A correlation between changes in gamma-aminobutyric acid metabolism and seizures induced by antivitamin B6.
The effects of DL-penicillamine (DL-PeA), hydrazine and toxopyrimidine (TXP, 2-methyl-6-amino-5-hydroxymethylpyrimidine) on gamma-aminobutyric acid (GABA) metabolism in mouse brain were studied. All these compounds inhibited the activity of glutamate decarboxylase [EC 188.8.131.52] (GAD) and slightly inhibited that of 4-aminobutyrate: 2-oxoglutarate aminotransferase [EC 184.108.40.206] (GABA-T). In contrast, very different effects were observed on GABA levels; hydrazine caused a marked increase, DL-PeA had no effect, and TXP caused a slight decrease in the content of the amino acid. These results could be described by an equation which related the excitable state to changes in the flux of the GABA bypass. Since the values obtained from the equation clearly reflect the seizure activity, it is suggested that the decreased GABA flux might be a cause of convulsions induced by these drugs. (+info)
Novel characteristic for distinguishing Lactococcus lactis subsp. lactis from subsp. cremoris.
Lactococcus lactis strains were examined for their ability to produce gamma-aminobutyric acid (GABA). Results showed that strains of L. lactis subsp. lactis were able to produce this acid, whereas L. lactis subsp. cremoris were not. GABA production thus represents another effective characteristic for distinguishing L. lactis subsp. lactis from L. lactis subsp. cremoris. (+info)
The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons.
We describe here the functional analysis of the C. elegans LIM homeobox gene lim-6, the ortholog of the mammalian Lmx-1a and b genes that regulate limb, CNS, kidney and eye development. lim-6 is expressed in a small number of sensory-, inter- and motorneurons, in epithelial cells of the uterus and in the excretory system. Loss of lim-6 function affects late events in the differentiation of two classes of GABAergic motorneurons which control rhythmic enteric muscle contraction. lim-6 is required to specify the correct axon morphology of these neurons and also regulates expression of glutamic acid decarboxylase, the rate limiting enzyme of GABA synthesis in these neurons. Moreover, lim-6 gene activity and GABA signaling regulate neuroendocrine outputs of the nervous system. In the chemosensory system lim-6 regulates the asymmetric expression of a probable chemosensory receptor. lim-6 is also required in epithelial cells for uterine morphogenesis. We compare the function of lim-6 to those of other LIM homeobox genes in C. elegans and suggest that LIM homeobox genes share the common theme of controlling terminal neural differentiation steps that when disrupted lead to specific neuroanatomical and neural function defects. (+info)
Autoantibody appearance and risk for development of childhood diabetes in offspring of parents with type 1 diabetes: the 2-year analysis of the German BABYDIAB Study.
The temporal development of autoantibodies was studied in 1,353 offspring of parents with type 1 diabetes. Islet cell antibodies (ICAs) and autoantibodies to insulin (IAAs), glutamic acid decarboxylase, and IA-2 were measured at birth, 9 months, 2 years, and 5 years of age. At birth, no offspring had islet autoimmunity other than maternally acquired antibodies, which were shown to influence antibody prevalence up to age 6 months. Antibodies detected thereafter were likely to represent a true de novo production, since prevalences were the same for offspring from mothers and fathers with diabetes, antibodies detected at 9 months were almost always confirmed in the 2-year sample and were associated with an increased likelihood of having or developing other antibodies. By 2 years of age, autoantibodies appeared in 11% of offspring, 3.5% having more than one autoantibody. IAAs were detected most frequently, and few had autoantibodies in the absence of IAAs. In 23 offspring with multiple islet autoantibodies, IAAs preceded other antibodies in 10 cases and were first detected concurrently with other antibodies in 12 and after detection of other antibodies in 1. Development of additional antibodies and changes in levels, including decline of IAAs at older age, was frequent. Nine children, all with IAAs and ICAs, developed diabetes. Overall cumulative risk for disease by 5 years of age was 1.8% (95% CI 0.2-3.4) and was 50% (95% CI 19-81) for offspring with more than one autoantibody in their 2-year sample. Autoimmunity associated with childhood diabetes is an early event and a dynamic process. Presence of IAAs is a consistent feature of this autoimmunity, and IAA detection can identify children at risk. (+info)
Major DQ8-restricted T-cell epitopes for human GAD65 mapped using human CD4, DQA1*0301, DQB1*0302 transgenic IA(null) NOD mice.
