Regulation of insulin synthesis in an insulin-producing cell line (RINm5F): Long-term experiments

Summary The insulin-producing cell line RINm5F, has been used in short-term experiments to evaluate insulin secretion. We sought to maintain the responsiveness of these cells to stimuli for up to 2 days. We examined the course of new insulin synthesis over this period by measuring at intervals immunoreactive insulin (IRI) in two parts: IRI in the medium (M) and IRI extracted from the cells (C). Control cells were incubated in RPMI 1640/2.8 mM glucose/10% fetal bovine serum/200 μg/ml bacitracin (to prevent insulin degradation). The addition of dibutyryl cAMP 10 mM to the experimental dishes significantly increased total (M+C) IRI at 48 hr to 37% above the insulin content of the control dishes (p<0.01). Theophylline 10 mM increased total (M+C) IRI by 24% over control (p<0.05) after 24 hrs. Glucose, glyceraldehyde, leucine, arginine, glucagon and tolbutamide, other stimulants of insulin production, had no effect. Under the experimental conditions reported here, including the use of bacitracin, IRI synthesis can be studied for up to 48 hr. Portions of this study have been published in abstract form for the 47th Annual Meeting of the American Diabetes Association, Indianapolis, Indiana, 1987. Supported in part by the American Diabetic Association, Maryland Affiliate.     

Differential Involvement of the Five RNA Helicases in Adaptation of Bacillus cereus ATCC 14579 to Low Growth Temperatures

Bacillus cereus ATCC 14579 possesses five RNA helicase-encoding genes overexpressed under cold growth conditions. Out of the five corresponding mutants, only the ΔcshA, ΔcshB, and ΔcshC strains were cold sensitive. Growth of the ΔcshA strain was also reduced at 30°C but not at 37°C. The cold phenotype was restored with the cshA gene for the ΔcshA strain and partially for the ΔcshB strain but not for the ΔcshC strain, suggesting different functions at low temperature.Bacillus cereus is a human pathogenic sporulated bacterium which is associated with emetic and diarrheal types of food-borne illnesses (4). B. cereus is widespread in the environment and in a wide range of foods. The growth domains of B. cereus strains range from psychrotrophic to nearly thermophilic and correlate with several phylogenetic clusters (15), which presumably permit B. cereus to colonize many different habitats with different thermal regimes. Many foods are stored refrigerated before consumption, and in such cases, B. cereus has to adapt to low-temperature conditions.B. cereus growth at low temperature takes place with a lag phase which may correspond to an adaptation phase (12). Cold is a stress which dramatically affects membrane fluidity, protein synthesis, and also the topology of nucleic acids (22). When exposed to low temperature, bacteria have to face a transient inhibition of protein synthesis mainly due to the presence of secondary structures in mRNA that are stabilized by cold conditions (16, 19). To overcome the translation interruption, cold-shocked cells synthesize cold-induced RNA helicases, which remove secondary structures from RNA duplexes in the presence of ATP, such as CsdA of Escherichia coli (19) or CshA of Bacillus subtilis (1). csdA and srmB deletion mutants of E. coli showed a cold-sensitive phenotype, and these RNA helicases have been described as involved in the biogenesis of the ribosomal 50S subunit at 20°C (10, 11). RNA helicases could also be involved in the degradation of mRNA by unwinding double-stranded mRNA, thereby allowing the action of RNase (8).We have recently shown that the deregulation of the expression of one RNA helicase gene of B. cereus ATCC 14579 increased the lag phase of B. cereus at a low temperature (7). In this context, our aim was to investigate the role of the five putative RNA helicases present in the genome of B. cereus ATCC 14579 in its adaptation at low temperature, close to the growth limit.     

A Major Determinant for Binding and Aminoacylation of tRNA in Cytoplasmic Alanyl-tRNA Synthetase Is Mutated in Dominant Axonal Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at θ = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNA^Ala aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features.     

