[Characteristics and clinical analysis of MLH1 c.463dupC gene mutation in a Lynch syndrome family].

In this study, a case of Lynch syndrome (LS) family line with a novel mutation site in the MLH1 c.463dupC gene was reported and the clinical and pathogenic genetic features of this family were analyzed. A 40-year-old female patient with colon cancer diagnosed at the First Affiliated Hospital of Kunming Medical University on October 2, 2020 was retrospectively included. The clinical data of the family were collected and the family lineage was drawn. The family tumor history met the Amsterdam Criteria Ⅱ and the diagnostic criteria of LS in Chinese, which was a typical LS family lineage. A germline code-shift missense mutation c.463dupC in the MLH1 gene located in exon 6, a possible pathogenic variant, was detected by second-generation sequencing (NGS) in the patient. Subsequently, Sanger sequencing was performed on a total of 20 direct lineage members of the family of the MLH1 gene, 7 cases were found to harbor the mutation and included in the LS high-risk control. Follow-up to October 2023 showed that the patient had endometrial and cervical polyps, one case had colorectal cancer, and two cases had intestinal polyps, all were treated with early intervention and therapy; two cases did not show any clinical symptoms. This study is the first to report a new mutation site for the potentially pathogenic MLH1 c.463dupC, providing a rationale for the pathogenicity of the mutation and standardized health management for familial carriers.

[Analysis of factors influencing lung function in patients with pneumoconiosis].

Objective: To analyze the influencing factors of lung function in pneumoconiosis patients, and to provide reference for clinical treatment. Methods: From July 2020 to December 2020, a questionnaire survey was conducted on pneumoconiosis patients in the jurisdiction by using the "Guangdong Province Occupational Disease Prevention and Control Institute" questionnaire, and the relevant items of patients were examined. The rate of counting data is expressed, and the measurement data is expressed by mean and standard deviation. Chi-square test was used for comparison between groups, trend chi-square test was used for trend analysis of ordered classified data. Multivariate analysis was carried out with binary logistic regression model. Results: A total of 1409 pneumoconiosis patients were enrolled. The abnormal rate of lung function in pneumoconiosis patients was 68.77%. The results of trend Chi-square test showed that the abnormal rate of lung function increased with the age of exposure to dust in different age groups (Chi Sqnare Trend=64.12、8.49、24.20, P<0.05) . In univariate analysis, there were statistical significance in different dust exposure age, working age, pneumoconiosis stage, complications and occupational pneumoconiosis diseases (P<0.05) . Multiple logistic regression results showed that age of exposure to dust, years of service, stage of pneumoconiosis and complications were the main influencing factors of lung function in pneumoconiosis patients (P<0.05) . Compared with patients aged 0-30 years, patients aged 50-70 years and older had a higher rate of abnormal lung function (OR=2.16, 95%CI: 1.12~4.16; OR=4.82, 95%CI: 2.05~11.35, all P<0.05) ; Compared with patients with 0~20 years of service, patients with 20~30 years of service and more than 30 years of service had a higher rate of abnormal lung function (OR=1.58, 95%CI: 1.10~2.25; OR=1.63, 95%CI: 1.28~2.40, P<0.05) ; Compared with stage Ⅰ patients, Stage Ⅱ and Stage Ⅲ patients had a higher rate of abnormal lung function (OR=1.62, 95%CI: 1.20~2.17; OR=2.23, 95%CI: 1.40~3.55, all P<0.05) ; Compared with patients without comorbidities, patients with comorbidities had a higher rate of abnormal lung function (OR=1.68, 95%CI: 1.20~2.38, P<0.05) . Conclusion: The factors such as age of exposure to dust, working age, stage of pneumoconiosis and complications may be the influencing factors of lung function in pneumoconiosis patients.

Differentiating infected focal liver lesions from malignant mimickers: value of ultrasound-based radiomics.