The 65KD isoform of GAD is considered to be a major target autoantigen in many humans with autoimmune prediabetes or diabetes. The major histocompatibility complex class II allele DQA1*0301, DQB1*0302, which encodes HLA-DQ8, confers susceptibility to type 1 diabetes and occurs in up to 80% of affected individuals. To map T-cell epitopes for GAD65 restricted to the diabetes-associated DQ8 heterodimer, we generated transgenic NOD mice expressing HLA-DQ8 and human CD4 while having the mouse class II gene (IA(beta)) deleted. These mice were immunized with full-length purified recombinant GAD65, and the fine specificity of T-cell responses was mapped by examining recall responses of bulk splenocytes to an overlapping set of 20-mer peptides encompassing the entire GAD65 protein. Four different peptides (P121-140, P201-220, P231-250, and P471-490) gave significant T-cell recall responses. P201-220 and P231-250 have been shown previously to bind DQ8, whereas the other two peptides had been classified as nonbinders. Interestingly, the peptide giving the greatest response (P201-220) encompasses residues 206-220 of GAD65, a region that has been shown to be a dominant T-cell epitope in wild-type IA(g7) NOD mice. Overlap in this T-cell epitope likely reflects structural similarities between DQ8 and IA(g7). The fine specificity of antibody responses in the GAD65-immunized mice was also examined by testing the antisera by enzyme-linked immunosorbent assay (ELISA) against the same overlapping set of peptides. The two dominant B-cell epitopes were P361-380 and P381-400; P121-140 and P471-490 appeared to correspond to both B- and T-cell epitopes. Although the NOD human CD4, DQ8, IA(null) transgenic mice generated in these studies do not develop autoimmune diabetes either spontaneously or after cyclophosphamide treatment, they can be used to map DQ8-restricted T-cell epitopes for a variety of human islet autoantigens. They can also be used to test T-cell-specific reagents, such as fluorescently labeled DQ8 tetramers containing GAD65 peptides or other beta-cell peptides, which we believe will be useful in analyzing human immune responses in diabetic and prediabetic patients. (+info)
Acid- and base-induced proteins during aerobic and anaerobic growth of Escherichia coli revealed by two-dimensional gel electrophoresis.
Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 proteins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may reverse alkalinization by metabolizing amino acids to produce acidic products. Also induced at high pH, but only in anaerobiosis, was glutamate decarboxylase (GadA). The gad system (GadA/GadBC) neutralizes acidity and enhances survival in extreme acid; its induction during anaerobic growth may help protect alkaline-grown cells from the acidification resulting from anaerobic fermentation. To investigate possible responses to internal acidification, cultures were grown in propionate, a membrane-permeant weak acid which acidifies the cytoplasm. YfiD, a homologue of pyruvate formate lyase, was induced to high levels at pH 4.4 and induced twofold more by propionate at pH 6; both of these conditions cause internal acidification. At neutral or alkaline pH, YfiD was virtually absent. YfiD is therefore a strong candidate for response to internal acidification. Acid or propionate also increased the expression of alkyl hydroperoxide reductase (AhpC) but only during aerobic growth. At neutral or high pH, AhpC showed no significant difference between aerobic and anaerobic growth. The increase of AhpC in acid may help protect the cell from the greater concentrations of oxidizing intermediates at low pH. Isocitrate lyase (AceA) was induced by oxygen across the pH range but showed substantially greater induction in acid or in base than at pH 7. Additional responses observed included the induction of MalE at high pH and induction of several enzymes of sugar metabolism at low pH: the phosphotransferase system components ManX and PtsH and the galactitol fermentation enzyme GatY. Overall, our results indicate complex relationships between pH and oxygen and a novel permeant acid-inducible gene, YfiD. (+info)
Islet cell and glutamic acid decarboxylase antibodies present at diagnosis of diabetes predict the need for insulin treatment. A cohort study in young adults whose disease was initially labeled as type 2 or unclassifiable diabetes.
