Mating behavior is controlled by acute changes in metabolic fuels

Mild food restriction for 48 h inhibits mating behavior in female musk shrews (Suncus murinus). However, mating behavior is restored after a 90-min feeding bout. In this series of experiments, we examined the role of metabolic fuels in this behavioral restoration. First, drugs reported to block glycolysis or fatty acid oxidation were given 2 h before mating. Both treatments inhibited mating in food-restricted females that were refed after treatment. Blood glucose levels were assessed in females that were fed ad libitum, food restricted, or food restricted and refed for 90 min. Food restriction significantly lowered blood glucose compared with ad libitum feeding or food restriction in combination with 90 min of refeeding. However, neither glucose nor fat alone could substitute for food and promote mating behavior in food-restricted females. In addition, analysis of ketone bodies and body composition in females demonstrated low or undetectable levels of these energy substrates. Our data suggest that musk shrews have relatively little stored energy. Therefore, female musk shrews rely on continuous food intake and monitor multiple cues acutely, including glucose availability and fatty acid oxidation. This ensures that mating does not occur when adequate energy is unavailable.     

Electrophoretic separation of gill proteins of the clam Codakia orbicularis

Marine organisms inhabiting the coastal environment of the Caribbean islands have attracted the attention of a number of research scientists. These organisms generally live in areas of high environmental stress and may, therefore, contain specialized proteins and enzymes which exhibit valuable biotechnological applications. Among them, a small number of clams have been studied. Our work relates to the clam Codakia orbicularis. This bivalve lives in areas of high hydrogen sulfide concentrations on the Caribbean island of Guadeloupe. Its enzymatic system must, therefore, have evolved to allow its adaptation to this high-stress environment. C. orbicularis also contains endosymbiotic bacteria which are housed in the bacteriocytes of the gills. Its protein content can, therefore, be expected to have an impact on this symbiotic relationship. We have analysed gill protein extracts of this clam by various biochemical techniques: SDS-PAGE, IEF, PAS, and Western blotting using a panel of lectins, in order to establish its protein and glycoprotein profiles. This biochemical analysis, the first of its kind, constitutes an important step in separating and characterizing the proteins involved in the biochemical pathways of this organism whose stock is in decline in Guadeloupe. Our results show the presence of three major proteins whose molecular weights vary between 14,000 and 24,000 Daltons, and some of which are glycoproteins with predominantly alpha-mannose and N-acetylgalactosamine moieties. Their pI values are in the range between 4.5 and 5.6. These protein profiles are different from those observed for Lucina pectinata, a clam which has been the subject of earlier studies in the literature.     

Interleukin 10 in disseminated lupus erythematosus

A number of years ago several groups reported that Interleukin 10 (IL10) was vigorously overproduced in disseminated lupus erythematosus (reviewed in Llorente et al., 2000). This hyperproduction obeys two different patterns. In one pattern, IL10 serum concentrations become elevated during disease and evolve in parallel with them. In the other, the patients' blood mononucleated cells spontaneously release increased IL10 quantities; contrary to serum variations in the first pattern, this increased output is not correlated with disease spurts. Interestingly an increase of this type is frequently found in disease-free parents of these patients (Llorente et al., 1997; Gr?ndal et al., 1999). Some studies relate this IL10 hyperproduction to a genetic origin (Mehrian et al., 1998; Mok et al., 1998), while others seem to indicate an environmental cause (Gr?ndal et al., 1999). Together these findings suggest two different possible origins for lupus IL10 overproduction. The first would be due to intrinsic anomalous IL10 production by some immune cells, the second would result from high IL10 output by tissues damaged by the inflammatory process. Considering the properties of IL10, which activates B lymphocytes and inhibits T lymphocytes, overproduction in lupus could explain the immune anomalies of this disease, which is characterized by anti-self antibodies production and by deficient T responses. Interestingly several of these anomalies are found independently from disease outbreaks and, in the case of some, in relatives of patients. The part played by IL10 in lupus has been inferred from the murine NZB-W model, in which an anti-IL10 monoclonal antibody prevents development of the disease (Ishida et al., 1994); this antibody also prevents the appearance of human anti-ADN autoantibodies in immune-deficient mice restored with patients' lymphocytes. A direct demonstration of the role of IL10 in the etiology of lupus symptoms has recently been adduced in a pilot study bearing on six lupus patients, refractory to anti-inflammatory and immuno-suppressive treatments, who were treated for 3 weeks with a murine anti-IL10 monoclonal antibody (Llorente et al., 2000). This treatment brought about a rapid amelioration of the clinical picture, in particular of the cutaneous and articular symptoms, which was maintained for six months after this trial. This symptomatic amelioration was accompanied by a decrease of the biological signs of immune system hyperactivity and a partial amelioration of T lymphocyte function. The better clinical control of the disease allowed a significant decrease of corticotherapy. While this was an open study, without a control group and without randomisation, the rapid and important evolution of clinical and biological parameters towards normal strongly pleads for a favorable effect of the treatment. While confirming previous hypotheses about the rote of IL10 in lupus, this study leaves several points unexplained. First the clinical and biological amelioration in these patients treated with anti-IL10 monoclonal antibody occurred independently of circulating anti-ADN auto-antibodies, indicating that the role of this interleukin in the disease is more complex than could be thought from the initial experiments in mouse. This unexpected result does not exclude that the beneficial effect of the antibody could be mediated by its action on B lymphocytes, since it is well known that these cells play important parts in development auto-immune processes other than only the production of auto-antibodies. It is also possible that the negative effect of IL10 in this disease exerts on other targets than B lymphocytes, in particular on fibroblasts and endothelial cells. Indeed it should be recalled that, while IL10 has often been held as an anti-inflammatory cytokine, its biological properties are more ambiguous, since it can immuno-stimulate some cell types. Among other remaining unknowns, the reason why some IL10 overproducing individuals, belonging to the family of lupus patients, do not develop lupus, is not understood. This fact underlines the multi-factorial origin of this disease, which must stem from associated genetic and environmental causes. IL10 overproduction, while it apparently promotes the symptoms, is certainly not sufficient. The efficiency of anti-IL 10 monoclonal antibodies during lupus, which are well tolerated, points to their usefulness in the therapeutic arsenal, especially in forms that are refractory to conventional anti-inflammatory and immunosuppressive treatments. In order to be validated, this hypothesis must be submitted to more in depth, randomized, studies. Moreover humanized antibodies should be developed, which would make it possible to reiterate the treatment during further outbreaks. Once all these conditions are fulfilled, the place of anti-IL10 treatment in lupus and the benefice-risk ratio should be compared to these of therapeutic approaches currently used in refractory forms. If further studies confirm the efficiency of- and tolerance to- IL10 neutralizing antibodies in lupus, it is well possible that these antibodies will eventually equate, for this disease, the recent use of TNF antagonists in the treatment of rheumatoid arthritis.     