AIM: To establish an ultrasound-based radiomics model through machine learning methods and then to assess the ability of the model to differentiate infected focal liver lesions from malignant mimickers. MATERIALS AND METHODS: A total of 104 patients with infected focal liver lesions and 485 patients with malignant hepatic tumours were included, consisting of hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), combined hepatocellular-cholangiocarcinoma (cHCC-CC), and liver metastasis. Radiomics features were extracted from grey-scale ultrasound images. Feature selection and predictive modelling were carried out by dimensionality reduction methods and classifiers. The diagnostic effect of the prediction mode was assessed by receiver operating characteristic (ROC) curve analysis. RESULTS: In total, 5,234 radiomics features were extracted from grey-scale ultrasound image of every focal liver lesion. The ultrasound-based radiomics model had a favourable predictive value for differentiating infected focal liver lesions from malignant hepatic tumours, with an area under the curve (AUC) of 0.887 and 0.836 (HCC group), 0.896 and 0.766 (CC group), 0.944 and 0.754 (cHCC-CC group), 0.918 and 0.808 (liver metastasis group), and 0.949 and 0.745 (malignant hepatic tumour group) for the training set and validation set, respectively. CONCLUSIONS: Ultrasound-based radiomics is helpful in differentiating infected focal liver lesions from malignant mimickers and has the potential for use as a supplement to conventional grey-scale ultrasound and contrast-enhanced ultrasound (CEUS).

Screening of drug metabolizing enzymes for fusidic acid and its interactions with isoform-selective substrates in vitro.

1. Fusidic acid (FA) is widely used for the treatment of infections of sensitive osteomyelitis or skin and soft tissue caused by bacteria. However, the role of cytochrome P450s (CYPs) in the metabolism of FA is unclear. In the present study, we screened the main CYPs for the metabolism of FA and studied its interactions with isoform-selective substrates in vitro. 2. The main CYP450s were screened according to the inhibitory effect of specific inhibitors on the metabolism of FA in human liver microsomes (HLMs) or recombinant CYP isoforms. Enzyme kinetic parameters including Ki, Ki', Vmax, and IC50 were calculated to determine the potential of FA to affect CYP-mediated metabolism of isoform-selective substrates. 3. FA metabolism rate was inhibited by 49.8% and 83.1% under CYP2D6, CYP3A4 selective inhibitors in HLMs. In recombinant experiment, the inhibitory effects on FA metabolism were 83.3% for CYP2D6 and 58.9% for CYP3A4, respectively. FA showed inhibition on CYP2D6 and CYP3A4 with Kis of 13.9 and 38.6 µM, respectively. Other CYP isoforms including CYP1A2, CYP2A6, CYP2C9, CYP2E1, and CYP2C19 showed minimal or no effect on the metabolism of FA. 4. FA was primarily metabolized by CYP2D6 and CYP3A4 and showed a noncompetitive inhibition on CYP2D6 and a mixed competitive inhibition on CYP3A4. Drug-drug interactions between FA and other chemicals, especially with substrates of CYP2D6 and CYP3A4, are phenomena that clinicians need to be aware of and cautious about.

Sodium tanshinone IIA sulfonate and its interactions with human CYP450s.

1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro. 2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme-substrate interactions. 3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS. 4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug-drug interactions with other CYP3A4 substrates.

Trpv6 mediates intestinal calcium absorption during calcium restriction and contributes to bone homeostasis.

Energy-dependent intestinal calcium absorption is important for the maintenance of calcium and bone homeostasis, especially when dietary calcium supply is restricted. The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is a crucial regulator of this process and increases the expression of the transient receptor potential vanilloid 6 (Trpv6) calcium channel that mediates calcium transfer across the intestinal apical membrane. Genetic inactivation of Trpv6 in mice (Trpv6(-/-)) showed, however, that TRPV6 is redundant for intestinal calcium absorption when dietary calcium content is normal/high and passive diffusion likely contributes to maintain normal serum calcium levels. On the other hand, Trpv6 inactivation impaired the increase in intestinal calcium transport following calcium restriction, however without resulting in hypocalcemia. A possible explanation is that normocalcemia is maintained at the expense of bone homeostasis, a hypothesis investigated in this study. In this study, we thoroughly analyzed the bone phenotype of Trpv6(-/-) mice receiving a normal (approximately 1%) or low (approximately 0.02%) calcium diet from weaning onwards using micro-computed tomography, histomorphometry and serum parameters. When dietary supply of calcium is normal, Trpv6 inactivation did not affect growth plate morphology, bone mass and remodeling parameters in young adult or aging mice. Restricting dietary calcium had no effect on serum calcium levels and resulted in a comparable reduction in bone mass accrual in Trpv6(+/+) and Trpv6(-/-) mice (-35% and 45% respectively). This decrease in bone mass was associated with a similar increase in bone resorption, whereas serum osteocalcin levels and the amount of unmineralized bone matrix were only significantly increased in Trpv6(-/-) mice. Taken together, our findings indicate that TRPV6 contributes to intestinal calcium transport when dietary calcium supply is limited and in this condition indirectly regulates bone formation and/or mineralization.

