Biomedical subjects
Publications and source records attributed to A L Zhang.
This is the first reported case where the diagnosis of hypertrophic pyloric stenosis (HPS) was entertained in the antenatal period and the neonate was followed up in the postnatal period on a prospective basis until the HPS became manifest.
From the chloroform/methanol extract of the fruiting bodies of the ascomycete Chinese truffle Tuber indicum Cooke et Massee, a new trihydroxylated monounsaturated fatty acid (1) has been isolated. The structure of this new linoleic acid-derived metabolite was established as 9,10,11-trihydroxy-(12Z)-12-octadecenoic acid by means of spectroscopic and chemical methods. The fatty acid composition of the chloroform-soluble fraction of this fungus was analyzed by gas chromatography-mass spectrometry. The content of the predominant unsaturated fatty acids (oleic and linoleic acids) is as high as 68%. The use of dimethyl disulfide adduct was effective in the determination of the position of the double bond, and the glycol oxidation fission reaction with sodium metaperiodate supported on silica gel was helpful in establishing the location of the trihydroxylic groups in the new fatty acid.
Cytosine arabinoside (AraC) is a nucleoside analog that produces significant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC-induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC-induced cell death was reduced by treatment with several different free-radical scavengers (N-acetyl-L-cysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the fluorescence of the dye 5-(6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate. AraC produced DNA single-strand breaks as measured by single-cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53-dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens.
A method for the liquid-liquid micro-extraction and gas chromatographic analysis of trace phenol in small amount of aqueous sample has been developed. The influences of acid and salts on recovery rate of phenol were examined. And, the influences of injection speeds and the conditions of silica wool in injection port on the quantitation precision were tested. With a direct injection port, an FID and a DB-1 capillary column of wide bore, a detection limit of 1 microgram.L-1 can be obtained employing 8 mL water sample, 160 microL ethyl acetate and 3.5 g ammonium sulfate. The added recoveries were in the range of 95.0%-98.5%. The relative standard deviations were in the range of 2.8%-3.3%. This method is simple, convenient, rapid, accurate and practical.
Schwann cell precursors, derivatives of the neural crest, generate Schwann cells in a process that is tightly timed, well characterized, and directly controlled by axonal signals, in particular beta-neuregulins. Here we provide evidence that endothelins (ETs) are also important for survival and lineage progression in this system. We show that ETs promote rat Schwann cell precursor survival in vitro without stimulation of DNA synthesis. Using ET receptor agonists and antagonists, we demonstrate that this action of ET is mediated by the ET(B) receptor. RT-PCR reveals the presence of ET and ET receptor mRNA in the developing rat PNS. We showed previously that in vitro beta-neuregulins promote the generation of Schwann cells from precursors on schedule and that this process can be accelerated by fibroblast growth factor 2. Here we show that although ETs promote long-term precursor survival the transition of precursors to Schwann cells is delayed. Moreover, ETs block the maturation effects of beta-neuregulins. In spotting lethal rats, in which functional ET(B) receptors are absent, we find accelerated expression of the Schwann cell marker S100 in developing nerves. These observations indicate that complex growth factor interactions control the timing of Schwann cell development in embryonic nerves and that ETs act as negative regulators of Schwann cell generation.
Ca(2+)mobilization induced by ATP, isoproterenol and the Ca(2+)-ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca(2+)-sensitive fluorescent indicator fura-2. ATP and isoproterenol increased cytosolic free Ca(2+)([Ca(2+)](i)) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca(2+)](i). However, ATP and isoproterenol were not able to elicit a further increase in [Ca(2+)](i)after exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca(2+)](i)in the presence of thapsigargin probably due to agonist-induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P(2)-purinergic receptors and IP(3), one opened by isoproterenol acting through beta2-adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.
The mechanisms by which extracellular nucleotides (ATP and UTP) regulate intracellular Ca2+ in cultured pig tracheal gland cells were studied. The calcium response induced by ATP or UTP was composed of a peak response and a steady plateau. In the absence of extracellular Ca2+, the peak response of the cells to both ATP and UTP was smaller, and no subsequent plateau was observed. After treatment of the cells with pertussis toxin, the peak response to UTP was significantly smaller and no plateau was seen even in the presence of extracellular Ca2+, but pertussis toxin did not change the effect of ATP. Pretreatment with U107, a phospholipase C inhibitor, almost abolished the calcium response to both ATP and UTP. Immunocytochemistry showed that in these cells, the IP3 receptor was localized in the cytoplasm (including the endoplasmic reticulum) of the cells. Our results indicate that both release of calcium from the intracellular store and Ca2+ influx across the cell membrane contribute to the mobilization of [Ca2+]i upon stimulation with nucleotides, that ATP and UTP regulate intracellular Ca2+ predominantly via the G protein-phospholipase C-IP3 pathway, and that ATP and UTP may act via distinct subtypes of P2Y receptors.
