Molecular organization of the human CD3 gene family on chromosome 11q23

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 , , and chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 and chain genes in vectors designed for rapid and efficient chromosome walking. The human CD3 gene was located in the region immediately downstream of the CD3 and genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 gene located 26 kb downstream of the CD3 and genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11g23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.     

The skeletal isotopic composition as an indicator of ecological and physiological plasticity in the coral genus<Emphasis Type="Italic"> Madracis</Emphasis>

Three species of the reef coral genus Madracis display skeletal isotopic characteristics that relate to depth, colony topography, and consequently to coral physiology. The joint interpretation of skeletal ¹³C and ¹⁸O provides information on the ecological plasticity and adaptation to depth of a coral species. Isotopic results are most easily understood in terms of kinetic effects, which reduce both ¹⁸O and ¹³C below isotopic equilibrium values, and metabolic effects, which only influence the skeletal ¹³C. Madracis mirabilis is adapted to depths shallower than 20 m, and shows the greatest range in kinetic effects and the strongest metabolic ¹³C enrichments caused by symbiont photosynthesis. Madracis formosa lives deeper than 40 m, and shows a reduced range of kinetic effects and relatively weak metabolic ¹³C enrichments. Madracis pharensis inhabits depths from 5 to >60 m, and does not attain the strength of kinetic effects of either of the other two species, apparently because it is not quite as well adapted to rapid growth at either extreme.     

Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Summary Plasmid-coded -glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl--d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The -glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the -glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of -glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK _m values for cellobiose and PNPG indicated that the -glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the -glucosidase fromE. coli pJS3 showed higher affinity for PNPG.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

The responses of one class of neurons in the optic tract of crayfish (Procambarus) to monochromatic light

Summary Spectral sensitivity curves for four sustaining neurons in the optic tracts of Procambarus clarkii were determined under dark-adapted and chromatic light-adapted conditions. The _max in the dark-adapted state is at 570 to 575 nm, and shifts to longer wavelengths in the violet-light-adapted state (Fig. 4). Red-light-adaptation suppresses the sensitivity of the yellow-green receptors of the eye and alters the discharge pattern of the sustaining neurons, thereby exposing an input with a _max at 445 nm from the blue-sensitive receptors (Fig. 5). Such data raise the possibility that sustaining neurons may carry information that functions in color vision.This work was supported by a predoctoral fellowship FO1-GM-31,230 and then a training grant 2TO1-GM00836 to D.L.T. and grant NB-05423 to J.L.L. and Biomedical Sciences Support Grant 5-S05 FR-07091, all from the U.S.P.H.S.     

“Garden hunting” in the American tropics

Using faunal analysis, this article outlines a coastal mammalian harvesting pattern involving a few terrestrial mammals whose biomass appears to have been greater when associated with man than under natural conditions. Archeological evidence suggests that these animals fed regularly on cultivated crops and were hunted in house gardens and cultivated fields. By concentrating the supply of both carbohydrates and animal protein, garden hunting may have eliminated seasonality and scheduling problems. And because it artificially increased the biomass of selected animals, it may have functioned as a substitute for animal domestication.     

Starch hydrolysing enzymes in the developing barley grain

Summary The enzymes -amylase (-1, 4-glucan 4-glucanohydrolase, 3.2.1.1), -amylase (-1,4-glucan maltohydrolase, 3.2.1.2) and phosphorylase (-1,4-glucan: orthophosphate glucosyltransferase, 2.4.1.1) were assayed in whole grains of barley throughout the maturation period. -amylase and phosphorylase had peaks of activity between 25 and 30 days after anthesis. On the other hand the activity of -amylase in both the available and latent forms reached a maximum value at 35 days after anthesis which did not decrease thereafter. -amylase activity was also assayed throughout development in the endosperm, aleurone, testa pericarp and embryo. Latent -amylase reached a constant maximum value in endosperm at 35 days but available -amylase reached a peak of activity at 25 days and then declined to zero at 45 days. Only latent -amylase was associated with the aleurone layer and activity rose to a maximum value at 35 days. The testa pericarp had mainly latent -amylase whose activity fell from an early maximum at 21 days to zero at 35 days. No hydrolytic activity was associated with the embryo. The phosphorylase activity was low and mainly associated with the endosperm fraction.     

Enzymatic basis for sialyl-Tn expression in human colon cancer cells

Sialyl-Tn antigen (SA2-6 GalNAc-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R 3-Gal-transferase (core 1 3-Gal-transferase) was lacking. Core 1 3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc1-6 (Gal1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 3-Gal-transferase activity. The lack of core 1 3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.     

Cellular redox state and activating protein-1 are involved in ascorbate effect on calcitriol-induced differentiation

Summary Ascorbate has been related to the differentiation of several mesenchymal cells including haematopoietic cells. We have previously demonstrated that ascorbate enhances the activity of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on monocytic differentiation of HL-60 cells. Here, we show that ascorbate-mediated modification of cellular redox state and AP-1 (activating protein-1) DNA binding during early phases are related to the enhancing effect of ascorbate on differentiation. Ascorbate, but not its fully oxidized form, dehydroascorbate, or an ascorbate analogue with a low rate of oxidation, ascorbate-2-phosphate, enhanced the differentiation induced by 1,25(OH)₂D₃, modified cytosolic reactive oxygen species levels and mitochondrial redox potential (_m), and modulated AP-1 DNA binding in HL-60 cells. Ascorbate itself increased AP-1 binding to DNA in noninduced cells, whereas it inhibited AP-1 binding in 1,25(OH)₂D₃-induced cells. However, ascorbate increased the mRNA levels ofc-jun, junB. andc-fos in 1,25(OH)₂D₃-induced cells. Taken together, these results suggest that the enhancing effect of ascorbate on HL-60 differentiation induced by 1,25(OH)₂D₃ is related to its effect on the cellular redox state and the modulation of AP-1 activity.Abbreviations 1,25-(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - _m mitochondrial transmembrane potential - AP-1 activating protein-1 - Asc-2-P ascorbate-2-phosphate - DHA dehydroascorbate - DiOC₆(3) 3,3-dihexyloxacarboxyanine iodide - EMSA electromobility shift assay - NBT nitroblue tetrazolium - ROS reactive oxygen species     