Cellular mechanisms of WNK4-mediated regulation of ion transport proteins in the distal tubule.

Gene mutations in WNK4 kinase cause a genetic form of hypertension by affecting multiple ion transport pathways through different mechanisms. Cai et al. report that the inhibitory effect of WNK4 on the thiazide-sensitive sodium-chloride cotransporter occurs through the lysosomal degradation pathway in mammalian cells.

The structure of ultrathin Langmuir-Blodgett films of cadmium behenate.

Grazing incidence x-ray-diffraction investigations of the structures of Langmuir-Blodgett films of cadmium behenate with 1, 2, 3, 5, and 21 monolayers are reported. The single monolayer film, deposited on a hydrophilic substrate, showed a hexagonal structure, whereas the bilayer film, deposited on a hydrophobic substrate, had a rectangular structure with herringbone orientation of the acyl chains. With multilayer films formed on a hydrophilic substrate, it was possible to detect that the hexagonal structure of the first layer was retained when additional layers were deposited and that the additional layers had the same rectangular structure as the bilayer.

Single-channel activities of the human epithelial Ca2+ transport proteins CaT1 and CaT2.

The human epithelial channels, CaT1 and CaT2, were expressed in oocytes, and their single-channel characteristics were compared. In the presence of Na+ and K+ as charge carriers in the pipette solutions, channel activities were observed only when the the extracellular sides of the patches were exposed to nominally Ca2+- and Mg2+-free solutions. In patches of both CaT1- and CaT2-expressing oocytes, multiple channel openings were observed, but the current levels were higher in CaT2-expressing oocytes, particularly at more negative voltages. With K+ as a charge carrier in patches of CaT1-expressing oocytes, the channel activity was low at -10 to -60 mV, but increased dramatically at more negative potentials. This voltage dependence was observed in the presence of both Na+ and K+. The channel activity with Na+, however, was higher at all potentials. Differences between the voltage dependencies for the two cations were also observed in CaT2-expressing oocytes, but the channel activities were higher than those in CaT1-expressing oocytes, particularly in the presence of Na+. We also found that low concentrations of extracellular Mg2+ (5-50 microm) elicited a strong inhibitory action on the CaT channels. Activation of the CaT1 and CaT2 channels by hyperpolarization and other factors may promote increased Ca2+ entry that participates in stimulation of intestinal absorption and renal reabsorption and/or other Ca2+ transport mechanisms in epithelial cells.

Inhibition of the glutamate transporter EAAC1 expressed in Xenopus oocytes by phorbol esters.

Recent evidence indicates that second messengers and protein kinases regulate the activity and expression of glutamate transporters. The aim of the present study was to determine if direct activation of protein kinases C or A modulates the activity of the sodium-dependent glutamate transporter EAAC1. EAAC1 modulation was studied in cRNA-injected Xenopus oocytes by measuring [3H]L-glutamate uptake or glutamate-evoked uptake currents. We found that activation of PKA was ineffective, whereas treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) caused a significant decrease in EAAC1 transport activity (IC(50)=44.7+/-12 nM). PMA-induced EAAC1 inhibition was PKC-mediated because the inhibition could be blocked by specific PKC inhibitors and incubation with the inactive 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) did not affect EAAC1. Saturation studies of glutamate-evoked uptake currents showed that PMA-mediated inhibition was due to a decrease in I(max) with no change in K(m). PMA simultaneously decreased membrane capacitance (C(m)) and transport-associated current and increased cytosolic accumulation of EAAC1 protein, compared to control. These results suggest that PKC activation inhibits EAAC1 by promoting its retrieval from the plasma membrane. PMA also significantly decreased glutamate uptake in a Madin-Darby canine kidney (MDCK) cell line stably transfected with EAAC1 but enhanced EAAC1-mediated glutamate uptake in the rat C6 glioma cells, consistent with previous observations. Because activation of PKC by phorbol esters leads to opposite effects on EAAC1 activity in different culture models, we conclude that the PKC-mediated regulation of EAAC1 is cell-type specific.

Structural conservation of the genes encoding CaT1, CaT2, and related cation channels.