The effects of cholinergic and alpha-adrenergic stimulation on calcium concentrations in rat submandibular gland acinar cells were evaluated at the subcellular level by electron probe X-ray microanalysis, and the subcellular distribution of receptors for inositol 1,4,5-trisphosphate (IP(3)Rs) was investigated by electron microscopic immunohistochemistry. For measurement of calcium concentrations, experiments were carried out both in vivo and in vitro. In in vivo experiments, fragments of glands were removed and slam-frozen 3 min after intraperitoneal injection of an agonist. In in vitro experiments, the gland tissue was enzymatically dispersed, treated with an agonist in the presence or absence of extracellular Ca(2+), and slam-frozen. The frozen specimens were cut into ultrathin cryosections, which were then freeze dried. Spectra were collected from secretory granules, the perinuclear cytoplasm containing endoplasmic reticulum (ER), and the nucleus of the acinar cells. A clear decrease in calcium content in secretory granules was observed after cholinergic stimulation both in vivo and in vitro in the presence or absence of extracellular Ca(2+). In the presence of extracellular Ca(2+), cholinergic stimulation following alpha-adrenergic stimulation reduced the calcium content in secretory granules to the same extent as cholinergic stimulation alone. No significant changes in the calcium content of the perinuclear cytoplasm and the nucleus were noticed after cholinergic stimulation. alpha-adrenergic stimulation did not significantly affect the calcium concentrations in any of the three compartments studied. IP(3)Rs were localized to ER lamellae, but not to secretory granule membranes or to nuclear membranes. Our findings suggest that: (1) in rat submandibular acini, Ca(2+) can be released from secretory granules by exposure to cholinergic, but not alpha-adrenergic, stimuli, (2) there are, however, no IP(3)Rs present in the granular membrane and the trigger for the Ca(2+) release remains under study, and (3) the response to either type of stimulus does not involve changes in the Ca(2+) content of the nucleus or of the perinuclear cytoplasm/ER, despite the presence of IP(3)Rs in the latter structure. This may be the result of limitation in the technique used, which cannot measure changes in the proper compartment of the ER.
Tracheal submucosal glands are of great relative importance in the secretion of chloride and water to the airway lumen. This study aimed to examine whether the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in cyclic adenosine monophosphate (cAMP) or Ca2+-activated Cl- secretion. Regulation of Cl- secretion in cell cultures derived from pig tracheal submucosal gland acini was investigated by X-ray microanalysis. With or without preincubation with CFTR antisense oligodeoxynucleotide (5 microM). A significant decrease in cellular Cl and K concentration was induced by 5 mM 8-bromo-adenosine 3': 5'-cyclic monophosphate (8-bromo-cAMP), 3 microM calcium ionophore ionomycin, 200 microM 5'-uridine triphosphate (UTP) and 200 microM 5'-adenosine triphosphate (ATP), respectively. The decrease in cellular Cl content was significantly inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB; 50 microm). Preincubation of the cells with CFTR antisense oligodeoxynucleotide significantly inhibited the 8-bromo-cAMP-induced decrease in Cl, whereas CFTR sense oligodeoxynucleotide had no effect. The effects of ionomycin, ATP or UTP were not blocked by either CFTR antisense oligodeoxynucleotide or CFTR sense oligodeoxynueleotide. To measure the cytosolic free calcium concentration ([Ca2+]i) the cells grown on glass coverslips were loaded with fura-2 tetraoxymethylester (fura-2 AM; 5 microM). The [Ca2+]i was measured as the fluorescence ratio of emission (340/380 nm). Ionomycin (3 microM) caused a rapid increase in [Ca2+]i followed by a sustained plateau, but 8-bromo-cAMP had a more complex effect on [Ca2+]i. Exposure to ATP or UTP caused a rapid increase in [Ca2+]i followed by a decrease. In conclusion, cystic adenosine monophosphate and ionomycin induced Cl- secretion through different intracellular pathways. Adenosine triphosphate and uridine triphosphate also induced Cl- secretion probably with Ca as an intracellular messenger. The cystic fibrosis transmembrane conductance regulator is not involved in Cl- secretion activated by extracellular adenosine triphosphate and uridine triphosphate.
