediates in the catabolism of 2,4-D and 3-Cba. Similarities in the nucleotide (nt) sequences of genes tfdC and clcA, encoding pyrocatechases, were reported earlier [Ghosal and You, Mol. Gen. Genet. 211 (1988a) 113-120]. Genes tfdD and clcB, encoding Clc-specific cycloisomerases, have been completely sequenced. The tfdD gene (1107 bp) is slightly smaller than gene clcB (1113 bp). Comparison of the two cycloisomerase-encoding genes reveals that the nt sequences are 63% homologous with 62% homology in the deduced amino acid (aa) sequences of the polypeptides they encode. Genes tfdD and tfdE are contiguous in the tfdCDEF operon, whereas the corresponding genes, clcB and clcD, of the clcABD operon, are known to be separated by a long open reading frame of unknown function. The predicted N-terminal aa sequences of the two hydrolase-encoding genes, tfdE and clcD, also show homology. The structural and nt homologies between the two Clc operons, tfdCDEF and clcABD, suggest their relatedness.

Nucleotide sequence of plasmid NAH7 gene nahR and DNA binding of the nahR product.

The nah and sal operons of the 80-kilobase-pair (kb) NAH7 plasmid specify catabolism of naphthalene and salicylate under positive regulation by gene nahR. A 1.75-kb fragment (PstI-HindIII) cloned into the pCP13 derivative of vector RK2 complemented in trans five nahR mutations. The fragment sequence contained a 1,122-base-pair open reading frame with a predicted sequence of 374 residues that was rich in basic amino acids with regions similar to known DNA-binding proteins. Clones from the nahR gene region were expressed in mexicells. Plasmid pY1923, carrying the 1.75-kb PstI-HindIII fragment, expressed a protein of Mr ca. 35,000 which bound to the upstream region of gene nahR in a gel electrophoresis DNA-binding assay. Other clones expressed proteins of currently unknown function; pY1311, with the 1.1-kb HindIII fragment, produced a polypeptide with an Mr of 23,000, and pY1812, with the 1.2-kb PstI-SphI fragment, produced a polypeptide (Mr 41,000) which appeared to be a fused nahR-lacZ product.

Gene duplication in haloaromatic degradative plasmids pJP4 and pJP2.

pJP2 and pJP4 are 2,4-dichlorophenoxyacetic acid catabolic plasmids, and they show DNA sequence homology. Most of the pJP2 molecules (80% or more) isolated from 2,4-dichlorophenoxyacetic acid grown cells of Alcaligenes eutrophus harbor a tandem duplication of a 25-kilobase (kb) segment encoding the catabolic functions. Unlike plasmid pJP4, pJP2 in A. eutrophus gives rise to a 3-chlorobenzoate phenotype without further genetic rearrangement. pJP4 under 3-chlorobenzoate selection contains an inverted duplication of 24.5 kb. Absence of selective pressure results in the prompt loss of one copy of the duplication in pJP4, but not of the tandem duplication in pJP2. In both pJP4 and pJP2, mutation of the duplicated copy, rather than gene dosage, is likely to be the basis of phenotypic change of catabolic functions. Experiments using the cloned DNA suggest that a tandem duplication is more stable than an inverted duplication.

Nucleotide homology and organization of chlorocatechol oxidation genes of plasmids pJP4 and pAC27.

