Plasmid maintenance and recombinant cell fitness explored in bacterial colonies

During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered -galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes.     

Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1) by denaturing high performance liquid chromatography

Background  

R122, the primary autolysis site of the human cationic trypsinogen (PRSS1), constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC) single nucleotide substitution, a c.365~366GC>AT (CGC>CAT) gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant.     

Thermal acclimation, growth, and burst swimming of threespine stickleback: enzymatic correlates and influence of photoperiod

Threespine sticklebacks (Gasterosteus aculeatus) that had been reared in the laboratory under natural photoperiods were acclimated to 23 degrees and 8 degrees C in late spring under increasing day lengths and again in late fall under decreasing day lengths. The parents of these fish were from the anadromous Isle Verte population. In the spring, cold- and warm-acclimated fish grew at the same rates and attained similar condition factors (mass L(-3)), although food intake was considerably higher at 23 degrees C. As both groups had similar increases in mass and condition, the higher axial muscle activities of citrate synthase and phosphofructokinase (measured at 20 degrees C) after cold acclimation were likely a direct response to temperature. Multiple regression analysis showed that axial muscle levels of cytochrome C oxidase and citrate synthase were correlated with the burst swimming speeds of the spring sticklebacks, while growth rates were positively correlated with lactate dehydrogenase levels in pectoral and axial muscles and creatine kinase levels in the axial muscle. In the fall, the fish in both acclimation groups grew little, although they fed at similar rates as in the spring experiment. Overall, the sticklebacks showed lower burst swimming speeds in the fall. In both spring and fall, the burst speeds of cold- and warm-acclimated sticklebacks only differed at warm temperatures. In the spring experiment, the cold-acclimated fish swam faster, whereas in the fall experiment the warm-acclimated fish swam faster despite their lower percentage of axial muscle. Swimming speeds were measured both at a fish's acclimation temperature and after 12 h at the other temperature. Cold-acclimated sticklebacks seem to have more facility in rapidly adjusting to warm temperatures when they have experienced increasing rather than decreasing day lengths, perhaps as a result of the requirements of the spring migration to the intertidal breeding grounds.     

New species of benthopelagic hydromedusae from the Weddell Sea

Four medusa species were collected by an epibenthic sledge during the "Polarstern" ANT XV/3 cruise carried out from January to March 1998 in the eastern Weddell Sea. The specimens were collected in the benthic boundary layer at depths ranging between 1,583 and 2,034 m; 2 of the species collected are new to science. The narcomedusa Sigiweddellia bathypelagica gen. nov. et sp. nov. is characterised by two types of marginal tentacles and closed marginal statocysts. The trachymedusa Voragonema laciniata sp. nov. (known only from the single holotype) is characterised by the number and irregular shape of the centripetal canals. These findings are the first to report benthopelagic hydromedusae in deep Antarctic waters. Examination of several specimens of Benthocodon pedunculata (Bigelow 1913) leads us to move it to the genus Voragonema Naumov 1971 because of the clear presence of centripetal expansions in the ring canal.     

Natriuretic peptide-dependent lipolysis in fat cells is a primate specificity

We have recently demonstrated that natriuretic peptides (NPs), which are known for regulation of blood pressure via membrane guanylyl cyclase (GC) receptors, are lipolytic in human adipose tissue. In this study, we compared the NP control of lipolysis in adipocytes from humans, nonhuman primates (macaques), rodents (rats, mice, hamsters), and nonrodent mammals (rabbits, dogs). Isolated adipocytes from these species were exposed to increasing concentrations of atrial NP (ANP) or isoproterenol (beta-adrenergic agonist). Although isoproterenol was lipolytic in all of the species, ANP only enhanced lipolysis in human and macaque adipocytes. In primate fat cells, NP-induced lipolysis involved a cGMP-dependent pathway. Binding studies and real-time quantitative PCR assays revealed that rat adipocytes expressed a higher density of NP receptors compared with humans but with a different subtype pattern of expression; type-A GC receptors predominate in human fat cells. This was also confirmed by the weak GC-activity stimulation and the reduced cGMP formation under ANP exposure in rat adipocytes compared with human fat cells. In conclusion, NP-induced lipolysis is a primate specificity, and adipocytes from ANP-nonresponsive species present a predominance of "clearance" receptors and very low expression of "biologically active" receptors.     