OBJECTIVE: To clarify the predictive value of islet cell antibody (ICA) and GAD65 antibody (GADA) present at diagnosis with respect to the need for insulin treatment 6 years after diagnosis in young adults initially considered to have type 2 or unclassifiable diabetes. RESEARCH DESIGN AND METHODS: The patient material was representative of the entire Swedish population, consisting of patients who were 15-34 years old at diagnosis of diabetes in 1987-1988 but were not considered to have type 1 diabetes at onset. At follow-up, 6 years after the diagnosis, it was noted whether the patient was treated with insulin. The presence of ICA was determined by an immunofluorescence assay, and GADAs were measured by a radioligand assay. RESULTS: Six years after diagnosis, 70 of 97 patients were treated with insulin, and 27 of 97 patients were treated with oral drugs or diet alone. At diagnosis, ICAs and GADAs were present in 41 (59%) of 70 patients and 41 (60%) of 68 patients, respectively, of those now treated with insulin, compared with only 1 (4%) of 26 patients and 2 (7%) of 27 patients who were still not treated with insulin. For either ICA or GADA, the corresponding frequencies were 50 (74%) of 68 for patients who were later treated with insulin and 3 (12%) of 26 for those who were still not treated with insulin, respectively. The sensitivity for later insulin treatment was highest (74%) for the presence of ICA or GADA, and the specificity was highest (100%) for ICA and GADA. The positive predictive value was 100% for the combination of ICA and GADA, 98% for ICA alone, and approximately 95% for GADA alone. CONCLUSIONS: Determination of the presence of ICA and GADA at diagnosis of diabetes improves the classification of diabetes and predicts the future need of insulin in young adults. (+info)
Expression of the Corynebacterium glutamicum panD gene encoding L-aspartate-alpha-decarboxylase leads to pantothenate overproduction in Escherichia coli.
The Corynebacterium glutamicum panD gene was identified by functional complementation of an Escherichia coli panD mutant strain. Sequence analysis revealed that the coding region of panD comprises 411 bp and specifies a protein of 136 amino acid residues with a deduced molecular mass of 14.1 kDa. A defined C. glutamicum panD mutant completely lacked L-aspartate-alpha-decarboxylase activity and exhibited beta-alanine auxotrophy. The C. glutamicum panD (panDC. g.) as well as the E. coli panD (panDE.c.) genes were cloned into a bifunctional expression plasmid to allow gene analysis in C. glutamicum as well as in E. coli. The enhanced expression of panDC.g. in C. glutamicum resulted in the formation of two distinct proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, leading to the assumption that the panDC.g. gene product is proteolytically processed into two subunits. By increased expression of panDC.g. in C. glutamicum, the activity of L-aspartate-alpha-decarboxylase was 288-fold increased, whereas the panDE.c. gene resulted only in a 4-fold enhancement. The similar experiment performed in E. coli revealed that panDC.g. achieved a 41-fold increase and that panDE.c. achieved a 3-fold increase of enzyme activity. The effect of the panDC.g. and panDE.c. gene expression in E. coli was studied with a view to pantothenate accumulation. Only by expression of the panDC.g. gene was sufficient beta-alanine produced to abolish its limiting effect on pantothenate production. In cultures expressing the panDE.c. gene, the maximal pantothenate production was still dependent on external beta-alanine supplementation. The enhanced expression of panDC.g. in E. coli yielded the highest amount of pantothenate in the culture medium, with a specific productivity of 140 ng of pantothenate mg (dry weight)-1 h-1. (+info)