Effect of lipid composition on the stability of cellular membranes during freeze-thawing of Lactobacillus acidophilus grown at different temperatures

Lactobacillus acidophilus CRL 640 grown at the optimal temperature of 37 degrees C (M37) appeared more sensitive to freeze-thawing than when it was grown at 25 degrees C (M25). In the first case, 87% of the cells died, in contrast to 33% for cells grown at 25 degrees C. All the surviving M37 cells showed sensitivity to NaCl. However, among the surviving M25 cells, only 85% were sensitive to NaCl. The rest of the cells were considered uninjured. Freeze-thawing in cells grown at 25 degrees C showed a liberation of nucleic acids and proteins. However, the leakage was higher in M37 cells after freeze-thawing. The greater fraction of damaged cells were observed in M25 culture after freeze-thawing. A relative increase of 81% in cardiolipid (CL), with respect to total phospholipids and 72% triglycosyldiglyceride (TGDG) with respect to the total glycolipids was observed in M37. In addition, a decrease of palmitoyl (C16:0), oleoyl (C18:0) fatty acids at CL, phosphatidylglycerol (PG), and diglicosyldiglyceride (DGDG) fractions and the increase of C19 cyc and C18:0, 10-OH fatty acids in neutral lipid, and CL fractions was also apparent. In M25 cells, the concentration of DGDG and PG was higher than in M37 cells. The difference in cryotolerance between the frozen cultures emphasizes the importance of selecting appropriate conditions of growth of microorganisms for use as dietary adjuncts.     

Expression analysis and chromosomal assignment of PRA1 and RILP genes

PRA1 (prenylated Rab acceptor) is a general regulator of Rab proteins, while RILP (Rab interacting lysosomal protein) is a specific effector for Rab7. It has been shown that PRA1 interacts with Rab proteins and with VAMP2. Therefore PRA1 is probably an important factor for membrane traffic, linking together the function of Rab proteins and SNAREs. RILP has a key role in the control of transport to degradative compartments together with Rab7 and probably links Rab7 function to the cytoskeleton. Here we have studied by Northern blot the expression of the two genes in several different human tissues. The 0.8-kb mRNA for human PRA1 is ubiquitously expressed, while the two mRNAs for RILP are differentially expressed. In addition, we have assigned the human PRA1 gene to chromosome 19q13.13-q13.2 and the human RILP gene to chromosome 17p13.3.     

Role of Glutamate Receptors and Glutamate Transporters in the Regulation of the Glutamate-Glutamine Cycle in the Awake Rat

In the present study we investigate the effects of a specific glutamate reuptake blocker, L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), on extracellular concentrations of glutamine and glutamate in the striatum of the freely moving rat. Intracerebral infusions of PDC (1, 2 and 4 mM) produced a dose-related increase in extracellular concentrations of glutamate and a dose-related decrease in extracellular concentrations of glutamine. These increases in extracellular glutamate and decreases in extracellular glutamine were significantly correlated. To investigate the involvement of ionotropic glutamate receptors in the decreases of extracellular glutamine produced by PDC, N-methyl-D-aspartate (NMDA) receptor antagonist and -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist were used. Perfusion of the NMDA receptor antagonist blocked the decrease of extracellular glutamine but had no effect on the increase of extracellular glutamate, both produced by PDC. Perfusion of the AMPA/kainate receptor antagonist attenuated the increase of extracellular glutamate and not only blocked the decrease of extracellular glutamine but also produced a significant increase of extracellular glutamine. The results reported in this study suggest that both NMDA and AMPA/kainate glutamatergic receptors are involved in the regulation of extracellular glutamine.     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac TM

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac^TM has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac^TM in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995–2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.     

