The complete nucleotide sequence of Pepper mottle virus-Florida RNA.

The Pepper mottle virus-Florida (PepMoV-FL) RNA genome was cloned and sequenced, and shown to consist of 9,717 nucleotides (nt) excluding the poly (A) tail. A single open reading frame was identified beginning at nucleotide position 169 encoding a polyprotein of 3068 amino acids. Phylogenetic sequence analysis revealed that of 44 full-length viral RNA genomes analyzed within the family Potyviridae, PepMoV-FL was most closely related to PepMoV-California (PepMoV-CA), Potato virus Y-H (PVY-H), PVY-N, PVY(o) and Potato virus V-DV42 (PVV-DV42). Using the PepMoV-FL sequence as a basis for comparison, the overall nucleotide sequence identity was highest between PepMoV-FL and PepMoV-CA at 93%, while the relationship was more distant with PVV-DV42 at 64% and for the PVY strains at 61%. A unique direct repeat sequence of 76 nucleotides was identified in the PepMoV-FL 3'-untranslated region (UTR), and this repeat sequence was confirmed not to occur in the PepMoV-CA sequence. Since the Florida isolate was among the first of the PepMoV isolates described, extensive biological and serological data on this isolate are available, and it has now been cloned and sequenced, we recommend that PepMoV-FL be recognized as the PepMoV type strain.

Complementary expression patterns of six nonessential Caenorhabditis elegans core 2/I N-acetylglucosaminyltransferase homologues.

The Caenorhabditis elegans genome contains 18 sequences related to mammalian core 2/I N-acetylglucosaminyltransferases. The six most closely related genes (gly-1 and gly-15 to gly-19) likely encode active enzymes, because are all transcribed and do not appear to be pseudogenes. Polypeptide divergence and the gene structures are both concordant with a common ancestor at the time of radiation from mammals that underwent three rounds of duplication and, most recently, a tandem duplication. Polypeptide alignments with mammalian homologues do not indicate whether the enzyme specificities are core 2, 4, or I-like or novel, but do clearly demonstrate the secondary structure characteristics of glycosyltransferases. The six homologues have essentially nonoverlapping expression patterns, unrelated by tissue type or cell lineage. The extent varies widely; gly-15 is expressed only in two gland cells, whereas gly-18 is broadly expressed in diverse cell types. gly-1, -15, -18 and -19 are expressed during adulthood; gly-16 and gly-17 appear to be restricted to embryonic or early larval stages. The parsimonious interpretation of the expression pattern and sequence data is that the catalytic activities are similar but with diverged promoters. Null alleles of three of the genes were generated without causing gross abnormality in homozygous animals. RNA-mediated interference experiments also failed to induce defects in the four genes tested. Nevertheless, the nematode has evolved six diverged core 2 GlcNAc-T-like genes, and we postulate that these arose in response to selection pressures to which C. elegans is not ordinarily subjected in the laboratory.

Genetic defects in N-glycosylation and cellular diversity in mammals.

Glycoproteins in mammalian cells are modified with complex-type aspargine-linked glycans of variable chain lengths and composition. Observations of mice carrying mutations in glycosyltransferase genes imply that N-glycan structures regulate T-cell receptor clustering and hence sensitivity to agonists. We argue that the heterogeneity inherent in N-glycosylation contributes to cellular diversity and, thereby, to adaptability in the immune system.

mab-20 encodes Semaphorin-2a and is required to prevent ectopic cell contacts during epidermal morphogenesis in Caenorhabditis elegans.

The Semaphorins are a family of secreted and transmembrane proteins known to elicit growth cone repulsion and collapse. We made and characterized a putative null mutant of the C. elegans gene semaphorin-2a (Ce-sema-2a). This mutant failed to complement mutants of mab-20 (Baird, S. E., Fitch, D. H., Kassem, I. A. A. and Emmons, S. W. (1991) Development 113, 515-526). In addition to low-frequency axon guidance errors, mab-20 mutants have unexpected defects in epidermal morphogenesis. Errant epidermal cell migrations affect epidermal enclosure of the embryo, body shape and sensory rays of the male tail. These phenotypic traits are explained by the formation of inappropriate contacts between cells of similar type and suggest that Ce-Sema-2a may normally prevent formation or stabilization of ectopic adhesive contacts between these cells.

Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Elevated levels of glycocojugates, commonly observed in the myocardium of diabetic animals and patients, are postulated to contribute to the myocardial dysfunction in diabetes. Previously, we reported that UDP-GlcNAc: Galbeta1-3GalNAcalphaRbeta1-6-N-acetylglucosaminyltransferas e (core 2 GlcNAc-T), a developmentally regulated enzyme of O-linked glycans biosynthesis pathway, is specifically increased in the heart of diabetic animals and is regulated by hyperglycemia and insulin. In this study, transgenic mice overexpressing core 2 GlcNAc-T with severe increase in cardiac core 2 GlcNAc-T activities were normal at birth but showed progressive and significant cardiac hypertrophy at 6 months of age. The heart of transgenic mice showed elevation of sialylated O-glycan and increases of c-fos gene expression and AP-1 activity, which are characteristics of cardiac stress. Furthermore, transfection of PC12 cells with core 2 GlcNAc-T also induced c-fos promoter activation, mitogen activated-protein kinase (MAPK) phosphorylation, Trk receptor glycosylation, and cell differentiation. These results suggested a novel role for core 2 GlcNAc-T in the development of diabetic cardiomyopathy and modulation of the MAP kinase pathway in the heart.-Koya, D., Dennis, J. W., Warren, C. E., Takahara, N., Schoen, F. J., Nishio, Y., Nakajima, T., Lipes, M. A., King, G. L. Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice.

Glycoprotein glycosylation and cancer progression.

Glycosylation of glycoproteins and glycolipids is one of many molecular changes that accompany malignant transformation. GlcNAc-branched N-glycans and terminal Lewis antigen sequences have been observed to increase in some cancers, and to correlate with poor prognosis. Herein, we review evidence that beta1, 6GlcNAc-branching of N-glycans contributes directly to cancer progression, and we consider possible functions for the glycans. Mgat5 encodes N-acetylglucosaminyltransferase V (GlcNAc-TV), the Golgi enzyme required in the biosynthesis of beta1,6GlcNAc-branched N-glycans. Mgat5 expression is regulated by RAS-RAF-MAPK, a signaling pathway commonly activated in tumor cells. Ectopic expression of GlcNAc-TV in epithelial cells results in morphological transformation and tumor growth in mice, and over expression in carcinoma cells has been shown to induce metastatic spread. Ectopic expression of GlcNAc-TIII, an enzyme that competes with GlcNAc-TV for acceptor, suppresses metastasis in B16 melanoma cells. Furthermore, breast cancer progression and metastasis induced by a viral oncogene expressed in transgenic mice is markedly suppressed in a GlcNAc-TV-deficient background. Mgat5 gene expression and beta1, 6GlcNAc-branching of N-glycans are associated with cell motility, a required phenotype of malignant cells.

Protein glycosylation in development and disease.

N- and O-linked glycan structures of cell surface and secreted glycoproteins serve a variety of functions related to cell-cell communication in systems affecting development and disease. The more sophisticated N-glycan biosynthesis pathway of metazoans diverges from that of yeast with the appearance of the medial-Golgi beta-N-acetylglucosaminyltransferases (GlcNAc-Ts). Tissue-specific regulation of medial- and trans-Golgi glycosyltransferases contribute structural diversity to glycoproteins in metazoans, and this can affect their molecular properties including localization, half-life, and biological activity. Null mutations in glycosyltransferase genes positioned later in the biosynthetic pathway disrupt expression of smaller subsets of glycan structures and are progressively milder in phenotype. In this review, we examine data on targeted mutations affecting glycosylation in mice and congenital mutations in man, with a view to understanding the molecular functions of glycan structures as modulators of glycoprotein activity. Finally, pathology associated with the expression of GlcNAc-Ts in cancer and diabetes-induced cardiac hypertrophy suggest that inhibitors of these enzymes may have therapeutic value.

Modification of CD43 and other lymphocyte O-glycoproteins by core 2 N-acetylglucosaminyltransferase.

CD43, the major leukocyte sialoglycoprotein, is expressed on T lymphocytes in two predominant glycoforms. CD43 115 kDa is a pan T cell marker and is specifically recognized by the monoclonal antibody S7. CD43 130 kDa is associated with T cell activation and is specifically recognized by the monoclonal antibody 1B11. The thymoma EL-4 has been identified to express mainly CD43 115 kDa and little or no CD43 130 kDa. Transfection of EL-4 cells with core 2 beta 1-->6N-acetylglucosaminyltransferase (C2GnT), an enzyme in the O-glycan biosynthesis pathway, resulted in an enhanced expression of the 1B11 epitope, CD43 130 kDa, and a loss of expression of the S7 epitope, CD43 115 kDa. Analysis of CD43 by SDS-PAGE revealed that CD43 in C2GnT transfected EL-4 cells has a molecular weight of 125 kDa compared to 115 kDa in nontransfected or control transfected EL-4 cells. SDS-PAGE analysis of three other lymphocyte O-glycoproteins, CD44, CD45, and RPTP alpha, revealed that C2GnT expression resulted in a molecular weight increase of approximately 3-5 kDa for each of these three cell surface glycoproteins. Our data indicate that, while CD43 may be a predominant substrate for C2GnT, other lymphocyte O-glycoproteins are also modified by this glycosyltransferase. Increased reactivity of cells with the monoclonal antibody 1B11, which specifically detects the expression of murine CD43 130 kDa, may thus be a marker of increases in branching of O-linked glycans generally.

