Characterization of a glucuronyltransferase: neolactotetraosylceramide glucuronyltransferase from rat brain

The properties of a rat brain glucuronyltransferase, which is presumed to be associated with the biosynthesis of the HNK-1 epitope on sulfoglucuronyl glycolipids, are described. The enzyme required divalent cations for reaction, with maximal activity at 10mm Mn²⁺, and exhibited a dual optimum at pH 4–5 and pH 6 depending upon the buffer used, with the highest activity at pH 4.5 in MES buffer. This enzyme strictly recognized the Gal1-4GlcNAc terminal structure, and was highly specific for neolacto (type 2) glycolipids as acceptor. The enzyme was localized specifically in the brain, and was barely detected in other issues, including the thymus, spleen, liver, kidney, lung, and sciatic nerve fibres. Phosphatidylinositol and phosphatidylserine increased the enzymatic reaction 4.4- and 2.3-fold, respectively, whereas phosphatidylcholine slightly decreased the rate.Abbreviations GlcA glucuronic acid - Lc-PA₁₄ lactotetraose-phenyl-C₁₄H₂₉ - nLc-PA₁₄ neolactotetraose-phenyl-C₁₄H₂₉ - nLcOse₄-Cer neolactotetraosylceramide - NP-40 Nonidet P-40 - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SGGL sulfoglucuronyl glycolipid     

Studies on the association of NIDDM in Japanese patients with hyperthyroid Graves' disease.

We attempted to analyze the association of hyperthyroid Graves' disease with non-insulin-dependent diabetes mellitus (NIDDM). Forty-nine patients (23 males and 26 females; 7.6%) of a total of 647 patients with hyperthyroid Graves' disease had NIDDM, several years before or after Graves' disease was diagnosed. Only 1 patient had insulin-dependent diabetes mellitus. Compared with the general Japanese population (n = 9,133), the incidence of NIDDM (n = 348; 3.9%) in patients with Graves' disease was higher in all age groups. Only 4 patients (8.2%) of the 49 hyperthyroid patients with NIDDM had a history of being overweight (body mass index > 25). In contrast, 276 (79.9%) of the 348 diabetic patients were currently or previously overweight. Moreover, the incidence of a family history of diabetes (13 of the 49 hyperthyroid Graves' patients with NIDDM; 26.5%) was also lower in the patients with NIDDM in the general Japanese population (50% incidence). The male:female ration in patients with Graves' disease and NIDDM was 1:1.1; much different from that in the total Graves' disease population (1:4.1). Analysis of the HLA loci A, B, C, DR and DQ (35 determinations) in 35 hyperthyroid patients with NIDDM and in 386 subjects from the general population revealed a highly significant difference between them in the incidence of HLA-Cw4, -DR2, -DQw1, -DQw3 and -DQw4. This study suggests that there was an association of Graves' disease with NIDDM. A significant association of HLA-DR and -DQ loci was observed in hyperthyroid Graves' patients with NIDDM.     

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Yeast glucan of Pneumocystis carinii cyst wall: an excellent target for chemotherapy.

Pneumocystis pneumonia is the most serious opportunistic infection in immunocompromised patients, particularly those with AIDS. Approved therapy is limited to pentamidine and inhibitors of folic acid synthesis, but these drugs show a high rate of adverse reactions in AIDS patients emphasizing the urgent need for additional effective therapies. Progress has, however, been hindered by lack of knowledge about this parasite's cellular characteristics. Previously we reported that beta (1,3)glucan is a major component of the Pneumocystis carinii cyst wall. This study shows that administration of aculeacin A, an inhibitor of beta (1,3)glucan biosynthesis, affects cyst wall formation, inhibits cyst maturation, and prevents severe pneumonia in steroid-treated rats. Thus this study not only demonstrates that beta (1,3)glucan is indispensable for growth of the parasite in rats, but suggests a new therapeutic strategy for human pneumocystosis.     

Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes.

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp41 contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp41 could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti-astrocyte antibodies, which were directed at a common epitope with the gp41 sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients.     

Ontogeny of absence-like and tonic seizures in the spontaneously epileptic rat

The ontogeny of epileptic seizures in spontaneously epileptic rats (SER; zi/zi, tm/tm) was studied by examining behaviour and electroencephalogram (EEG) simultaneously. Weight gain and survival time were also studied. Compared with the control Kyo:Wistar rats, SER showed a much smaller increase in body weight. All male and female SER died before 20 and 18 weeks of age, respectively. Body tremor was observed at 2 weeks of age but disappeared after 11 weeks. Staggering gait appeared after 7 weeks of age, and intensified with age. Absence-like seizures characterized by paroxysmal appearance of 5-7 Hz spike-wave-like complexes were observed in the cortical or hippocampal EEG after 5 weeks of age, and tonic seizures with low voltage fast waves were observed after 6 weeks of age. All SER exhibited both absence-like and tonic seizures with high frequencies from 12 weeks of age. Differences with other spontaneous rat models of epilepsy and application methods for estimating seizure-inhibitory effects of anti-epileptic drugs are discussed.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Characterization and Intracellular Distribution of Pea Phytochrome I Polypeptides Expressed in E. coli

The pea phytochrome I (PI) cDNA clone, pPP1001, was expressedin E. coli. The plasmid pPP1001 contains pea PI cDNA which coversthe entire coding region with the Shine-Dalgarno consensus sequencejoined upstream of the cDNA in an expression vector pNUT6. ThepPP1001 transformants formed typical inclusion bodies when culturedat 32?C. However, when cultured at 37?C or in the presence ofisopropyl-ß-D-thiogalactopyranoside (IPTG) at 32?C,the bacteria lysed before inclusion body formation. Immuno-stainingwith anti-PI monoclonal antibody, mAP5, of transformants fixedby cold methanol showed that stainable materials were distributedin whole cytoplasmic region. When the inclusion bodies wereobserved clearly, the regions corresponding to the inclusionbodies became difficult to stain. Western blot analysis, however,showed that a ca. 100 kDa PI polypeptide was detected in thefraction from inclusion bodies and a ca. 90 kDa PI polypeptidefrom the soluble fraction. The amino acid sequence analysisof purified 100 kDa PI sample indicated that its amino terminusis blocked. However, minor signals in one experiment yieldeda sequence corresponding to the expected amino terminus of peaPI except for the initiation methionine. One of the anti-peaPI monoclonal antibodies, mAP9, that recognizes the near N-terminusof pea phytochrome was reactive to the 100 kDa polypeptide. (Received June 22, 1990; Accepted November 18, 1990)     

Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)