Long-term maintenance of high rates of very-low-density-lipoprotein secretion in hepatocyte cultures. A model for studying the direct effects of insulin and insulin deficiency in vitro.

High rates of hepatic cellular triacylglycerol synthesis and very-low-density-lipoprotein (VLDL) triacylglycerol output were maintained in vitro for at least 3 days when hepatocytes were cultured in a medium lacking insulin but supplemented with 1 microM-dexamethasone, 10 mM-lactate, 1 mM-pyruvate and 0.75 mM-oleate (supplemented medium). Under these conditions VLDL output remained constant, whereas cell triacyglycerol content increased 10-fold over 3 days, suggesting that the secretory process was saturated. Insulin, present during the first 24 h period, enhanced the storage of cellular triacylglycerol by inhibiting the secretion of VLDL. This stored triacyglycerol was subsequently released into the medium as VLDL if insulin was removed. With the supplemented medium the increased rate of VLDL secretion after insulin removal exceeded that observed under 'saturating' conditions, suggesting that pre-treatment with insulin enhanced the capacity for VLDL secretion. In contrast with the short-term (24 h) effects of insulin, longer-term exposure (greater than 48 h) to insulin enhanced the secretion of VLDL compared with insulin-untreated cultures. Under these conditions, insulin increased the net rates of triacylglycerol synthesis. The results suggest that insulin affects the secretion of VLDL triacylglycerol by two distinct and opposing mechanisms: first, by direct inhibition of secretion; second by increasing triacylglycerol synthesis, which stimulates secretion. The net effect at any time depends upon the relative importance of each of these processes.     

RecD function is required for high-pressure growth of a deep-sea bacterium

A genomic library derived from the deep-sea bacterium Photobacterium profundum SS9 was conjugally delivered into a previously isolated pressure-sensitive SS9 mutant, designated EC1002 (E. Chi and D. H. Bartlett, J. Bacteriol. 175:7533-7540, 1993), and exconjugants were screened for the ability to grow at 280-atm hydrostatic pressure. Several clones were identified that had restored high-pressure growth. The complementing DNA was localized and in all cases found to possess strong homology to recD, a DNA recombination and repair gene. EC1002 was found to be deficient in plasmid stability, a phenotype also seen in Escherichia coli recD mutants. The defect in EC1002 was localized to a point mutation that created a stop codon within the recD gene. Two additional recD mutants were constructed by gene disruption and were both found to possess a pressure-sensitive growth phenotype, although the magnitude of the defect depended on the extent of 3' truncation of the recD coding sequence. Surprisingly, the introduction of the SS9 recD gene into an E. coli recD mutant had two dramatic effects. At high pressure, SS9 recD enabled growth in the E. coli mutant strain under conditions of plasmid antibiotic resistance selection and prevented cell filamentation. Both of these effects were recessive to wild-type E. coli recD. These results suggest that the SS9 recD gene plays an essential role in SS9 growth at high pressure and that it may be possible to identify additional aspects of RecD function through the characterization of this activity.     

Effects of flutamide on testicular involution induced by an antagonist of gonadotrophin-releasing hormone and on stimulation of spermatogenesis by follicle-stimulating hormone in rats.