A method to decrease the detection limit of trace benzene in CS2 extract from coastal water by gas chromatography has been studied. A direct injection port (Shimadzu WBI-17) and a 2 m x 2 mm i.d. column packed with Chromosorb W(AW-DMCS) coated with 10% SE-30 was used. It is simpler and has low detection limit, small sample amount and high repeatability. The experiment showed that the trace water in the organic phase and the too small purge flow of the direct injection port could cause serious tailing of CS2 peak. There was an optimum value of the purge flow (purge flow/total flow = 5%). The minimum detectable limit of benzene was 4 micrograms/L. The repeatability (RSD) was better than 6% and the average recovery was 96.7%.
Lethal white foal syndrome (LWFS) is a congenital anomaly of horses characterized by a white coat colour and aganglionosis of the bowel, which is similar to Hirschsprung disease (HSCR). We decided to investigate possible mutations of the endothelin-B receptor gene ( EDNRB ) in LWFS as recent studies in mutant rodents and some patients have demonstrated EDNRB defects. First, we identified a full-length cDNA for horse EDNRB . This cDNA fragment contained a 1329 bp open reading frame which encoded 443 amino acid residues. The predicted amino acid sequence was 89, 91 and 85% identical to human, bovine and mouse as well as rat EDNRB respectively, but only 55% identical to the human, bovine and rat endothelin A receptor (EDNRA). Secondly, sequence analysis, together with allele-specific PCR and the amplification-created restriction site (ACRS) technique, revealed a dinucleotide TC-->AG mutation, which changed isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. This was associated with LWFS when homozygous and with the overo phenotype when heterozygous.
The submucosal glands of the airway may contribute more to the airway fluid than the surface epithelium. The cellular mechanisms underlying the regulation of electrolyte and water transport in airway submucosal glands are, however, still poorly understood. Therefore, we attempted to establish a cell culture system to facilitate study of this regulation. Acinar cells were isolated by enzymatic disaggregation from pig tracheal submucosal tissue and cultured on a plastic substrate coated with human placental collagen. The fine structure of the cells in confluent culture was studied by conventional transmission electron microscopy. The elemental content in resting cells and stimulated cells grown on a permeable substrate was studied by X-ray microanalysis. The cultured cells retained structural characteristics (microvilli, secretory granules and desmosomes) of in situ epithelia. The total intracellular Cl and K concentrations significantly decreased after stimulation with the cholinergic agonist acetylcholine, the predominantly alpha-adrenergic agonist norepinephrine, or the beta-adrenergic agonist isoproterenol. Both ionomycin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP) caused a marked decrease of the intracellular Cl, K and Na concentrations. Cl- secretion induced by acetylcholine was inhibited by Cl- channel blockers anthracene-9-carboxylic acid and 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), but Cl- efflux induced by 8-bromo-cAMP was blocked only by NPPB. The intracellular Cl and Na content significantly increased and the cellular K content markedly decreased after treatment with ouabain. These results indicate that the cultured acinar cells maintained the structural and functional characteristics of in situ tissue and that this system is suitable for studying aspects of ion and water transport by the airway submucosal gland cells.
This review of 303 patients with infantile hypertrophic pyloric stenosis (IHPS) concentrates on the influence of clinical audit on diagnosis, complications, and factors contributing to hospital stay. Although the audit has enabled improvement in care by pediatric surgeons, there has been less change in areas controlled by other specialities. During a 12-year period, the number of patients diagnosed solely by clinical examination decreased from 74% to 28%, and the use of diagnostic tests increased (ultrasonography from 16% to 65% and barium meal from 12% to 28%). This trend continued throughout the series despite a review after 8.5 years, which recommended fewer tests. Although there may be some benefit from earlier confirmation of IHPS (the percentage of patients with a serum chloride value of less than 85 mmol/L decreased from 26% to 15%), the need for diagnostic tests could be reduced by expectant management. Better improvement occurred with surgical complications; the incidence of mucosal perforation decreased from 7 of 151 (4.6%) in the first 6 years to 0 of 152 in the last 6 years, and wound dehiscence was reduced from 3 to 0. The wound infection rate decreased from 9% to 4%, but had fluctuations. The average length of stay was reduced from 3.7 to 3.2 days. Further reductions in hospital stay will depend on earlier operation for patients with normal electrolyte values at the time of admission (61%) and a preparedness to confidently discharge patients even if there is vomiting. From the data available, early operation on the day of admission and discharge the next day would be a reasonable strategy for the majority of patients whose admission electrolyte values are normal. Complications such as mucosal perforation and wound dehiscence should be rare. This is not to suggest that IHPS is a condition of minor consequence; the surgeon must be skilled and care meticulous. If electrolytes are disturbed at the time of operation or if unrecognized mucosal perforation occurs, what should be an uneventful illness can result in disaster.
