Dominant role of smooth muscle L-type calcium channel Cav1.2 for blood pressure regulation

Blood pressure is regulated by a number of key molecules involving G-protein-coupled receptors, ion channels and monomeric small G-proteins. The relative contribution of these different signaling pathways to blood pressure regulation remains to be determined. Tamoxifen-induced, smooth muscle-specific inactivation of the L-type Cav1.2 Ca2+ channel gene in mice (SMAKO) reduced mean arterial blood pressure (MAP) in awake, freely moving animals from 120 +/- 4.5 to 87 +/- 8 mmHg. Phenylephrine (PE)- and angiotensin 2 (AT2)-induced MAP increases were blunted in SMAKO mice, whereas the Rho-kinase inhibitor Y-27632 reduced MAP to the same extent in control and SMAKO mice. Depolarization-induced contraction was abolished in tibialis arteries of SMAKO mice, and development of myogenic tone in response to intravascular pressure (Bayliss effect) was absent. Hind limb perfusion experiments suggested that 50% of the PE-induced resistance is due to calcium influx through the Cav1.2 channel. These results show that Cav1.2 calcium channels are key players in the hormonal regulation of blood pressure and development of myogenic tone.     

Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.     

Differential analysis of protein expression of Bifidobacterium grown on different carbohydrates

We observed recently that colonic fermentation of lactose might be a major factor in the pathophysiology of lactose intolerance. Proteomic techniques could be helpful in interpreting the metabolic pathways of lactose fermentation. The objective of this study was to explore proteomic methodologies for studying bacterial lactose metabolism that can be used to detect and identify proteins associated with the onset of intolerance symptoms. Differential expression of cytoplasmic proteins of Bifidobacterium animalis, Bifidobacterium breve and Bifidobacterium longum grown on different carbohydrates (lactose, glucose, galactose) was analyzed with surface-enhanced laser desorption ionization-time of flight (SELDI-TOF) MS and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After fractionation by SDS-PAGE, differentially-expressed proteins were identified with LC-MS/MS. The three strains grown on the same carbohydrate or the same strain grown on glucose or lactose showed differences in SELDI-TOF MS protein profiles. Differences in protein expression were observed in B. breve grown on glucose, galactose or lactose as analyzed with SDS-PAGE. With LC-MS/MS, proteins from Bifidobacterium were identified, which included enzymes for metabolism of lactose, glucose and galactose. In conclusion, the applied techniques can discern differences in protein expression of bacteria metabolizing different carbohydrates. These techniques are promising in studying metabolism of lactose and other substrates in a complex bacterial ecosystem such as the colonic microbiota.     

The primacy of trust within romantic relationships: Evidence from conjoint analysis of HEXACO-derived personality profiles

Mate preference research often focuses on traits that indicate a romantic partner's personal worth (e.g., their physical attractiveness, resource potential) rather than their tendency to leverage that worth for mutual vs. zero-sum benefit (i.e., their trustworthiness). No one has assessed the contribution of trustworthiness to perceived mate value relative to other personality dimensions. Here we examined the desirability of a partner's trustworthiness relative to five other personality indicators of mate quality during initial partner selection. Participants (n = 918) ranked multivariate partner profiles constructed from the HEXACO model of personality (i.e., honesty-humility, emotionality, extraversion, agreeableness, conscientiousness, and openness to experience) and provided partner ratings for each trait. Using conjoint analysis, we found that honesty-humility influenced participants' ranking decisions substantially more than each other characteristic (all Cohen's ds > 0.62). This was true for both long- (i.e., committed) and short-term (i.e., purely sexual) partner evaluations, though honesty-humility was relatively more important for long- vs. short-term contexts. There were no sex differences. A different pattern, including sex differences, emerged for partner ratings. Based on these findings, we hypothesize that the challenge of avoiding romantic interpersonal predation may have been a relatively stronger selection pressure during the evolution of human mate preference than has the challenge of identifying other valuable partner traits.     

Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions

The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.     

