Human hybrid immunoglobulin M containing immunoglobulin A

Summary Some hybridoma clones made by fusion of a human lymphoblastoid cell line, HO323 with human B lymphocytes, secreted not only IgA but also IgM-like immunoglobulin molecules. The IgM-like immunoglobulin had a molecular size of 900 K which corresponded to that of IgM. Immunochemical analyses revealed that the IgM-like immunoglobulin contained two monomeric IgA and three monomeric IgM molecules. In the IgA moieties, half of original light chains were replaced withx chains derived from the IgM, and vice versa.     

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     

Immunohistochemical localization of Na+-dependent glucose transporter in rat jejunum

Summary Glucose is actively absorbed via a Na⁺-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564–575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.     

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.     

Analysis of intergenic spacer regions in the nuclear rDNA of Pharbitis nil.

The nucleotide sequence of a 4.2-kb EcoRI fragment from the intergenic region between the genes for 25S and 18S ribosomal RNA of Pharbitis nil Choisy was determined. The region contained a unique repetitive family of DNA sequences, called the RsaI family, composed of 32-bp units. The 32-bp unit was tandemly repeated in the intergenic region, and four subfamilies of repeating units were clustered as discrete blocks. The RsaI family of repeats was shown to be specific to the genus Pharbitis by Southern blot hybridization.     

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)     

Correspondence of Apoproteins of Light-Harvesting Chlorophyll a/b Complexes Associated with Photosystem I to cab Genes: Evidence for a Novel Type IV Apoprotein

Apoproteins of spinach and pea light-harvesting chlorophylla/b complexes associated with photosystem I (LHCI) were identifiedby their chlorophyll fluorescence spectra and protein sequences.Spinach LHCI holocomplex consisted of four apoproteins of 25kDa, 23 kDa, 21 kDa and 20.5 kDa. LHCI subcomplex isolated bysucrose density gradient centrifugation fluoresced at 680 nmwith a shoulder around 700–710 nm at 77 K. It containedthe 23 kDa protein of which the N-terminal sequence correspondedto Type II gene of LHCI. Another LHCI subcomplex isolated bygel electrophoresis emitted at 679 nm and contained the 25 kDaprotein, of which the N-terminus was blocked. Its internal sequenceswere determined after protease treatment and found to be homologousto Type III gene of LHCI. An oligomeric subcomplex of LHCI isolatedby gel electrophoresis emitted at 726 nm and consisted of the21 kDa and 20.5 kDa apoproteins. N-terminal sequence of the20.5 kDa component corresponded to the Type I gene of LHCI.The 21 kDa component did not have any clear homologue, but itsN-terminal sequence was weakly but significantly homologousto all LHC components particularly to Type I LHCI among others.It was, thus, concluded that the 21 kDa protein is the fourthtype of LHCI apoprotein. Similar sequence homology was foundfor pea LHCI apoproteins. (Received September 10, 1990; Accepted November 22, 1990)     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.     

13C-n.m.r. analysis of some sulphate derivatives of chitosan.

Positions of substitution with sulphate in three water-soluble sulphated derivatives of chitosan were analysed by 13C n.m.r. The structures of N-acetylchitosan 3,6-O-disulphate, sodium chitosan N-, 6-O-disulphate, and sodium chitosan 6-O-monosulphate were confirmed.