Changes in the elemental composition of cells during isolation of glandular epithelia were studied by electron probe X-ray microanalysis. Fine chopping of rat submandibular gland followed by enzymatic treatment for 15 min caused marked increases in Na and Cl and a decrease in K concentrations in acinar cells. After enzymatic treatment for 50 min, Na, Cl and K concentrations returned to close to the control level. Mechanical disaggregation of the acinar clumps following enzymatic treatment resulted again in minor increases in Na and Cl and a marked decrease in K concentration. Exposure of isolated acini to cholinergic stimulation in vitro resulted in secretion of Cl and K from the acinar cells. Dissection of the sweat gland from human skin caused a decrease in the K/Na ratio. Incubation of the gland for 30-45 min with collagenase gave rise to a gradual decrease in the K/Na ratio. After mechanical separation of the gland into the secretory coil and reabsorptive duct, a further reduction of the K/Na ratio was seen. However, the duct cells had a much lower K/Na ratio and higher Ca concentration than the coil cells. In primary cultures, the K/Na ratios of the coil and duct cells returned to the in situ level. The elemental composition of sweat gland cells incubated in collagenase-containing medium was no different from that in cells incubated in collagenase-free medium. In the intact collagenase-isolated tissue, Cl- secretion in the coil was elicited by carbachol but not by cAMP, whereas in the duct cells the reverse was the case. In primary cell cultures, Cl- efflux in both coil and duct cells could be elicited by both carbachol and cAMP. In conclusion, although changes in elemental composition of gland cells during the isolation procedure occur, physiological responses can be detected. When primary cell cultures are used, it should be borne in mind that cultured cells may have physiological properties different from those of the intact tissue.
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The autosomal recessive spotting lethal (sl) rat phenotype is characterized by absence of intramural ganglion cells in the entire colon and distal small bowel, thus resembling the human Hirschsprung (HSCR) disease. A strategy for identifying the gene responsible for this rat defect was initiated by backcrossing DA x sl rats. After excluding linkage with two candidate genes, RET and Endothelin-3 (EDN3), a highly significant lod score (Z = 47.05 at theta = 0) was found between the Endothelin-B Receptor (EDNRB) gene and the sl phenotype. The exon-intron structure of this rat gene was reconstructed and each exon of the sl rat was screened for possible mutations. A 301 bp interstitial deletion, encompassing the distal half of the first coding exon (exon 2) and the proximal part of the adjacent intron, was demonstrated. This deletion results in two transcriptional products, 270 and 238 bp shorter than wild type cDNA. The discovery of the molecular defect underlying the sl rat phenotype should contribute to the understanding of the genetic heterogeneity of HSCR in man.
The number of GABA-like immunoreactive (LI) cells in lamina I-III of the rat spinal cord was significantly decreased bilaterally 48-72 h after photochemical induction of transient spinal cord ischemia compared to sham-operated controls. No significant changes in the number of GABA-LI cells were observed at cervical level. The number of GABA-LI cells was restored 2 weeks after ischemia. These data, together with recent behavioral and electrophysiological findings, suggest that decreased intraneuronal GABA levels after spinal cord ischemia may underlie the development of the temporary pain-like response to innocuous mechanical stimuli (allodynia) in rats after transient spinal cord ischemia.
A structured questionnaire was sent to the parents of 120 children from a cohort 192, who had undergone a pyloromyotomy for pyloric stenosis at Westmead Hospital during the years 1984-91. Information was gained from 85 children in 82 families regarding gastrointestinal complaints and family history of pyloric stenosis. There were few gastrointestinal problems, no reports of recurrent abdominal pain, but two reports of clinically significant vomiting (2.3%) and one of infantile colic. Of the 85 individuals studied, 21 (24.7%) had a positive family history of pyloric stenosis, a higher frequency than previously reported.