Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.     

A strategy to study <Emphasis Type="Italic">tyrosinase</Emphasis> transgenes in mouse melanocytes

Background  

A number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalise melanocyte cell cultures from postnatal mouse skin.     

A genomewide exploration suggests a new candidate gene at chromosome 11q23 as the major determinant of plasma homocysteine levels: results from the GAIT project

Homocysteine (Hcy) plasma level is an independent risk marker for venous thrombosis, myocardial infarction, stroke, congestive heart failure, osteoporotic fractures, and Alzheimer disease. Hcy levels are determined by the interaction of genetic and environmental factors. The genetic basis is still poorly understood, since only the MTHFR 677 C-->T polymorphism has been consistently associated with plasma Hcy levels. We conducted a genomewide linkage scan for genes affecting variation in plasma Hcy levels in 398 subjects from 21 extended Spanish families. A variance-components linkage method was used to analyze the data. The strongest linkage signal (LOD score of 3.01; genomewide P = .035) was found on chromosome 11q23, near marker D11S908, where a candidate gene involved in the metabolism of Hcy (the nicotinamide N-methyltransferase gene [NNMT]) is mapped. Haplotype analyses of 10 single-nucleotide polymorphisms within this gene found one haplotype associated with plasma Hcy levels (P = .0003). Our results, to our knowledge, represent the first genomic scan for quantitative variation in Hcy plasma levels. They strongly suggest that the NNMT gene could be a major genetic determinant of plasma Hcy levels in Spanish families. Since this gene encodes an enzyme involved in Hcy synthesis, this finding would be consistent with known biochemical pathways. These data could be relevant in determining the relationships between Hcy level, cardiovascular disease, osteoporosis, and Alzheimer disease.     

Exploring the interaction of the surfactant N-terminal domain of gamma-Zein with soybean phosphatidylcholine liposomes

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

The epidemiological investigation of Trichinella infection in brown rats (Rattus norvegicus) and domestic pigs in Croatia suggests that rats are not a reservoir at the farm level

Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering.     

LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy

The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H(2)O(2). A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H(2)O(2) accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.     

Geographic differentiation in the pollination system of the columnar cactus Pachycereus pecten -aboriginum

The pollination biology of the cactus Pachycereus pecten-aboriginum was studied in a tropical location in western Mexico (ca. 18° N latitude) to compare with data from a northern population (ca. 28° N latitude). Throughout this range, the nectar-feeding bat Leptonycteris curasoae is resident within the tropics but migratory in its northern range. The hypothesis was tested that if a predictable bat presence has been an important force in the evolution of pollination systems in columnar cacti, P. pecten-aboriginum will have a specialized pollination system within the tropics and a generalized pollination system in northern populations. In both areas, pollination experiments showed that P. pecten-aboriginum has a self-incompatible, hermaphroditic breeding system. In the tropical area, flowers open at night and close early in the morning. Nectar is secreted only during the night, and flowers are exclusively pollinated by three species of nectar-feeding bats, indicating a specialized pollination system. In contrast, anthesis and nectar secretion in northern populations occur during the night and day, allowing visitation and effective pollination by both nocturnal and diurnal pollinators. This study provides evidence of divergence mediated by pollinator predictability (resident vs. migrant), through shifts from short to long anthesis and nectar production periods from southern to northern populations.