[Temporal and spatial variations and driving factors of phytoplankton in Yongjiang River estuary]. / ç”¬æ±Ÿå£æµ®æ¸¸æ¤ç‰©æ—¶ç©ºåˆ†å¸ƒç‰¹å¾åŠé©±åŠ¨å› å­.

To explore the temporal and spatial variations in phytoplankton community in small estuaries, we collected surface water samples from Yongjiang River estuary during wet, normal, and dry seasons and determined the main driving factors of phytoplankton community. A total of 358 species belonging to nine phyla and 123 genera were identified in all seasons. During wet, normal, and dry seasons, species number was 276, 154 and 151, and the abundance was (170.45±225.43)×103, (51.92±30.28)×103 and (31.65±12.79)×103 cells·L-1, respectively. Diatoms dominated the phytoplankton community, and the main dominant species were Cyclotella meneghiniana, Skeletonema costatum, and Paralia sulcata. Shannon diversity and Pielou evenness indices decreased from inside mouth to outside mouth in wet season, but there was no obvious spatial difference in normal season or dry season. Results of non-metric multidimensional scaling analysis and analysis of similarities showed that phytoplankton community composition differed significantly among different regions (inside, at and outside mouth) and different seasons. In wet season, phytoplankton abundance was significantly positively correlated with temperature, dissolved inorganic nitrogen, and dissolved reactive phosphorus, but significantly negatively correlated with salinity. In normal season, phytoplankton abundance was significantly negatively correlated with temperature. In dry season, it was not significantly correlated with environmental factors. Results of redundancy analysis showed that temperature, salinity, ammonium and dissolved reactive phosphorus explained the variations in phytoplankton community by 19.5%, 11.9%, 9.4% and 8.2%, respectively. These results revealed high dominance of diatoms and the main driving factors (temperature, salinity and nutrients) of phytoplankton community in Yongjiang River estuary.

Antibodies to cryptic epitopes on HLA class I and class II heavy chains bound to single antigen beads: Clinically relevant?

Cell surface HLA class I consists of trimers, i.e., alpha - heavy chain, beta - 2 - microglobulin, and a peptide, termed closed conformers (CC) on non-activated lymphocytes. HLA class I and class II may also exist, respectively, as alpha-chain only or alpha and beta - chain only on activated cells termed open conformers (OC). We extend previous studies using an OC-specific monoclonal antibody that demonstrate LABScreen HLA class I and II single antigen beads (SABs) contain a mixture of open and closed conformers. LIFECODES SABs have bound CC only. More HLA class I and class II LABScreen SABs were reactive than LIFECODES SABs due to the presence of OC on LABScreen SABs. We hypothesized that antibody against OC on HLA B antigens would not be detected in cell based cross matches (XMs) with typical lymphocyte targets since anti-HLA OC antibodies would not react with native HLA CC on the cell surface. To test this hypothesis, we performed flow cytometry XM (FCXM) assays with sera of sufficient strength that most laboratories would likely predict positive FCXMs. Sera that reacted strongly with LABScreen SABs (>13,000 MFI) but weakly or not at all with LIFECODES SABs (<1000 MFI) gave negative T and B cell FCXMs. In contrast, sera that reacted with LIFECODES SABs (>13,000 MFI) but weakly with LABScreen SABs (<2100 MFI) exhibited positive FCXMs. Detection of antibodies directed against OC in SAB assays, may lead to inappropriate listing of unacceptable antigens, a decision not to XM or pre-or post - transplant desensitization procedures.

Unraveling the Novel Effect of Patchouli Alcohol Against the Antibiotic Resistance of Helicobacter pylori.

The long-term colonization of Helicobacter pylori can cause various gastrointestinal diseases, and its high genetic variability is prone to antibiotic resistance and leads to failure of clinical treatment. Intracellular survival also contributes to the drug tolerance of H. pylori. Patchouli alcohol (PA) shows a highly efficient activity against H. pylori in vitro and in vivo. And this study aims to explore whether PA can reduce the resistance of H. pylori and determine the underlying mechanism. Checkerboard and time-kill bactericidal curve assay reveal that the combination of PA and clarithromycin (CLR) promoted the inhibition and bactericidal effect against H. pylori. Stimulation of CLR leads to the internalization of H. pylori, but PA can effectively inhibit the invasion induced by CLR. Compared with antibiotics, PA remarkably eradicated the intracellular H. pylori, and this intracellular sterilized ability was further improved in combination with antibiotics (CLR and metronidazole). The expression of H. pylori efflux pump genes (hp0605, hp1327, and hp1489) was dose-dependently downregulated by PA. Digital droplet PCR indicated that the H. pylori mutant of A2143G can be inhibited by PA. Cellular uptake and transport assays showed that PA is rapidly absorbed, which promotes its activity against intracellular bacteria. Therefore, PA can act synergistically with CLR as a candidate treatment against drug-resistant H. pylori.