Rpa12p, a conserved RNA polymerase I subunit with two functional domains

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.     

The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

An in vitro study of the sub-cellular distribution of nicotinic receptors

Nicotinic and serotoninergic 5HT3 receptors share important sequence identities except for their cytoplasmic loop. Both ends of this loop display conserved 3D helical structures with distinct primary sequences. We decided to check whether these two helices named F and G play a role in the sub-cellular distribution of different nicotinic receptors. We systematically exchanged each helix with the equivalent sequence of neuronal nicotinic and alpha4, beta2 and alpha7 subunits in the functional chimeric alpha7-5HT3 receptor used as a model system. The new chimeras were expressed in vitro in polarized epithelial cells from pig kidney. We quantified synthesis and export of the receptors to the cell surface by measuring alpha-bungarotoxin binding sites. Immunogold labelling was used, at the electron microscope level, to determine the amount of each chimera present at either domain, apical and/or basolateral, of these cells. We noticed that in epithelial cells the majority of alpha-bungarotoxin binding sites remained sequestered in the cytoplasm as already observed in neurons in vivo. The majority of the pentamers present at the cell surface were located at the apical domain. Our results suggest that helix F and G differently regulate assembly and export to the cell surface of alpha-bungarotoxin binding receptors.     

The RNA-binding protein TIA-1 is a novel mammalian splicing regulator acting through intron sequences adjacent to a 5' splice site

Splicing of the K-SAM alternative exon of the fibroblast growth factor receptor 2 gene is heavily dependent on the U-rich sequence IAS1 lying immediately downstream from its 5' splice site. We show that IAS1 can activate the use of several heterologous 5' splice sites in vitro. Addition of the RNA-binding protein TIA-1 to splicing extracts preferentially enhances the use of 5' splice sites linked to IAS1. TIA-1 can provoke a switch to use of such sites on pre-mRNAs with competing 5' splice sites, only one of which is adjacent to IAS1. Using a combination of UV cross-linking and specific immunoprecipitation steps, we show that TIA-1 binds to IAS1 in cell extracts. This binding is stronger if IAS1 is adjacent to a 5' splice site and is U1 snRNP dependent. Overexpression of TIA-1 in cultured cells activates K-SAM exon splicing in an IAS1-dependent manner. If IAS1 is replaced with a bacteriophage MS2 operator, splicing of the K-SAM exon can no longer be activated by TIA-1. Splicing can, however, be activated by a TIA-1-MS2 coat protein fusion, provided that the operator is close to the 5' splice site. Our results identify TIA-1 as a novel splicing regulator, which acts by binding to intron sequences immediately downstream from a 5' splice site in a U1 snRNP-dependent fashion. TIA-1 is distantly related to the yeast U1 snRNP protein Nam8p, and the functional similarities between the two proteins are discussed.