Olfactory responses ofIps plastographus maritimus lanier (coleoptera: Scolytidae) to insect and host-associated volatiles in the laboratory.

Attraction of both sexes ofIps plastographus maritimus Lanier to bark-phloem-xylem discs of Monterey pine,Pinus radiata D. Don, was demonstrated in the laboratory. Increasing concentrations of male and female volatiles trapped separately and released in a one-to-one ratio decreased attraction for both sexes combined. Attraction of both sexes to volatiles derived from males and females tunneling together in a one-to-one ratio increased with increasing concentration of extract. Attraction of males and females to male-infested discs and to trapped male volatiles increased with increasing dose of males or male extract. Attraction of males and females to female-infested discs and to trapped female volatiles was also demonstrated. The presence of females in male galleries reduced the attractiveness of infested disks to both sexes combined. Increasing numbers of females, tunneling separately from males in the same disc, reduced attraction of males, but not females. When a constant attractive dose of male volatiles was released with increasing doses of female volatiles, there was no difference in response of either sex when female volatiles were present compared with the response to male volatiles alone. When a constant attractive dose of male volatiles was released with increasing concentrations of volatiles derived from males and females tunneling together in a one-to-one ratio, attraction ofI. p. maritimus decreased. Response of females was frequently higher than that of males to the same attractant source. Hence, both sexes produce an attractant, and both sexes tunneling together in the same gallery reduce attraction of males and females to an attractive dose of male attractant.

GlcNAc-transferase V and core 2 GlcNAc-transferase expression in the developing mouse embryo.

UDP-GlcNAc:Manalpha1-6Manbeta-R beta1-6-N-acetylglucosaminyltransferase V (GlcNAc-TV) and UDP-GlcNAc:Galbeta1-3GalNAc-R beta1-6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) are Golgi enzymes that catalyse the biosynthesis of beta1-6GlcNAc-branched intermediates in the N- and O-linked biosynthesis pathways, respectively. The activities of these enzymes change during haematopoiesis, embryo-carcinoma cell differentiation and following malignant transformation, but little is known about their expression in normal adult tissues and during embryogenesis. We have examined the expression of GlcNAc-TV and core 2 GlcNAc-T in sections of post-implantation mouse embryos by in situ RNA hybridization. The two enzymes showed distinct temporal and spatial patterns of expression. Core 2 GlcNAc-T mRNA was widely expressed at embryonic day (E) 7, and became restricted to a subset of mucin- and cartilage-producing tissues at E11.5 through to E17.5. GlcNAc-TV transcripts were absent at E7, became expressed throughout E9.5 embryos, and then progressively restricted to regions of the developing central nervous system and to specialized epithelia of skin, intestine, kidney, endocrine tissues and respiratory tract. In the adult gonads, GlcNAc-TV transcripts were excluded from germ cells, but were detected in the follicular and testicular cells. Leukoagglutinin (L-PHA)-reactive oligosaccharides co-localized with GlcNAc-TV transcripts in skin, kidney and intestine, but brain showed unexpectedly low overall staining punctuated by bright staining of the vascular endothelium. A common feature of cells in basal epithelia and in the cortical neural epithelium is the capacity to migrate, a cellular function which may require GlcNAc-TV-dependent glycoconjugates.

Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue.

Primary cardiac abnormalities have been frequently reported in patients with diabetes probably due to metabolic consequences of the disease. Approximately 2,000 mRNA species from the heart of streptozotocin-induced diabetic and control rats were compared by the mRNA differential display method, two of eight candidate clones thus isolated (DH1 and 13) were confirmed by Northern blot analysis. The expression of clone 13 was increased in the heart by 3.5-fold (P < 0.05) and decreased in the aorta by twofold (P < 0.05) in diabetes as compared to control. Sequence analysis showed that clone 13 is a rat mitochondrial gene. DH1 was predominantly expressed in the heart with an expression level 6.8-fold higher in the diabetic rats than in control (P < 0.001). Insulin treatment significantly (P < 0.001) normalized the expression of DH1 in the hearts of diabetic rats. DH1 expression was observed in cultured rat cardiomyocytes, but not in aortic smooth muscle cells or in cardiac derived fibroblasts. The expression in cardiomyocytes was regulated by insulin and glucose concentration of culture media. The full length cDNA of DH1 had a single open-reading frame with 85 and 92% amino acid identity to human and mouse UDP-GlcNAc:Gal beta 1-3GalNAc alpha R beta 1-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), respectively, a key enzyme determining the structure of O-linked glycosylation. Transient transfection of DH1 cDNA into Cos7 cells conferred core 2 GlcNAc-T enzyme activity. In vivo, core 2 GlcNAc-T activity was increased by 82% (P < 0.05) in diabetic hearts vs controls, while the enzymes GlcNAc-TI and GlcNAc-TV responsible for N-linked glycosylation were unchanged. These results suggest that core 2 GlcNAc-T is specifically induced in the heart by diabetes or hyperglycemia. The induction of this enzyme may be responsible for the increase in the deposition of glycoconjugates and the abnormal functions found in the hearts of diabetic rats.

