Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Uptake and metabolism of α-aminoadipic acid by Penicillium chrysogenum wis 54-1255

The uptake of 1-¹⁴C-dl--aminoadipate in resting mycelium of Penicillium chrysogenum Wis 54-1255 and its metabolism during benzylpenicillin formation were studied. The pH optimum for uptake at 25°C was 6.4. Over a range of concentrations from 0.01–1.0 mM, approximately 45% of 1-¹⁴C-dl--aminoadipate was taken up by carbon-starved mycelium. ¹⁴CO₂ was formed at a low rate, and the total formed amounted to only 1–3% of the 1-¹⁴C-dl--aminoadipate supplied. The intracellular pool of -aminoadipate appears to be expandable, depending on the concentration of -aminoadipate in the medium. The rate of penicillin synthesis depended on the intracellular concentration of -aminoadipate. Penicillin biosynthesis achieved half of the maximum rate at an intracellular concentration of 0.06 nmol -aminoadipate/mg dry cell weight. This low concentration, the result of adding 0.01 mM dl--aminoadipate to the medium, was sufficient to reverse the inhibition of penicillin biosynthesis caused by 10 mM extracellular l-lysine. Aminoadipate appears to be recycled during penicillin formation. Labeled -ketoadipate was formed from -aminoadipate to the extent of about 25%.Abbreviation DCW dry cell weight     

Stablizing crude and ultrafiltrated α-amylase and α-glucosidase from Aspergillus oryzae by trehalose

Thermal resistance of freeze-dried -amylase and -glucosidase in trehalose matrices (1 to 20 % w/v) stored at 90 °C and relative humidities (RH) between 0 and 44 % was studied. At RH values up to 33 %, 10 % (w/v) trehalose was necessary to retain at least 50 % of -amylase activity. For -glucosidase, 10 % (w/v) trehalose was effective only at 0 % RH. Ultrafiltration of the crude enzymatic fermentation extracts enhanced enzyme stability per se. However, ultrafiltration in combination with 1 % (w/v) trehalose retained 74 % of -glucosidase and 95 % of -amylase activities. © Rapid Science Ltd. 1998     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Synthesis of New 15-Ketosterol Analogues from Ergosterol

Ergosteryl acetate was converted through three stages into 3-acetoxy-24-methyl-5-cholesta-8(14),22-diene-15-one in 32% overall yield. The product was transformed to 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, 3-hydroxy-24-methyl-5-cholesta-8(14),22-diene-15-one, and 24-methyl-5-cholesta-8(14),22-diene-3,15-dione. The compounds were characterized by ¹H and ¹³C NMR spectra.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

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.     

Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12

The immune responses of the intestine mucosa feature the noninflammatory type, such as IgA production and oral tolerance. Th2 type cytokines have been implicated in the induction of these noninflammatory responses. In the present study, cytokine responses of CD8+ and CD4+ TCR+ intestinal intraepithelial lymphocyte ( iIEL) subsets to TCR stimulation under the influence of IL-12, IL-4, or CD28 costimulation were examined. IL-12 enhanced production of IL-10 and IFN- by the CD8+ iIEL significantly but only marginally affected the CD8+ subset, whereas IL-4 induced IL-4, IL-5, and IL-10 production and augmented TGF- production by both subsets. CD28 costimulation induced production of Th2 cytokines by CD4+ iIEL in the absence of exogenous IL-4. Unlike lymph node CD4+ cells, the CD28 costimulation-induced Th2 differentiation of CD4+ iIEL was not inhibited by IFN-. These results demonstrate active cytokine production by CD4+, CD8+, as well as CD8+ iIEL. The Th2-skewed cytokine profile of CD8+ iIEL and the IFN--resistance of Th2 differentiation of the CD4+ iIEL suggest that both iIEL subsets contribute to the induction of noninflammatory mucosal immune responses.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Protein synthesis in tissues cultured from the bovine hoof

Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone ( and II subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the - and II-subunits shows that colocalization of the - and II-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, -immunolabeling is absent in the large globules, even though II labeling is abundant throughout the period of seasonal gametogenesis. The -specific antiserum recognizes the intact -subunit as well as the reduced and deglycosylated -subunits by immunoblotting. These results indicate that an accumulation of the II-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of II-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained II-subunits remains unknown.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Ontogeny of estrogen receptor (ER) alpha and its co-localization with pituitary hormones in the pituitary gland of chick embryos

Estrogen is involved in regulating the development and hormone secretion of the anterior pituitary gland following its binding to estrogen receptors (ERs) expressed on pituitary cells. However, the pituitary is comprised of several cell types, and to date, there is no data about the specific cell types expressing ERs in embyonic chick pituitary. We therefore followed, by immunohistochemistry, the ontogeny of the pituitary ER alpha (ER), and the cell types expressing ER throughout chick embryo development. ER immunoreacitivity was restricted to the nuclei of pituitary cells. ER-immunopositive (ER⁺) cells were first detected at embryonic day 6.5 (E6.5), after which ER⁺ cells were consistently detected throughout the anterior pituitary gland, although the density of ER⁺ cells in the caudal lobe of the pars distalis was higher than that in the cephalic lobe. The proportion of ER⁺ cells in the pituitary was about 6% at E8.5; expression increased to 22% by E18.5 of gestation, with no additional change until hatching. Double-labeling of ER and pituitary hormones showed that the dominant cell types expressing ER were gonadotrophs immunopositive for luteinizing hormone (LH); the proportion of ER⁺ cells expressing LH increased throughout gestation and reached approximately 57% at hatching. About 2%–6% of thyroid-stimulating-hormone-immunopositive and 1%–2% prolactin-immunopositive cells expressed ER at later stages of embryonic development, but no growth-hormone-positive or adrenocorticotropic-hormone-positive cells expressed ER during the embryonic period. Thus, gonadotrophs are the main cell population expressing ER in the anterior pituitary gland of chick embryo, and ER is involved in regulating the development of the pituitary gland and the maturation of the hormone-secreting function.This work was supported by grants from the Natural Science Foundation for Outstanding Young Scientists of China (30325034) and the Natural Science Foundation of China (30170693, 30471264).     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

Cytokines in Brain Ischemia—The Role of TNFα

1. The role of cytokines and other inflammatory mediators in the progression of ischemic brain injury is a new and exciting era of research. Evidence in support for a role for TNF in this respect is emerging as evidence on de novo upregulation of TNF following ischemia is now well established.2. TNF administered directly to the brain parenchyma elicits local microvascular injury in the form of pericapillary edema and leukocyte adhesion to cerebral capillaries.3. TNF administered into the cerebroventricular space prior to ischemia augment the extent of tissue damage and neurological deficits.4. Specific and potent inhibitors of TNF synthesis or TNF receptors must be developed and tried to prove firmly a role for TNF in ischemic brain injury.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation

The interaction of heme-free (^o) and heme-containing (^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^–1 s^–1. This was 100-fold more rapid than the reported rate of combination of ^h and ^h proteins. This encounter-like rate of semi--hemoglobin (^oh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled ^o protein superimposed over native ^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the ₁₁ interface, was apparently responsible for the enhanced association properties of the ^o protein.     

Generation and characterization of an HLA-DRα-specific monoclonal antibody using L-cell transfectants expressing human and mouse class II major histocompatibility dimers

Among the HLA-DR-specific monoclonal antibodies (mAbs) that have been characterized previously, there is a marked shortage of DR chain-specific mAbs which stain unfixed cells. This may result from the high degree of sequence similarity between the ₁ domains of HLA-DR and H-2E leading to a state of cross-tolerance to DR in H-2-expressing mice. BALB/b (E-negative) mice were immunized with DR1-transfected mouse L cells. The chain specificity of the resulting DR-specific mAb was determined using a panel of transfectants expressing hybrid mouse/human class II heterodimers. A DR-specific mAb was generated which was capable of immunoprecipitating DR dimers and inhibiting the anti-DR alloresponse of human T-cell clones. The present study demonstrates that, with the selection of suitable recipient strain, transfectants can be useful in the generation and definition of chain-specific mouse mAbs.