Alginate as a support ligand for enhanced colloidal liquid aphron immobilization of proteins and drug delivery

Research within the field of colloidal liquid aphrons (CLAs) for enzyme immobilization has often used ionic surfactants for the retention of enzymes. Although these charged interactions allow for enhanced immobilization, they can often lead to denaturation of enzyme activity, and even release of the protein. Sodium alginate has been used in drug delivery applications due to its low toxicity and charged interactions that allow for encapsulation. Hence, alginate systems can be used as an alternative to ionic surfactants in CLA immobilization. This paper presents, for the first time, the use of sodium alginate as potential ligand for enhanced CLA immobilization. The use of five model proteins; lysozyme, bovine serum albumin, ovalbumin, insulin, and α-chymotrypsin, of various pIs and hydrophobicities, showed the relevance of electrostatic interactions in promoting binding with sodium alginate when the pH < pI, with 100% immobilization attributed to alginate incorporated CLAs over general nonionic formulations. Furthermore, above their pI, >80% protein recovery was observed, with activity and conformation comparable to their native counterparts. Finally, the use of proteolysis showed that as the degree of ionic bonding increased between the protein and sodium alginate, the degree of protease resistance decreased due to conformational changes experienced during binding.     

Protein electrophoresis in Andean condors (Vultur gryphus): Reference values and differences between wild and rehabilitating individuals

The study of wildlife health greatly contributes to understanding population dynamics and detecting conservation threats. The determination of the different fractions of plasma proteins (proteinogram) is an important laboratory tool to study wildlife health. The aim of this study was to characterize protein electrophoresis in wild Andean condors (Vultur gryphus) from north‐western Patagonia and to evaluate differences according to age and sex classes. Once reference values of wild, apparently healthy individuals, were established, we compared these values to those of individuals received at the Buenos Aires Zoo in Argentina for rehabilitation due to various health problems. Reference proteinograms from wild Andean condors differed only in the α 1 and β 2‐fractions between sex categories. Males showed higher concentrations of these protein fractions than females. We found clear differences between wild birds and rehabilitating individuals. Total proteins, globulins, α 1‐globulins, total α‐globulins, β 2‐globulins, total β‐globulins, and γ‐globulins were significantly higher in rehabilitating than in wild individuals, whereas albumin, α 2, and β1‐globulins were similar between these groups. The albumin/globulin ratio, as a general indicator of health, was significantly lower in rehabilitating than in wild individuals. The results indicate the effects on different protein fractions of pathologic processes occurring in individuals undergoing rehabilitation. Our results provide useful insights, contributing to improving diagnoses and prognoses in this species. This information may also be useful to assess the health status of Andean condors in studies of wild populations and for comparisons with other bird species.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants

To test the hypothesis that human milk fucosyloligosaccharides are part of an innate immune system, we addressed whether their expression (1) depends on maternal genotype and (2) protects breastfed infants from pathogens. Thus the relationship between maternal Lewis blood group type and milk oligosaccharide expression and between variable oligosaccharide expression and risk of diarrhea in their infants was studied in a cohort of 93 Mexican breastfeeding mother-infant pairs. Milk of the 67 Le(a-b+) mothers contained more LNF-II (Le(a)) and 3-FL (Le(x)) (oligosaccharides whose fucose is exclusively alpha 1,3- or alpha 1,4-linked) than milk from the 24 Le(a-b-) mothers; milk from Le(a-b-) mothers contained more LNF-I (H-1) and 2'-FL (H-2), whose fucose is exclusively alpha 1,2-linked. The pattern of oligosaccharides varied among milk samples; in each milk sample, the pattern was summarized as a ratio of 2-linked to non-2-linked fucosyloligosaccharides. Milks with the highest ratios were produced primarily by Le(a-b-) mothers; those with the lowest ratios were produced exclusively by Le(a-b+) mothers (p<0.001). Thus maternal genetic polymorphisms expressed as Lewis blood group types are expressed in milk as varied fucosyloligosaccharide ratios. The four infants who developed diarrhea associated with stable toxin of Escherichia coli were consuming milk with lower ratios (4.4 +/- 0.8 [SE]) than the remaining infants (8.5 +/- 0.8; p<0.001). Furthermore, the 27 infants who developed moderate to severe diarrhea of any cause were consuming milk with lower ratios (6.1 +/- 0.9) than the 26 who remained healthy (10.5 +/- 1.9; p = 0.042). Thus, milk with higher 2-linked to non-2-linked fucosyloligosaccharide ratios affords greater protection against infant diarrhea. We conclude that specific oligosaccharides constitute a major element of an innate immune system of human milk.     

Predation Pattern and Phylogenetic Analysis of <Emphasis Type="Italic">Bdellovibrionaceae</Emphasis> from the Great Salt Lake,Utah

The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria. The prey susceptibility pattern is frequently used to characterize new isolates. The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing. To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey. After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced. Water samples were also plated for isolation of heterotrophic bacteria. The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined. The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake. This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.     

