Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density

Total cell density in growing insect cell suspension cultures was accurately estimated from both off-line and on-line measurements of optical density (OD). The latter measurements were done with an in-situ autoclavable OD sensor. The ability to continuously monitor cell density in insect cell cultures may be useful for the development of a large-scale process for recombinant protein production using baculovirus expression vectors.     

Specific DNA probes for the identification of the fish pathogen,Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Germination and regeneration mechanisms in Mediterranean degenerate forests

Abstract. Germination and developmental behaviour during juvenile stages are of importance in relation to succession mechanisms acting on plant communities. This is particularly so in the Mediterranean region, where the climatic summer drought causes stress. We investigated and compared germination and development of four tree species in two parts of the French Mediterranean region where the original dominant tree species (Quercus pubescens) has been largely replaced by Quercus ilex in the southern part, and Castanea sativa in the northern part. Observed differences in germination rates and developmental behaviour, especially shootroot ratio and the role of water supply, suggest that other species may eventually replace the current dominant species.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Rapid recovery of photosynthetic cell suspension cultures following heterotrophic bleaching

Summary Seven suspension-cultured lines of five different species (Amaranthus powellii Datura innoxia, Glycine max, Gossypium hirsutum, andNicotiana tabacum × Nicotiana glutinosa fusion hybrid), which had been grown under photomixotrophic conditions, were placed under heterotrophic conditions (darkness and media with 3% sucrose or starch) where the chlorophyll levels declined to near zero. After three transfers over a 70-d period, the cells were placed back into photomixotrophic or photoautotrophic conditions where regreening occurred rapidly and continued growth was observed. This rapid adaptation to photosynthetic conditions contrasts with the original initiation process for these cultures, which required many months and an apparent selection since many of the original cells died. Thus, these seven photosynthetic cell suspension cultures appear to be different from the original cultures due possible to genetic or adaptive changes.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Effects of magnesium and iron on lipid peroxidation in cultured hepatocytes

In primary cultures of rat hepatocytes, the effects of extracellular Mg²⁺ and Fe on lipid peroxidation (LPO) as measured by means of malondialdehyde (MDA) formation were investigated.Incubation of hepatocytes at decreasing extracellular Mg²⁺ concentration enhanced LPO, depending on extracellular Fe. About 96% of MDA accumulated in the culture medium. Addition of desferrioxamine prevented LPO.Additionally, the formation of oxygen free radicals was determined by fluorescence reduction of cis-parinaric acid. With this method, an immediate decay of fluorescence was found after addition of Fe²⁺. Fluorescence reduction was completely prevented by desferrioxamine, indicating the function of extracellular Fe. This mechanism may operate additionally to the increase in intracellular Fe and intracellular formation of oxygen free radicals during Mg deficiencyin vivo.