The Clostridium botulinum GerAB germination protein is located in the inner membrane of spores

Clostridium botulinum dormant spores germinate in presence of l-alanine via a specific receptor composed of GerAA, GerAB and GerAC proteins. In Bacillus subtilis spores, GerAA and GerAC proteins were located in the inner membrane of the spore. We studied the location of the GerAB protein in C. botulinum spore fractions by Western-blot analysis, using an antipeptidic antibody. The protein GerAB was in vitro translated and used to confirm the specificity of the antibodies. GerAB was not present in a coat and spore outer membrane fraction but was present in a fraction of decoated spores containing inner membrane. These results strongly suggest that the protein GerAB is located in the inner membrane of the spore.     

In vivo specific binding of [3H]1-nicotine in the mouse brain

[3H] 1-Nicotine was used as a receptor ligand in the intact mouse. It was injected i.v., and radioactivity in brain regions was assayed. Nonspecific binding was estimated by pretreatment with unlabelled 1-nicotine. Radioactivity entered the brain rapidly, was heterogeneously distributed, and declined after 5 min. Estimated specific binding was highest in the medial and posterior cortex, midbrain, thalamus/hypothalamus and medulla/pons; intermediate in the cerebellum, caudate/putamen, frontal and frontoparietal cortex; and lowest in the hippocampus and olfactory bulb. Autoradiography showed similar patterns. Coinjection of unlabelled 1-nicotine reduced specific binding so that it approached estimated nonspecific binding. Nicotinic agonists reduced radioactivity in the thalamus/hypothalamus, but nicotinic antagonists were less active. Non-nicotinic drugs did not reduce brain radioactivity. The results suggest that radiolabelled nicotine may be used for in vivo receptor studies despite problems in estimating nonspecific binding.     

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group

Aims: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. Methods and Results: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l -alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l -alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. Conclusions: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l -alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. Significance and Impact of the Study: The greater ability of some strains to germinate in response to l -alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l -alanine.     

The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (T_min). CasKR was also involved in optimal cold growth above T_min and in cell survival below T_min. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at T_min. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains.     

Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses

Haemolysin BL (HBL) and non-haemolytic enterotoxin (Nhe), each consisting of three components, represent the major enterotoxins produced by Bacillus cereus. To evaluate the expression of these toxins, a set of 100 B. cereus strains was examined. Molecular biological characterization showed that 42% of the strains harboured the genes for HBL and 99% for Nhe. The production of all Nhe and HBL components were analyzed using specific antibodies and, in culture supernatants, detectable levels of HBL and Nhe were found for 100% of hbl-positive and 96% of nhe-positive strains. The concentrations of the HBL-L(2) and NheB component ranged from 0.02 to 5.6 microg mL(-1) and from 0.03 to 14.2 microg mL(-1), respectively. Comparison of the amount of NheB produced by food poisoning and food/environmental strains revealed that the median value for all food poisoning strains was significantly higher than for the food/environmental isolates. The data presented in this study provide evidence that specific and quantitative determination of the enterotoxins is necessary to evaluate the toxic potential of B. cereus. In particular, the level of Nhe seems to explain most of the cytotoxic activity of B. cereus isolates and may indicate a highly diarrheic potential.     

Evidence for deficiencies in perceptual and semantic olfactory processes in Parkinson's disease

Olfactory deficits have been reported in Parkinson's disease (PD) and are thought to represent a sensitive marker of the disease. The aim of the present study was to examine the differential contribution in olfactory dysfunction of perceptual and semantic processes of odours in PD patients. Twenty-four PD patients (12 males and 12 females) and 24 control subjects (12 males and 12 females) were tested. The experiment included two sessions. Initially, 12 odorants were delivered, one per minute. For each odour, subjects were asked to rate intensity, pleasantness, familiarity and edibility using linear rating scales. The odorants were again presented and the subjects were asked to identify them. The four olfactory judgements and odour identification were severely disturbed in PD patients when compared to control subjects. These findings demonstrate major deficits for all cognitive tasks of olfactory judgement in PD, and suggest that PD is associated with disruption of olfactory areas situated in the temporal lobes and also in the prefrontal cortex.