Lipid and carbohydrate metabolism in mice with a targeted mutation in the IL-6 gene: absence of development of age-related obesity

Obesity-related insulin resistance may be caused by adipokines such as IL-6, which is known to be elevated with the insulin resistance syndrome. A previous study reported that IL-6 knockout mice (IL-6(-/-)) developed maturity onset obesity, with disturbed carbohydrate and lipid metabolism, and increased leptin levels. Because IL-6 is associated with insulin resistance, one might have expected IL-6(-/-) mice to be more insulin sensitive. We examined body weights of growing and older IL-6(-/-) mice and found them to be similar to wild-type (IL-6(+/+)) mice. Dual-energy X-ray absorptiometry analysis at 3 and 14 mo revealed no differences in body composition. There were no differences in fasting blood insulin and glucose or in triglycerides. To further characterize these mice, we fed 11-mo-old IL-6(-/-) and IL-6(+/+) mice a high- (HF)- or low-fat diet for 14 wk, followed by insulin (ITT) and glucose tolerance tests (GTT). An ITT showed insulin resistance in the HF animals but no difference due to genotype. In the GTT, IL-6(-/-) mice demonstrated elevated postinjection glucose levels by 60% compared with IL-6(+/+) but only in the HF group. Although IL-6(-/-) mice gained weight and white adipose tissue (WAT) with the HF diet, they gained less weight than the IL-6(+/+) mice. Total lipoprotein lipase activity in WAT, muscle, and postheparin plasma was unchanged in the IL-6 (-/-) mice compared with IL-6(+/+) mice. There were no differences in plasma leptin or TNF-alpha due to genotype. Plasma adiponectin was approximately 53% higher (71.7 +/- 14.1 microg/ml) in IL-6(-/-) mice than in IL-6(+/+) mice but only in the HF group. Thus these data show that IL-6(-/-) mice do not demonstrate obesity, fasting hyperglycemia, or abnormal lipid metabolism, although HF IL-6(-/-) mice demonstrate elevated glucose after a GTT.     

The sensitive giant: the role of titin-based stretch sensing complexes in the heart

Every heart beat is not equal. As physiological demands of the cardiovascular system change, cardiac myocytes modulate contractile parameters including the rate and force of contraction. Adaptive responses require the sensing of biomechanical signals involving the interface between the contractile cytoskeleton (myofibrils) and the sarcolemma at specialized cell-cell junctions (intercalated discs) and cell-substrate adhesion complexes (costameres). Recent studies have shed insight into how protein complexes within cardiac myocytes sense biomechanical signals, processes required for normal adaptive or pathological responses. This new evidence suggests that complexes associated with the giant, myofibrillar protein titin sense myocyte stretch. Here, we discuss evidence supporting titin being an ideal biomechanical sensor.     

Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells

Immunostimulatory DNA sequences (ISS) containing CpG motifs induce interferon-α (IFN-α) and interferon-γ (IFN-γ) from human peripheral blood mononuclear cells and stimulate human B cells to proliferate and produce IL-6. We studied the motif and structural requirements for both types of activity using novel chimeric immunomodulatory compounds (CICs), which contain multiple heptameric ISS connected by non-nucleoside spacers in both linear and branched configurations. We found that the optimal motifs and structure for IFN-α production versus B cell activation differed. IFN-α production was optimal for CICs containing the sequences 5′-TCGXCGX and 5′-TCGXTCG, where X is any nucleotide. The presentation of multiple copies of these heptameric ISS with free 5′-ends via long, hydrophilic spacers, such as hexaethylene glycol, significantly enhanced the induction of IFN-α. Conversely, human B cell activity was predominately dependent on ISS motif, with 5′-TCGTXXX and 5′-AACGTTC being the most active sequences. Thus, we found CICs could be ‘programmed’ for IFN-α production or B cell activation as independent variables. Additionally, CICs with separate human- and mouse-specific motifs were synthesized and these were used to confirm in vivo activity in mice. CICs may offer unique advantages over conventional ISS because identification of the optimal motifs, spacers and structures for different biological properties allows for the assembly of CICs exhibiting a defined set of activities tailored for specific clinical applications.     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.