Transglycosylation reactions by exoglycosidases from the termite Macrotermes subhyalinus

The ability of four exoglycosidases (-galactosidase, -glucosidase, -glucosidase and invertase) from the termite Macrotermes subhyalinus to catalyse tranglycosylation reactions was tested using lactose, cellobiose, maltose and sucrose as glycosyl donors and 2-phenylethanol as glycosyl acceptor. The experimental conditions were optimized in relation to the time course of the reaction, pH and concentrations of glycosyl donor and acceptor. Whereas the hydrolytic activity was largely predominant over the transferase activity with -galactosidase and -glucosidase, the transglycosylation activity represented 68% with -glucosidase. In addition, as demonstrated by the transglycosylation product formed, the hydrolysis of sucrose was catalysed by -glucosidase and not by invertase. On the basis of this work, -glucosidase from M. subhyalinus appears to be a valuable tool for the preparation of neoglycoconjugates.     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

Summary When lignocellulosic materials are hydrolysed using cellulase fromTrichoderma reesei, cellobiose accumulates due to a deficiency in -glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo -1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased -glucosidase activity and greatly improved hydrolytic potential.     

Molecular analysis of plants regenerated from embryogenic cultures of hybrid sugarcane cultivars (Saccharum spp.)

Summary The genomic stability of tissue culture regenerants of sugarcane (Saccharum spp. hybrids, cvs CP721210, CP68-1067 and B43-62) was analyzed by DNA restriction fragment length polymorphism (RFLP). Plants regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts were used. Total DNA isolated from 19 different sources was digested with EcoRI, HindIII, BamHI, BamHI, EcoRI and PstI and probed with six known maize mitochondrial genes (coxI, coxII, atpA, atp6, atp9 and rrn18-rrn5), three random maize mitochondrial cosmid clones, two random maize chloroplast cosmid clones and a wheat Nor locus clone. Hybridization patterns indicated that the variation observed was minor and appeared only in the secondcycle regenerants. No differences were observed among the three cultivars and the regenerants from calli, suspension culture, cryopreserved suspension culture and protoplasts. Mitochondrial DNA (mtDNA) isolated from CP72-1210 plants and its embryogenic cell suspensions, and bulk samples from all CP72-1210 regenerants pooled together were digested with EcoRI, HindIII, PstI, BamHI and SalI and probed with three recombinationally active wheat mtDNA clones, K, K3 and X2. No variation in the mtDNA restriction patterns was observed between the CP72-1210 plants and its regenerants. However, restriction pattern variation was observed only from EcoRI digestion, and hybridization patterns of K3, K and X2 revealed minor variations in the mtDNA of cell suspensions when compared with the DNA of the CP72-1210 plant. Except for a qualitative variation detected by the X2 probe and minor stoichiometric variations detected by the K3 probe, sugarcane DNAs were found to be stable after plant regeneration.Florida Agriculture Experiment Station Journal Series No. R-02703     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Labdane diterpene derivatives from Holwaya mucida

Clumps of white crystals present in 40-day-old malt agar cultures of Holwaya mucida were isolated as long white needles by crystallization from ethanol following short extraction with chloroform. The levorotary compound ([] ₂₈₉ ²¹ =-193.8°) was recognized as a -lactone (C₁₇H₂₀O₅) by infrared and mass spectrometry. It was identified as 7-methoxy-3a, 10b-dimethyl-1, 2, 3, 3a, 5a, 7, 10b, 10c-octahydro-4H, 9H-furo[2, 3, 4 : 4, 5]naphtho[2, 1-c]pyran-4, 9-dione, a labdane-derived compound known as antibiotic LL-Z1271. Preparative thin-layer chromatography of the mother liquor afforded 2 minor metabolites. One was identified as LL-Z1271, the demethylated analogue of LL-Z1271. The other one named LL-Z1271, was recognized as a compound related to and : its structure could not be fully elucidated. H. mucida (anamorph: Crinula calciiformis) has no taxonomic relationship with two other LL-Z1271 producing species viz. Acrostalagmus sp. (= Acremonium cf. atrogriseum) and Oidiodendron truncatum.     

Micromatabilin, a new biliverdin conjugate in the spider,Micromata rosea (Sparassidae)

Summary Micromatabilin, the green pigment ofMicromata rosea, is characterized as a mixture of biliverdin conjugates. The two isolated fractions have identical absorption bands (free base: 620–630 m, hydrochloride: 690 m, zinc complex: 685–690 m). Chromic acid degradation yields imides I, II, IIIa, and IIIb. Differences in the non-hydrolytic degradation and in polarity lead to the conclusion that fraction 1 is a monoconjugate and fraction 2a diconjugate of biliverdin.     

Mechanism of substrate hydrolysis by a thermophilic endoglucanase from Thermotoga maritima†

A cellulase from the thermophile, Thermotoga maritima, hydrolyzed oligosaccharide substrates by an exoglucanase mode of action but acted as an endoglucanase to rapidly reduce the viscosity of the soluble polysaccharides carboxymethylcellulose and barley -glucan. The V _max for hydrolysis of the substrate, p-nitrophenyl -d-cellobioside, was 42 mol min^–1 (mg protein)^–1, while that for barley -glucan was 637. The enzyme had little activity on crystalline cellulose.