We report here the genomic structures of the genes encoding human calcium transport proteins CaT1 and CaT2, which belong to a recently identified class of highly selective calcium entry channels. The mRNA for CaT1 was expressed more abundantly than that for CaT2 in three major tissues involved in transcellular calcium transport, namely intestine, kidney, and placenta, as determined by quantitative PCR. The genes encoding CaT1 and CaT2, ECAC2 and ECAC1, respectively, are completely conserved in terms of exon size in the coding regions. They also share similar intron-exon structures with the genes encoding the closely related, nonselective cation channels VR1, VRL-1, OTRPC4 (also known as VR-OAC, Trp12, and VRL-2), and a hypothetical protein, VRL-3. We conclude that ECAC2 and ECAC1, which encode calcium selective channels, share a common ancestral gene with the genes encoding the related nonselective cation channels.

The structures of Langmuir-Blodgett films of fatty acids and their salts.

Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-trans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern. Multilayer films of the acids show only a single-step order-disorder transition at the melting point. This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached. Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed. In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains.

CaT1 expression correlates with tumor grade in prostate cancer.

Ca(2+) signaling is important for growth and survival of prostatic carcinoma (PCa) cells. Here we report that the gene for CaT1, a channel protein highly selective for Ca(2+), is expressed at high levels in human PCa and in the LNCaP PCa cell line. CaT1 mRNA levels were elevated in PCa specimens in comparison to benign prostatic hyperplasia (BPH) specimens and positively correlated with Gleason grade in a PCa series. CaT1 mRNA was suppressed by androgen and was induced by a specific androgen receptor antagonist in LNCaP cells, suggesting that the gene is negatively regulated by androgen. These findings are the first to implicate a Ca(2+) channel in PCa progression and suggest that CaT1 may be a novel target for therapy.

CaT1 manifests the pore properties of the calcium-release-activated calcium channel.

The calcium-release-activated Ca2+channel, ICRAC, is a highly Ca2+-selective ion channel that is activated on depletion of either intracellular Ca2+ levels or intracellular Ca2+ stores. The unique gating of ICRAC has made it a favourite target of investigation for new signal transduction mechanisms; however, without molecular identification of the channel protein, such studies have been inconclusive. Here we show that the protein CaT1 (ref. 4), which has six membrane-spanning domains, exhibits the unique biophysical properties of ICRAC when expressed in mammalian cells. Like ICRAC, expressed CaT1 protein is Ca2+ selective, activated by a reduction in intracellular Ca2+ concentration, and inactivated by higher intracellular concentrations of Ca2+. The channel is indistinguishable from ICRAC in the following features: sequence of selectivity to divalent cations; an anomalous mole fraction effect; whole-cell current kinetics; block by lanthanum; loss of selectivity in the absence of divalent cations; and single-channel conductance to Na+ in divalent-ion-free conditions. CaT1 is activated by both passive and active depletion of calcium stores. We propose that CaT1 comprises all or part of the ICRAC pore.

Transport function of the naturally occurring pathogenic polycystin-2 mutant, R742X.

Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations truncating polycystin-1 (PC1) or polycystin-2 (PC2), products of the PKD1 and PKD2 genes, respectively. A third member of the polycystin family, polycystin-L (PCL), was recently shown to function as a Ca(2+)-modulated nonselective cation channel. More recently, PC2 was also shown to be a nonselective cation channel with comparable properties to PCL, though the membrane targeting of PC2 likely varies with cell types. Here we show that PC2 expressed heterologously in Xenopus oocytes is targeted to intracellular compartments. By contrast, a truncated form of mouse PC2 corresponding to a naturally occurring human mutation R742X is targeted predominantly to the plasma membrane where it mediates K(+), Na(+), and Ca(2+) currents. Unlike PCL, the truncated form does not display Ca(2+)-activated transport activities, possibly due to loss of an EF-hand at the C-terminus. We propose that PC2 forms ion channels utilizing structural components which are preserved in the R742X form of the protein. Implications for epithelial cell signaling are discussed.

Polycystin-2 is a novel cation channel implicated in defective intracellular Ca(2+) homeostasis in polycystic kidney disease.

Mutations in polycystins-1 and -2 (PC1 and PC2) cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by progressive development of epithelial renal cysts, ultimately leading to renal failure. The functions of these polycystins remain elusive. Here we show that PC2 is a Ca(2+)-permeable cation channel with properties distinct from any known intracellular channels. Its kinetic behavior is characterized by frequent transitions between closed and open states over a wide voltage range. The activity of the PC2 channel is transiently increased by elevating cytosolic Ca(2+). Given the predominant endoplasmic reticulum (ER) location of PC2 and its unresponsiveness to the known modulators of mediating Ca(2+) release from the ER, inositol-trisphosphate (IP(3)) and ryanodine, these results suggest that PC2 represents a novel type of channel with properties distinct from those of the other Ca(2+)-release channels. Our data also show that the PC2 channel can be translocated to the plasma membranes by defined chemical chaperones and proteasome modulators, suggesting that in vivo, it may also function in the plasma membrane under specific conditions. The sensitivity of the PC2 channel to changes of intracellular Ca(2+) concentration is deficient in a mutant found in ADPKD patients. The dysfunction of such mutants may result in defective coupling of PC2 to intracellular Ca(2+) homeostasis associated with the pathogenesis of ADPKD.