The alpha1H Ca2+ channel subunit is expressed in mouse jejunal interstitial cells of Cajal and myocytes.

T-type Ca(2+) currents have been detected in cells from the external muscular layers of gastrointestinal smooth muscles and appear to contribute to the generation of pacemaker potentials in interstitial cells of Cajal from those tissues. However, the Ca(2+) channel alpha subunit responsible for these currents has not been determined. We established that the alpha subunit of the alpha(1H) Ca(2+) channel is expressed in single myocytes and interstitial cells of Cajal using reverse transcription and polymerase chain reaction from whole tissue, laser capture microdissected tissue and single cells isolated from the mouse jejunum. Whole-cell voltage clamp recordings demonstrated that a nifedipine and Cd(2+) resistant, mibefradil-sensitive current is present in myocytes dissociated from the jejunum. Electrical recordings from the circular muscle layer demonstrated that mibefradil reduced the frequency and initial rate of rise of the electrical slow wave. Gene targeted knockout of both alleles of the cacna1h gene, which encodes the alpha(1H) Ca(2+) channel subunit, resulted in embryonic lethality because of death of the homozygous knockouts prior to E13.5 days in utero. We conclude that a channel with the pharmacological and molecular characteristics of the alpha(1H) Ca(2+) channel subunit is expressed in interstitial cells of Cajal and myocytes from the mouse jejunum, and that ionic conductances through the alpha(1H) Ca(2+) channel contribute to the upstroke of the pacemaker potential. Furthermore, the survival of mice that do not express the alpha(1H) Ca(2+) channel protein is dependent on the genetic background and targeting approach used to generate the knockout mice.

Exogenous serotonin regulates proliferation of interstitial cells of Cajal in mouse jejunum through 5-HT2B receptors.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) are required for normal gastrointestinal motility. Loss of ICC is associated with several motility disorders. The mechanisms modulating ICC survival and proliferation are poorly understood. This study aimed to establish whether 5-hydroxytryptamine (5-HT) plays a role in regulating ICC proliferation. METHODS: Expression of 5-HT receptor mRNA was investigated in muscle strips, in purified populations of ICC, and in identified single cells. The effect of 5-HT(2B) receptor ligands on ICC numbers was studied in primary cell cultures. Proliferation of ICC was determined by counting Ki67-positive cells in culture. RESULTS: Of the 5-HT receptors known to be involved in proliferation, 5-HT(2B) receptor mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in jejunal muscle, whereas 5-HT(1A), 5-HT(1D), and 5-HT(2C) receptor mRNAs were not. 5-HT(2B) receptor mRNA was found in single ICC and cells purified by flow cytometry. Exogenous 5-HT (1 micromol/L) increased (66% +/- 9%, P < .005) ICC numbers in culture. The 5-HT(2) receptor antagonist, ritanserin, and the 5-HT(2B) receptor antagonist, SB204741, inhibited the effect of 5-HT. The 5-HT(2B) receptor agonist BW 723C86 induced a concentration-dependent increase in ICC number (50% +/- 6% at 50 nM, P < .04) and increased ICC proliferation (25% +/- 3% vs 19 +/- 1% in controls, P < .03). CONCLUSIONS: These studies establish that 5-HT(2B) receptors are expressed on ICC. Exogenous 5-HT regulates ICC numbers through 5-HT(2B) receptors in part by increasing ICC proliferation. The 5-HT(2B) receptor may serve as a novel pathway to regulate ICC numbers.

Effect of mibefradil on sodium and calcium currents.

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (Na(V)1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel alpha(1)3.3b + beta(2), the human intestinal L-type Ca2+ channel subunits alpha(1C) + beta(2), or Na(V)1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations > or = 0.1 microM (IC(50) = 0.29 microM), L-type Ca2+ current at > 1 microM (IC(50) = 2.7 microM), and Na+ current at > or = 0.3 microM (IC(50) = 0.98 microM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.