The effects of combined treatment with an antagonist of gonadotrophin-releasing hormone (ANT) and the antiandrogen flutamide (FL) on spermatogenesis were studied in the presence and absence of exogenous follicle-stimulating hormone (FSH). After treatment for 2 weeks, the combination of ANT (RS 68439, 450-500 micrograms/kg per day, s.c.) with 10, 20 or 40 mg FL/day, s.c. was as effective as ANT plus the Leydig cell toxin ethane dimethane sulphonate (75 mg/kg per week, i.p.) in terms of reduction in weight of testes, epididymides and seminal vesicles. Thus, a daily dose of 10 mg FL/kg was sufficient to block the androgen action in the testes of ANT-treated rats. In a second experiment, rats received ANT and ANT+FL (10 mg/kg) alone or in combination with a highly purified human FSH preparation (5 or 10 iu, twice a day) for 2 weeks. FSH did not affect testosterone concentration or weight of epididymides and seminal vesicles, but ANT+FL markedly enhanced the ANT-induced reduction of testis weight, seminiferous tubule diameter and numbers of germ cells, as revealed by qualitative and quantitative analysis of testis histology. In the absence of FL, testis size and numbers of germ cells, including elongated spermatids, were increased by FSH. In the presence of FL, the effects of FSH were less pronounced with respect to the germ cells, in terms of both numbers of cells and the effective dose of FSH. Irrespective of treatment with FL, exogenous FSH increased the inhibin concentrations in serum, indicating that Sertoli cells remained responsive to FSH. From the present study it is concluded that (i) FL accelerates ANT-induced testicular involution, (ii) FSH has a role in adult spermatogenesis and (iii) the effects of FSH on advanced germ cells are influenced by androgens.     

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its precursor expressed in Escherichia coli are associated with groEL protein

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is synthesized in the cytoplasm as a precursor which is transported into the chloroplast. During or after transport the precursor is processed to its mature size by removal of an amino-terminal transit peptide. Eight small subunits and eight large subunits (synthesized in the chloroplast) assemble to form the holoenzyme. We have expressed the precursor of the small subunit in Escherichia coli as a fusion to the carboxyl terminus of staphylococcal protein A'. The fusion protein was recovered from the bacterial lysate by chromatography on IgG-agarose. A 58-kDa protein copurified with the fusion protein in approximately equal amounts. Much less of the 58-kDa protein copurified with a fusion in which the transit peptide was deleted, and it did not copurify with protein A'. The 58-kDa protein was identified as the E. coli groEL gene product with antibodies directed against a homologous mitochondrial heat shock protein. This finding is particularly interesting because a chloroplast protein involved in the assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase also is homologous to the groEL protein. These homologs could modulate protein-protein interactions during folding and assembly of subunits into native complexes.     

Modelling nonlinear dynamics of Crassulacean acid metabolism productivity and water use for global predictions

Crassulacean acid metabolism (CAM) crops are important agricultural commodities in water‐limited environments across the globe, yet modelling of CAM productivity lacks the sophistication of widely used C3 and C4 crop models, in part due to the complex responses of the CAM cycle to environmental conditions. This work builds on recent advances in CAM modelling to provide a framework for estimating CAM biomass yield and water use efficiency from basic principles. These advances, which integrate the CAM circadian rhythm with established models of carbon fixation, stomatal conductance and the soil–plant‐atmosphere continuum, are coupled to models of light attenuation, plant respiration and biomass partitioning. Resulting biomass yield and transpiration for Opuntia ficus‐indica and Agave tequilana are validated against field data and compared with predictions of CAM productivity obtained using the empirically based environmental productivity index. By representing regulation of the circadian state as a nonlinear oscillator, the modelling approach captures the diurnal dynamics of CAM stomatal conductance, allowing the prediction of CAM transpiration and water use efficiency for the first time at the plot scale. This approach may improve estimates of CAM productivity under light‐limiting conditions when compared with previous methods.     

Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds

Concurrent osmotic and chaotropic stress make MgCl₂-rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl₂ brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl₂-rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl₂-saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.     

The isolation of FOS-1, a gene encoding a putative two-component histidine kinase from Aspergillus fumigatus

In fungi, two-component histidine kinases are involved in response mechanisms to extracellular changes in osmolarity, resistance to dicarboximide fungicides, and cell-wall assembly. In the human opportunistic fungus, Candida albicans, each of the three histidine kinases plays a role in virulence. Here, we identify, for the first time, a gene, FOS-1, from the human pathogenic fungus Aspergillus fumigatus that predicts a protein with homology to two-component histidine kinases. The predicted FOS-1 protein is highly homologous to bacterial and other fungal histidine kinases in several functional domains, but is divergent at the amino- and carboxy-termini. A mutant lacking the FOS-1 locus, DeltaFOS-1, did not exhibit a detectable defect in either hyphal growth or morphology when grown on solid or liquid medium. However, in liquid medium, conidiophore development of the DeltaFOS-1 mutant was delayed. Compared to wild type, the DeltaFOS-1 strain was neither osmotically sensitive nor sensitive or resistant to a number of nondicarboximide antifungal drugs, but was highly resistant to dicarboximide fungicides and resistant to novozym 234, suggesting that FOS-1p may play a role in the regulation of cell-wall assembly.     