Glycine intake decreases plasma free fatty acids, adipose cell size, and blood pressure in sucrose-fed rats

The study investigated the mechanism by which glycine protects against increased circulating nonesterified fatty acids (NEFA), fat cell size, intra-abdominal fat accumulation, and blood pressure (BP) induced in male Wistar rats by sucrose ingestion. The addition of 1% glycine to the drinking water containing 30% sucrose, for 4 wk, markedly reduced high BP in sucrose-fed rats (SFR) (122.3 +/- 5.6 vs. 147.6 +/- 5.4 mmHg in SFR without glycine, P < 0.001). Decreases in plasma triglyceride (TG) levels (0.9 +/- 0.3 vs. 1.4 +/- 0.3 mM, P < 0.001), intra-abdominal fat (6.8 +/- 2.16 vs. 14.8 +/- 4.0 g, P < 0.01), and adipose cell size were observed in SFR treated with glycine compared with SFR without treatment. Total NEFA concentration in the plasma of SFR was significantly decreased by glycine intake (0.64 +/- 0.08 vs. 1.11 +/- 0.09 mM in SFR without glycine, P < 0.001). In control animals, glycine decreased glucose, TGs, and total NEFA but without reaching significance. In SFR treated with glycine, mitochondrial respiration, as an indicator of the rate of fat oxidation, showed an increase in the state IV oxidation rate of the beta-oxidation substrates octanoic acid and palmitoyl carnitine. This suggests an enhancement of hepatic fatty acid metabolism, i.e., in their transport, activation, or beta-oxidation. These findings imply that the protection by glycine against elevated BP might be attributed to its effect in increasing fatty acid oxidation, reducing intra-abdominal fat accumulation and circulating NEFA, which have been proposed as links between obesity and hypertension.     

Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2

Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus (X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability (P_o) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance () was increased by 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH₂-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in P_o (with a small change in ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains. cystic fibrosis; chloride channel; protein kinases; ATP binding cassette proteins     

Mechanism of regulation of the gap junction protein connexin 43 by protein kinase C-mediated phosphorylation

Phosphorylation of the gap junction protein connexin 43 (Cx43) by protein kinase C (PKC) decreases dye coupling in many cell types. We report an investigation of the regulation by PKC of Cx43 gap junctional hemichannels (GJH) expressed in Xenopus laevis oocytes. The activity of GJH was assessed from the uptake of hydrophilic fluorescent probes. PKC inhibitors increased probe uptake in isolated oocytes expressing recombinant Cx43, indicating that the regulatory effect occurs at the hemichannel level. We identified by mutational analysis the carboxy-terminal (CT) domain sequences involved in this response. We found that 1) Ser368 is responsible for the regulation of Cx43 GJH solute permeability by PKC-mediated phosphorylation, 2) CT domain residues 253-270 and 288-359 are not necessary for the effect of PKC, and 3) the prolinerich CT region is not involved in the effect of phosphorylation by PKC. Our results demonstrate that Ser368 (but not Ser372) is involved in the regulation of Cx43 solute permeability by PKC-mediated phosphorylation, and we conclude that different molecular mechanisms underlie the regulation of Cx43 by intracellular pH and PKC-mediated phosphorylation. protein kinase C blocker; dye loading; hemichannel     

Characterization of bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, as determined by 16S rDNA analysis

The bacterial diversity in pulque, a traditional Mexican alcoholic fermented beverage, was studied in 16S rDNA clone libraries from three pulque samples. Sequenced clones identified as Lactobacillus acidophilus, Lactobacillus strain ASF360, L. kefir, L. acetotolerans, L. hilgardii, L. plantarum, Leuconostoc pseudomesenteroides, Microbacterium arborescens, Flavobacterium johnsoniae, Acetobacter pomorium, Gluconobacter oxydans, and Hafnia alvei, were detected for the first time in pulque. Identity of 16S rDNA sequenced clones showed that bacterial diversity present among pulque samples is dominated by Lactobacillus species (80.97%). Seventy-eight clones exhibited less than 95% of relatedness to NCBI database sequences, which may indicate the presence of new species in pulque samples.     

Molecular analysis of the eighteen most frequent mutations in the BRCA1 gene in 63 Chilean breast cancer families

BRCA1 gene mutations account for nearly all families with multiple cases of both early onset breast and/or ovarian cancer and about 30% of hereditary breast cancer. Although to date more than 1,237 distinct mutations, polymorphisms, and variants have been described, several mutations have been found to be recurrent in this gene. We have analyzed 63 Chilean breast/ovarian cancer families for eighteen frequent BRCA1 mutations. The analysis of the five exons and two introns in which these mutations are located was made using mismatch PCR assay, ASO hybridization assay, restriction fragment analysis, allele specific PCR assay and direct sequentiation techniques. Two BRCA1 mutations (185delAG and C61G) and one variant of unknown significance (E1250K) were found in four of these families. Also, a new mutation (4185delCAAG) and one previously described polymorphism (E1038G) were found in two other families. The 185delAG was found in a 3.17% of the families and the others were present only in one of the families of this cohort. Therefore these mutations are not prominent in the Chilean population. The variant of unknown significance and the polymorphism detected could represent a founder effect of Spanish origin.