The 2,4-dichlorophenoxyacetate (2,4-D) catabolic plasmid pJP4 of Alcaligenes eutrophus JMP134 contains two sets of nonidentical chlorocatechol oxidation gene sequences physically separated by a 7 kb DNA region. We determined the nucleotide sequence of the 1.6 kb HindIII fragment containing the known genes tfdC and tfdD (Don et al. 1985) which encode pyrocatechase and cycloisomerase, respectively. The 1.3 kb BglII-HindIII segment of recombinant plasmid pDC25 containing at least three chlorocatechol (clc) oxidation genes of the pAC27 plasmid in Pseudomonas putida AC867 (Ghosal et al. 1985a; Frantz and Chakrabarty 1986), was also sequenced. When the tfdC gene of the pJP4 plasmid was compared with gene clcA of plasmid pAC27, which encodes the chlorocatechol specific pyrocatechase (pyrocatechase II), the two genes showed 63% nucleotide sequence homology with 60% homology in their amino acid sequences. In both plasmid pJP4 and pAC27, the two genes encoding the pyrocatechase and the cycloisomerase showed a 4 bp overlap spanning the initiation codon of the cycloisomerase gene and the termination codon of the pyrocatechase gene. The sizes of the polypeptides encoded by the isofunctional genes tfdC and clcA are very similar and thus reflect their functional homology.

Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO.

The enzyme catechol 2,3-dioxygenase (C23O) encoded by the nahH gene of plasmid NAH7 converts catechol to alpha-hydroxymuconic epsilon-semialdehyde in Pseudomonas putida. We have cloned this structural gene into vectors pUC18 and pKT240, determined the nucleotide sequence and deduced the amino acid sequence. In comparison to the gene xylE of the TOL plasmid pWWO which encodes a similar C230 enzyme [Nakai et al. J. Biol. Chem. (1983b), 2923-2928], the respective G + C contents were 55% and 57%, the nucleotide sequences 81% homologous, and the amino acid homology 85%. The flanking sequences of the two C23O-coding genes also show homology. Clones of the nahH gene in Escherichia coli overproduce the protein product at least ten fold and the gene product was identified by polyacrylamide gel electrophoresis.

Regulation of the nah and sal operons of plasmid NAH7: evidence for a new function in nahR.

The catabolism of naphthalene and salicylate is specified by two operons on an 80 Kb metabolic plasmid, NAH7. These operons, nah and sal, are carried on the contiguous 30 Kb EcoRI-A, C fragments, and are under positive control of a regulator region, nahR. Five Nah Sal Tn5 insertion mutants form two complementation groups: A = nahR203, nahR204; and B = nahR201, nahR202, nahR205. The physical and genetic maps assign the nahR location to the 15.7-17.2 Kb region of the EcoRI-A fragment, with suggestion of more than one control gene.

Microbial degradation of halogenated compounds.

The mode of degradation of various halogenated compounds in isolated pure cultures and the disposition of the degradative genes have been studied. In many cases the degradative genes are found to be clustered on plasmids and appear to be under positive control. Genetic selection in vivo and genetic manipulations in vitro have allowed construction of strains having wider biodegradative potentials than their natural counterparts. Molecular cloning of the degradative gene clusters for halogenated compounds in vectors with a broad host range also allows the transfer of such genes to a large number of Gram-negative bacteria. The application of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-degrading microorganisms has demonstrated the effectiveness of this strain in removing large amounts of 2,4,5-T from contaminated soil within a short period, and such soil has been shown to support the growth of plants normally sensitive to low concentrations of 2,4,5-T. The two major challenges that must be addressed in the near future are the development of appropriate microbial technology for the decontamination of soil containing hazardous halogenated compounds, and the promulgation of appropriate regulations to ensure the safety and well-being of the public during the application of genetically improved strains in an open environment.

Genes specifying degradation of 3-chlorobenzoic acid in plasmids pAC27 and pJP4.

All of the structural genes for 3-chlorobenzoate degradation are clustered in a 4.2-kilobase (kb) region of plasmid pAC25 (or pAC27) in Pseudomonas putida. An approximate 10-kb DNA segment containing three structural genes for chlorocatechol metabolism present on plasmid pJP4 in Alcaligenes eutrophus shows homology with the above 4.2-kb region of pAC27. In spite of the detectable sequence homology in the structural genes present on both plasmids, the regulation of their expression seems quite different; unlike pAC27, structural rearrangements are prerequisite for efficient expression of the 3-chlorobenzoate genes on plasmid pJP4. Structural features such as stem-loop structures present on plasmid pJP4 are most likely the starting materials for such rearrangements.