Inhibition of Escherichia coli cytidine deaminase by a phosphapyrimidine nucleoside

The nature of the interaction between Escherichia coli cytidine deaminase and the phosphapyrimidine nucleoside 1 has been studied kinetically and spectrophotometrically. Compound 1 was designed as a transition-state analog, and is a potent, slow-binding inhibitor of cytidine deaminase (Ashley, G. W., and Bartlett, P. A. (1982) Biochem. Biophys. Res. Commun. 108, 1467-1474). We present evidence that the binding of 1 is reversible, with no covalent linkage between the enzyme and 1. At pH 6, the rate of recovery of enzyme activity from dissociation of the E X I complex is strongly dependent on the concentration of E X I, indicating that the inhibitor dissociates reversibly. UV difference spectroscopy reveals that the chromophore of 1 is unaltered on binding to the enzyme, thus eliminating the possibility of reversible, covalent modification of the enzyme. For the binding of the active beta-anomers of 1 to cytidine deaminase, the following kinetic parameters were determined at pH 6: kon = 8300 M-1 S-1, koff = 7.8 X 10(-6) S-1, Ki = 0.9 nM. We were also able to observe and characterize time-dependent inhibition of E. coli cytidine deaminase by tetrahydrouridine, 3. This interaction involves involves initial formation of a loose complex (KD = 1.2 microM), followed by isomerization in a slow step to give a more tightly bound complex (Ki = 0.24 microM) with forward and reverse rate constants kf = 3.81 min-1 and kr = 0.95 min-1, respectively.     

Lenalidomide enhances antibody-dependent cellular cytotoxicity of solid tumor cells in vitro: influence of host immune and tumor markers

We evaluated the effect of combining lenalidomide with therapeutic antibodies on antibody-dependant cell-mediated cytotoxicity (ADCC) of solid tumor cells, and the requirement for expression of natural killer (NK) cell-activating receptors and their solid tumor surface ligands. Twenty-three human tumor cell lines (colon, breast, lung, head and neck, ovary, and bone sarcoma) were analyzed. NK effector cells were isolated from healthy donors, pre-treated with and without lenalidomide, and incubated with antibody-coated tumor cells to determine ADCC. In blocking experiments, NK cells were pre-incubated with anti-DNAM-1 or anti-NKG2D antibodies, and target colorectal cells were pre-incubated with anti-CD155 (PVR), anti-MIC-A/B, or anti-ULBP 3 antibodies. Differences between groups were assessed using unpaired and paired Student’s t test and one-way ANOVA. Lenalidomide enhanced NK cell-mediated ADCC of trastuzumab- and cetuximab-coated tumor cells. Activity against colorectal cancer cells was dependent on target antigen expression, but independent of KRAS status and FcγRIIIa genotype. The extent of ADCC and its enhancement by lenalidomide correlated with NK cell expression of NKG2D and DNAM-1, and tumor cell expression of PVR and MIC-A. Blocking of NKG2D and, to a lesser extent, DNAM-1 inhibited ADCC. Anti-MIC-A/B monoclonal antibody blocked natural cytotoxicity, but not ADCC. Lenalidomide enhances the ability of IgG1-isotype antibodies to mediate ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D–NKG2D ligand interactions, but appears to be independent of MIC-A/B. This provides a rationale for exploratory clinical studies and an assessment of potential biomarkers predictive of clinical benefit.