Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Rco-3, a Gene Involved in Glucose Transport and Conidiation in Neurospora Crassa

Macroconidiation in Neurospora crassa is influenced by a number of environmental cues, including the nutritional status of the growing organism. Conidia formation is normally observed when the fungus is exposed to air. However, carbon limitation can induce conidiation in mycelia submerged in an aerated liquid medium. A mutant was previously isolated that could conidiate in submerged culture without imposing nutrient limitation and the gene responsible for this phenotype (rco-3) has now been cloned. RCO3 exhibits sequence similarity to members of the sugar transporter gene superfamily, with greatest similarity to glucose transporters of yeast. Consistent with this structural similarity, we find that glucose transport activity is altered in the mutant. However, growth of the mutant in media containing alternate carbon sources does not suppress conidiation in submerged culture. The properties of the mutant suggest that RCO3 is required for expression of glucose transport activity, glucose regulation of gene expression, and general carbon repression of development.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Discrimination by male ring-tailed Lemurs (Lemur catta) between the scent marks of male and those of female conspecifics

Olfaction plays an important role in the social communication of all prosimians. (The experiment reported in this paper forms part of an intensive chemobehavioral study of olfaction in Lemur catta (ring-tailed lemur) being carried out in this laboratory.) Five male Lemur cattawere tested on their behavioral responses to paired scent stimuli. Responses measured were (1) total investigation time, (2) arm-marking, (3) ABO/BO rubbing, and (4) flehmen. Males showed a strong discrimination between the scent stimuli,giving higher levels of response to female scent on measures 1, 3, and 4. This response suggests an olfactory-related preference by males for female scent under controlled conditions. This preference may be a consequence of the females’ dominance over males and the brevity of estrus in L. catta,both of which would favor such choice behavior.     

Identification of lipopolysaccharides and phospholipids of Escherichia coli in polyacrylamide gels.

Polyacrylamide gel electrophoresis of unfractionated lysates of radioactively labeled cells resolves not only proteins and polynucleotides into discrete bands but also cellular lipopolysaccharides and phospholipids. This allows a determination of the intracellular amounts of all of these macromolecules. In addition, this technique is sensitive enough to detect mutational alterations in lipopolysaccharide structure. Polyacrylamide gel electrophoresis is herein shown to be a useful tool for investigations into the structure of lipopolysaccharides and the synthesis of lipopolysaccharides and phospholipids.     

Perturbation analysis of coupled diffusion and reaction with high-order kinetics

Asymptotic solutions for the effectiveness factor and the concentration profile are obtained for mth-order chemical reactions inside a slab catalyst pellet with Robin boundary condition at the pellet's outer surface. Using perturbation analysis in the limit of large reaction order m, the effectiveness factor and the concentration profile are explicitly determined up to O(1/m). Higher-order solutions can be obtained in a systematic way if desired.     

Identification and genetics of horse lymphocyte alloantigens

Six hundred horses were tested with lymphocytotoxic antisera derived from 550 parous mares and 58 antisera produced by alloimmunization with horse blood cells. Seven equine lymphocyte specificities were identified using correlation analysis of the test data, absorption analysis and lysostripping. These specificities are expressed on lymphocytes and platelets, but not on red blood cells (RBC). Therefore, these specificities do not appear to be products of any of the eight known blood group systems of the horse. The distribution of these specificities in 113 Thoroughbred horses and 57 Arabian horses is presented. Two specificities are subtypic to two other specificities reported here. Family studies indicated that all of these specificities are products of one genetic system. However, it is not clear whether the system consists of one or more loci.     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.     

The chemistry of the collagen cross-links. Characterization of the products of reduction of skin, tendon and bone with sodium cyanoborohydride.

Reduction of tissues with sodium cyanoborohydride at pH7.4 gave results identical with those obtained by KBH4 treatment. On reduction with sodium cyanoborohydride at pH 4.4, however, a previously undetected basic compound was formed and was identified by mass spectrometry and chemical degradation techniques as dihydrohydroxymerodesmosine. Histidino-hydroxymerodesmosine was not present, and further analysis confirmed that reduced aldol, a mojor product of reduction with KBH4 at the lower pH, was also absent. These results, together with an analysis of the time course of the reduction, support previous assertions that histidino-hydroxymerodesmosine is an artifact [robins *Bailey (1973) Biochem. J. 135, 657-665] and suggests that the non-reduced form of hydroxymerodesmosine probably does not constitute a major intermolecular bond in vivo.