Structure of the HLA-A*0204 antigen,found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A^*0204 (isoelectric focusing variant A2.A) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-lenght cDNA clones encoding A^*0204 were analyzed to obtain a consensus sequence for this subtype. A^*0204 differs from A^*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A^*0204 suggests that it could have arisen from A^*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A^*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent -strands on the floor of the peptide binding site of the molecule.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X57954. Address correspondence and offprint requests to: J. A. López de Castro.     

Nuclear location of phosphoglycerate mutase BB isozyme in rat tissues

Summary We have previously reported (Ureña et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.Abbreviations PGAM phosphoglycerate mutase - PGAM-M(M) muscle specific subunit (isozyme) of PGAM - PGAM-B(B) brain type subunit (isozyme) of PGAM - ssDNA single stranded DNA - PBS 0.001 M phosphate buffer, pH 7.4, containing 0.15 M NaCl - kDa kilodalton     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

Biological control of Pythium damping‐off by seed‐treatment with pseudomonas putida: Relationship with ethanol production by pea and soybean seeds

The relationship between biocontrol activity of Pseudomonas putida strain N1R against Pythium ultimum on pea and soybean seeds and the reduction in ethanol evolution by imbibed seeds was investigated under different treatment conditions, including temperature and numbers of seed‐applied cells of the bacterium. Treatment with strain N1R increased emergence at all temperatures, except for soybean at 12 °C and reduced ethanol concentration in the spermosphere of imbibed seeds at several temperatures. The concentration of bacterial cells in the seed treatment suspension also significantly affected biocontrol efficiency and reduced ethanol production, especially in pea seeds. In contrast, the duration (0–7 h) of submergence of seeds in bacterial suspension had little effect on biocontrol activity of N1R, although submergence of soybean seeds reduced their emergence even in the absence of the pathogen or biocontrol agent. Competition for seed‐derived compounds, including ethanol, is suggested to be one possible mechanism of biocontrol of Pythium by strain N1R, which is not known to produce antifungal antibiotics.     

Chimeric molecules with multiple neurotrophic activities reveal structural elements determining the specificities of NGF and BDNF.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two members of a family of neurotrophic factors which show both overlapping and distinct neurotrophic activities. Using site-directed mutagenesis, chimeric molecules were constructed where different combinations of sequences from BDNF replaced the corresponding sequences in NGF. The resulting molecules were transiently expressed in COS cells and conditioned media containing the chimeric proteins were assayed for biological activity in explanted chick sympathetic, spinal and nodose ganglia. Our results show that the biological specificities of the two proteins are obtained by specific combinations of a set of sequences that differ between the two molecules. Some of these combinations allowed us to engineer molecules which display multiple neurotrophic activities recruited from both the NGF and BDNF proteins.     

Glutamic acid decarboxylase of embryonic avian retina cells in culture: Regulation byγ-aminobutyric acid (GABA)

1. Retina-cell aggregate cultures expressed glutamate decarboxylase activity (L-glutamate 1-carboxylase; EC 4.1.1.15) as a function of culture differentiation. 2. Glutamic acid decarboxylase (GAD) activity was low in the initial phases of culture and increased eight-fold until culture day 7, remaining high up to day 13 (last stage studied). 3. The addition of GABA to the culture medium 24 h after cell seeding almost totally prevented the expression of GAD activity. 4. In association with decreased enzyme activity, aggregates exposed to GABA did not display immunoreactivity for GAD, suggesting that GAD molecules were either lost from GABAergic neurons or significantly altered with GABA treatment. 5. Control, untreated aggregates showed intense GAD immunoreactivity in neurons. Positive cell bodies were characterized by a thin rim of labeled cytoplasm with thickest labeling at the emergence of the main neurite. 6. Heavily labeled patches were also observed throughout the aggregates, possibly reflecting regions enriched in neurites. 7. The GABA-mediated reduction of GAD immunoreactivity was a reversible phenomenon and could be prevented by picrotoxin.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.