Development of Heavy Metal-Resistant Mutants of Phosphate Solubilizing <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NBRI 4014 and Their Characterization

Pseudomonas sp. NBRI 4014 is a potent phosphorus solubilizer (284 μg/ml). It also produced significant levels of siderophore (143.87 μg/ml) and IAA (5.6 μg/ml). Siderotyping indicated it was P. aeruginosa siderovar 1. Cadmium (180 μM), nickel (420 μM), and chromium (370 μM) resistant mutants were developed and characterized for their PGPR properties. Mutants were stable under non-selective pressure. In cases of nickel and cadmium, there were reductions of the siderophore levels. However, they were able to promote root and shoot elongation in soybeans (Glycine max PK 564) at a significant level (p < 0.05) in the presence of metals unfamiliar to the wild type. The persistence and stability of mutants were evident in rhizospheric soil, thus their exploitation for polluted/contaminated sites was supported. Received: 27 December 2001 / Accepted: 28 January 2002     

Homologues of amino acid permeases: cloning and tissue expression of XAT1 and XAT2

The L-type (LAT) family of amino acid transporters is composed of exchangers for neutral, cationic, and anionic amino acids. They form functional heterodimers with membrane glycoproteins, rBAT or 4F2hc/CD98, to which they are linked by a disulphide bond. We report the molecular cloning and tissue expression of new mouse and human homologues of the LAT family, termed mXAT1, mXAT2 and hXAT2. The latter two proteins may correspond to ortholog genes in mouse and human. The hXAT2 gene is located on chromosome 8q21.3. The cloned X amino acid transporter (XAT) cDNAs are predicted to encode proteins of about 50 kDa. From a phylogenetic point of view, the three XAT proteins cluster together, but sequence comparison and secondary structure prediction show that they are also related to the members of the LAT family. Like these transporters, the XAT proteins show 12 transmembrane domains and a conserved cysteine residue, located in the second extracellular loop. This conserved cysteine is involved in the disulphide bond formed between the known members of the LAT family and 4F2hc or rBAT. The mXAT1 and hXAT2 mRNAs are expressed in the kidney but they are not detectable in a variety of other tissues. The corresponding proteins were efficiently translated following transfection of their cDNAs in Chinese hamster ovary (CHO) cells. However, cDNA transfection in CHO cells did not induce amino acid uptake, even when cotransfected with vectors expressing 4F2hc or rBAT. This could be related to the fact that mXAT1 and hXAT2 did not form detectable disulphide-linked heterodimers with 4F2hc or rBAT when they were co-expressed in CHO cells. Identification of other putative partner(s) of these LAT family-related transporters may be necessary to understand their role in renal physiology.     

Ratings of different olfactory judgements in schizophrenia

We assessed the influence of schizophrenia on different olfactory tasks. Forty patients with schizophrenia (20 males and 20 females) and 40 control subjects (20 males and 20 females) were tested. The experiment included two sessions. Initially, 12 odorants were presented at a rate of one per minute. The subjects were asked to rate intensity, pleasantness, familiarity and edibility for each odour using linear rating scales. The odorants were then presented a second time and the subjects were asked to identify them. The results showed that the scores for pleasantness, familiarity, edibility and identification but not intensity were disturbed in patients when compared with control subjects. Furthermore, the familiarity judgement of male patients was more often deficient than that of female patients and they rated odorants as being inedible when the women judged them as neutral. Considered together, these data show that our olfactory test may be used in patients with schizophrenia for evidencing various dysfunctions specific to different types of olfactory processing that represent steps in the odour name identification process.     

Glyconeogenic pathway in isolated skeletal muscles of rats

Although the conversion of lactate to glycogen (glyconeogenesis) in muscle was demonstrated a long time ago, the biochemical reactions responsible for this process are still a controversial matter. In the present study, advantage was taken from the specific inhibition induced by phenylalanine on muscle pyruvate kinase (PK) to investigate the role of reverse PK activity in muscle glyconeogenesis. Addition of phenylalanine to the incubation medium of a preparation of isolated, intact skeletal muscles that maintain metabolic activity for several hours reduced by 50% the rate of incorporation of [14C]lactate or [14C]bicarbonate into muscle glycogen. Muscle extracts presented high levels of maximal activity of PK in the reverse direction, which was completely blocked in the presence of phenylalanine. In contrast, mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), did not affect the incorporation of 14C from either lactate or bicarbonate into muscle glycogen. Maximal PEPCK activity was much lower in muscle extracts than in gluconeogenic or glyceroneogenic tissues and was suppressed in the presence of mercaptopicolinic acid. The data suggest that a reversal of the metabolic flux through the reaction catalyzed by PK contributes to the accumulation of lactate-derived glycogen that occurs in skeletal muscle under certain physiological conditions.