Inhibition of CaT1 channel activity by a noncompetitive IP3 antagonist.

A newly cloned, human epithelial Ca2+ transport protein (CaT1) was expressed in Xenopus laevis oocytes, and its single channel characteristics were examined. The CaT1 channel shows a strong dependence upon hyperpolarizing voltages, being activated by very negative voltages. The probability of channel opening and mean open times increase substantially at more negative voltages in the range of -90 to -160 mV. In addition, CaT1 channel activity was markedly inhibited by micromolar levels of a noncompetitive antagonist of the IP3 receptor originally isolated from a marine sponge, Xestospongin C. This inhibitory effect could be mediated indirectly via the binding of Xestospongin C to the inositol-trisphosphate (IP3) receptor or, alternatively, by a direct action on the CaT1 channel itself. Independent of its mechanism of action in inhibiting CaT1, Xestospongin C will provide a useful tool for elucidating the physiological role(s) of this novel epithelial Ca2+ channel.

Human calcium transport protein CaT1.

Transcellular calcium transport occurs in many epithelial tissues including intestine, kidney, and placenta. We identified the human ortholog (hCaT1) of a recently cloned rat calcium transport protein, CaT1, that mediates intestinal calcium uptake. hCaT1 messenger RNA is present in the gastrointestinal tract, including esophagus, stomach, duodenum, jejunum, ileum, and colon. High levels of hCaT1 transcripts are also present in pancreas, placenta, prostate, and salivary gland, while moderate levels are present in liver, kidney, and testis. hCaT1 mRNA is also expressed in the colorectal cancer cell line, SW480, and the chronic myelogenous leukemia cell line, K-562. The hCaT1 gene was assigned to the long arm of chromosome 7, bands q33-34, by fluorescence in situ hybridization. When expressed in Xenopus laevis oocytes, hCaT1 promotes saturable Ca(2+) uptake with a Michaelis constant of 0.25 mM. Our studies suggest a role for hCaT1 in cellular calcium uptake in a variety of tissues, including the transcellular calcium transport pathway in intestine.

A rat kidney-specific calcium transporter in the distal nephron.

Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CaT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D(28K) and the sodium calcium exchanger 1, NCX1, by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D(28K) were colocalized in the same cells. CaT2 mediated saturable calcium uptake with a Michaelis constant (K(m)) of 0.66 mm when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca(2+). Sr(2+) and Ba(2+) but not Mg(2+) also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd(2+) elicited inward current in CaT2-expressing oocytes with a K(m) of 1.3 mm. Cd(2+), however, also potently inhibited CaT2-mediated Ca(2+) uptake with an IC(50) of 5.4 micrometer. Ca(2+) evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd(2+) also provides a potential explanation for Cd(2+)-induced hypercalciuria and resultant renal stone formation.

[Cloning and analyzing of members of excitatory amino acid transporter family from neonatal mouse brain].

Excitatory amino acid transporter family (EAAT) contains several structure-related membrane proteins. They are essential for the removal of glutamate released from pre-synaptic terminal to terminate its action of synaptic transduction and maintaining the normal concentration of neurotransmitters in nerve system. To study these proteins in single animal model, we cloned several members of EAAT family, named mGLAST-1, mGLT-1, mEAAC1 and mASCT1, from a neonatal mouse brain cDNA library. The cDNA sequence of mASCT1 was firstly reported in mouse, it is composed of 3787 bp which has an open reading frame (ORF) encoding a protein of 532 amino acid residues. The mASCT1 protein was expressed in Xenopus oocyte and the function was characterized by 3H-Ser uptaking. The homology between human ASCT1 and mouse ASCT1 is 89.3%. The DNA sequence data shows the variance in length and composition exists in the sequence of 5'UTR and 3'UTR of mRNA in the family members of EAAT. This phenomenon may indicate a post-transcription regulation mechanism might exist in the gene expression of mouse EAAT family members.