Sodium current in human intestinal interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) generate the electrical slow wave required for normal gastrointestinal motility. The ionic conductances expressed in human intestinal ICC are unknown. The aim of this study was to determine expression of a Na+ current in human intestinal ICC and to determine the effects of the Na+ current on the slow wave. Visually identified, freshly dissociated, single ICC were verified by the presence of c-kit mRNA by using single-cell RT-PCR. Standard whole cell currents were recorded from patch-clamped ICC held at -100 mV between pulse protocols. A Na+ current was identified in human intestinal ICC. The current activated at -55 mV and peaked at -30 mV. Extracellular N-methyl-d-glucamine abolished and QX-314 (500 microM) blocked the Na+ current, but nifedipine and Ni2+ did not. The Na+ current was activated by shear stress. Single-cell RT-PCR detected mRNA for the Na+ alpha-subunit SCN5A in single human intestinal ICC. Lidocaine (200 microm) and QX-314 (500 microM) decreased slow wave frequency, and stretch increased slow wave frequency. A mechanosensitive Na+ channel current is present in human intestinal ICC and appears to play a role in the control of intestinal motor function.

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells.

A Na(+) current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na(+) current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs(+) in the pipette to block K(+) currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na(+) current recorded from jejunal smooth muscle cells by 27 +/- 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na(+) current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na(+) current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na(+) current.

Syntrophin gamma 2 regulates SCN5A gating by a PDZ domain-mediated interaction.

SCN5A encodes the alpha subunit of the cardiac muscle and intestinal smooth muscle mechanosensitive Na(+) channel. Mechanosensitivity in the intestine requires an intact cytoskeleton. We report, using laser capture microdissection, single cell PCR, and immunohistochemistry, that syntrophins, scaffolding proteins, were expressed in human intestinal smooth muscle cells. The distribution of syntrophin gamma 2 was similar to that of SCN5A. Yeast two-hybrid and glutathione S-transferase pull-down experiments show that SCN5A and syntrophin gamma 2 co-express and that the PDZ domain of syntrophin gamma 2 directly interacts with the C terminus of SCN5A. In native cells, disruption of the C terminus-syntrophin gamma 2 PDZ domain interaction using peptides directed against either region result in loss of mechanosensitivity. Co-transfection of syntrophin gamma 2 with SCN5A in HEK293 cells markedly shifts the activation kinetics of SCN5A and reduces the availability of Na(+) current. We propose that syntrophin gamma 2 is an essential Na(+) channel-interacting protein required for the full expression of the Na(+) current and that the SCN5A-syntrophin gamma 2 interaction determines mechanosensitivity and current availability.

The Kv2.2 alpha subunit contributes to delayed rectifier K(+) currents in myocytes from rabbit corpus cavernosum.

K(+) currents are known to regulate the excitability of corpus cavernosum myocytes and therefore to play a role in the control of penile erection and detumescence. We used electrophysiology and molecular cloning techniques to identify ion channel proteins that contribute to K(+) currents in rabbit cavernosal myocytes. Currents were recorded from freshly isolated myocytes using whole-cell patch clamp techniques. Cavernosal myocytes expressed a delayed rectifier voltage-gated K(+) current that appeared to contribute to the resting membrane potential. This voltage-gated K(+) (K(v)) current was inhibited by the nonselective compounds 4-aminopyridine (1-10 mM), (+)-fenfluramine (10 micro M-1 mM), and Grammostola spatulata venom (1:100) in a dose-dependent and reversible fashion. Hanatoxin-1 (1 micro M), a selective Kv2 channel inhibitor, partially inhibited the current, but alpha-dendrotoxin (200 nM), a Kv1 channel blocker, had no effect. The nucleotide sequence of K(+) channel subunits was determined by polymerase chain reaction-based cloning techniques using RNA derived from cavernosal muscle strips and single identified myocytes. Molecular cloning techniques identified the full-length sequence of the rabbit ortholog of the Kv2.2 alpha subunit. This sequence contains 911 amino acid residues and is 92% identical to the recently revised human Kv2.2 sequence. Identified cavernosal myocytes of the type used in physiological recordings expressed Kv2.2 messenger RNA. We conclude that Kv2.2 alpha subunits contribute to whole-cell currents in rabbit canvernosal myocytes. Further, K(v) currents play a role in regulating membrane potential and hence excitability in rabbit cavernosal myocytes.

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.

Sodium current in human jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study was to determine if a Na(+) current is present in human jejunal circular smooth muscle cells. METHODS: Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for alpha subunits of Na(+) channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened. RESULTS: A tetrodotoxin-insensitive Na(+) channel was present in 80% of cells patch-clamped. Initial activation was at -65 mV with peak inward current at -30 mV. Steady-state inactivation and activation curves revealed a window current between -75 and -60 mV. The Na(+) current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A, the alpha subunit of the cardiac Na(+) channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection. CONCLUSIONS: Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na(+) channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.