Glycosylation.

The majority of candidate recombinant therapeutics are glycoproteins. Four aspects of glycobiology are requisite if the full potential of such reagents is to be realised: an understanding of glycan biosynthesis and its regulation, rapid and sensitive oligosaccharide analytical techniques, determination of structure-function relationships in protein-linked carbohydrate; and manufacturing systems where product glycosylation is manipulable and consistent. The past year has seen significant progress in all of these areas, exemplified by the approval of glycosylation-engineered glucocerebrosidase for clinical use in replacement therapy of Gaucher's disease.

Antisense and sense cDNA expression cloning using autonomously replicating vectors and toxic lectin selection.

CHOP2 cells, a subline of the Chinese hamster ovary (CHO) cell glycosylation mutant Lec2 which expresses polyoma virus large T antigen, was used as the host cell line to select cDNAs conferring resistance to the toxic effects of ricin. Glycoconjugates in CHOP2 cells are deficient in sialic acid and therefore the cells are hypersensitive to ricin, a galactose-binding lectin. CHOP2 cells acquiring cDNA that either corrected the Lec2 mutation or created a Lec2/Lec1 phenotype were expected to show a selective growth advantage in ricin-containing medium. After a single cycle of transfection with a lymphoid cDNA library in pCDM8 followed by ricin selection, the the predominant cDNA recovered from the cells was antisense N-acetyl-glucosaminyltransferase I (GlcNAc-TI) which conferred a Lec2/Lec1 phenotype. cDNA encoding GlcNAc-TI in a sense orientation was also enriched by transfecting a cDNA library into CHOP-1 cells, a mutant deficient in this enzyme, and selecting in medium containing ConA lectin. The results show that cell selection with toxic agents can be used to expression-clone both antisense and sense cDNA sequences from bi-directional cDNA libraries in the pCDM8.

Environmental factors associated with continuers and terminators in adult out-patient psychotherapy.

Two separate studies examined five areas in the patient's environment (e.g. life adjustment, external support, alternative counsel, intercurrent stress events, and other factors such as transport availability, and difficulty obtaining time off work) which may influence continuation or premature termination from psychotherapy. In the first study, a mail questionnaire was completed by 69 adult clients (32 terminators and 37 continuers) who attended for therapy at a state-controlled psychiatric out-patient clinic. Multivariate analysis of variance on each set of environmental variables failed to achieve significance. Univariate analysis, however, showed the effect of alternative counsel in the community such as the number of people with whom clients could discuss their problems outside of the treatment setting. Continuers, for example, had access to more individuals in the community and actually received more help from specific individuals (e.g. spouses) than did terminators. A replication study was conducted on 45 clients (21 terminators and 24 continuers) who attended for treatment during a further period. Results from the second study showed similar patterning in the data as for Study 1. The role of informal support in conjunction with therapy cannot be overlooked as possibly assisting clients to continue towards their desired goals of treatment.

Conceptual frameworks and the philosophical foundations of general living systems theory.

The continued expansion and fragmentation of biological disciplines impede education, communication, and efficient advance of biological research. Reversing these trends may require a unification of theories and concepts from all levels of biological organization. One form of this unification is the statement of generalizations that apply to all living systems. We explore the philosophical foundations of general theories of living systems by analyzing conceptual frameworks as they apply to biology. This analysis examines the relation of empirical observation, theories, and conceptual frameworks within the context of an individual scientist's conceptual continuum. Also presented are a small set of translevel generalizations that articulate our conceptual framework of living systems in terms of organismic system organization, the environmental system-organismic system dyad, system capacity, and system incorporation. A set of procedural rules are stated which suggest minimum criteria for the evaluation of the explanatory adequacy of biological theories. The relation of this work to other similar analyses and syntheses of biological knowledge is discussed.