A phosphorylation-dependent export structure in ROMK (Kir 1.1) channel overrides an endoplasmic reticulum localization signal

The cell surface density of functional Kir1.1 (ROMK, KCNJ1) channels in the renal collecting duct is precisely regulated to maintain potassium balance. Here, we explore the mechanism by which phosphorylation of Kir1.1a serine 44 controls plasmalemma expression. Studies in Xenopus oocytes, expressing wild-type, phosphorylation mimic (S44D), or phosphorylation null (S44A) Kir1.1a, revealed that phosphorylation of serine 44 is required to stimulate traffic of newly synthesized channels to the plasma membrane through a brefeldin A-sensitive pathway. ROMK channels were found to acquire mature glycosylation in a serine 44 phosphorylation-dependent manner, consistent with a phosphorylation-dependent trafficking step within the endoplasmic reticulum/Golgi. Serine 44 neighbors a string of three "RXR" motifs, reminiscent of basic trafficking signals involved in directing early transport steps within the secretory pathway. Replacement of the arginine residues with alanine (R35A, R37A, R39A, R41A, or all Arg to Ala) did not restore cell surface expression of the phospho-null S44A channel, making it unlikely that phosphorylation abrogates a nearby RXR-type endoplasmic reticulum (ER) localization signal. Instead, analysis of the compound S44D phospho-mimic mutants revealed that the neighboring arginine residues are also necessary for cell surface expression, identifying a structure that determines export in the biosynthetic pathway. Suppressor mutations in a putative dibasic ER retention signal, located within the cytoplasmic C terminus (K370A, R371A), restored cell surface expression of the phospho-null S44A channel to levels exhibited by the phospho-mimic S44D channel. Taken together, these studies indicate that phosphorylation of Ser44 drives an export step within the secretory pathway to override an independent endoplasmic reticulum localization signal.     

Mutational spectrum in the neurofibromatosis type 2 gene in sporadic and familial schwannomas

Using a heteroduplex approach and direct sequencing, we have completed the screening of approximately 88% of the neurofibromatosis type 2 (NF2)-coding sequence of DNA extracted from 33 schwannomas from NF2 patients and from 29 patients with sporadic schwannomas. The extensive screening has resulted in the identification of 33 unique mutations. Similarly to other human genes, we have shown that the CpG sites are more highly mutable in the NF2 gene. The frequency, distribution, and types of mutations were shown to differ between the sporadic and familial tumors. The majority of the mutations resulted in protein truncation and were consistent with more severe phenotype, however three missense mutations were identified during this study and were all associated with milder manifestations of the disease. Received: 25 September 1995 / Revised: 19 December 1995     

Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples.

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.     

Antheridiogen,dark spore germination,and outcrossing mechanisms in Bommeria (Adiantaceae)

Isozymic analyses of the patterns of genetic variability in sporophyte populations have demonstrated that most fern species have outcrossing breeding systems. However, because fertilization takes place during the ephemeral, diminutive gametophyte generation, direct observation of breeding systems in nature has not been possible. Recent discoveries of soil-bound spore banks suggested that genetic diversity could be stored beneath the surface and subsequently released by appropriate chemical cues. Previous studies demonstrated that Bommeria sporophytes are the product of outcrossing, that their gametophytes carry high levels of genetic load, and that they produce and respond to antheridiogen. Research reported here demonstrated that Bommeria spores can survive long-term storage but will not germinate in the dark. Antheridiogen, however, will release spores from this light requirement and stimulate germination. Higher concentrations of antheridiogen result in higher germination rates. Gametophytes grown in the dark on antheridiogen-enriched agar form antheridia and release actively swimming sperms. Thus, spores housed beneath the soil surface could remain dormant until stimulated to germinate by antheridiogen secreted by surface-dwelling, archegoniate gametophytes. Sperm released from these subterranean gametophytes could fertilize eggs on the surface. Because spores housed in the soil are likely to be genetically different than those at the surface, heterozygous sporophytes would be more likely to result. Discovering that Bommeria species contain all of the prerequisites for this proposed outcrossing mechanism provides an explanation for the maintenance of genetic diversity in some fern populations.     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.