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Biomedical subjects

S H Lin

Publications and source records attributed to S H Lin.

At least 145 records · Page 8Linked to original sources
PubMed1996

Cloning, direct expression, and purification of a snake venom cardiotoxin in Escherichia coli.

The cardiotoxin analogue III (CTX III), isolated from the Taiwan cobra (Naja naja atra) venom, is a sixty-amino acid, all beta-sheet protein. We report the direct expression of CTX III from its synthetic gene as inclusion bodies in Escherichia coli. The yield of the expressed protein is about 40 mg/liter of the culture. CTX III trapped as inclusion bodies is dissolved and refolded by the slow refolding technique. The refolded protein is purified by reverse phase high performance liquid chromatography. The purified and refolded CTX III sample is further characterized by SDS-PAGE, circular dichroism, two-dimensional NMR spectroscopy and haemolytic activity. To our knowledge, this is the first report of the direct expression and purification of snake venom cardiotoxins.

Amino Acid Sequence↗
PubMed1996

Cloning of cDNAs from a mammalian expression library by a direct selection-amplification method.

A sensitive method was devised for cloning cDNAs from a mammalian expression library based on single-cell detection and selection of transfected cells. The method is applicable for cloning cell-surface or cytoplasmic proteins for which a detection assay, such as immunofluorescence or immunohistochemical reactivity, exists. The widely used eukaryotic expression vector cdm8 is utilized, and the method is demonstrated using the gene for C-CAM, a liver glycoprotein adhesion molecule. After transfection, cells are plated out on a collagen gel substrate to allow retrieval of selected cells. Simultaneous fixation and permeabilization with acetone permits immunological and histochemical detection of cell-surface and cytoplasmic proteins without loss of plasmid vector. Inserts of interest are recovered by PCR with vector primers.

Adenosine Triphosphatases↗
PubMed1996

Fatal Aeromonas hydrophila bacteremia in a hemodialysis patient treated with deferoxamine.

A 49-year-old woman undergoing long-term hemodialysis and treated with deferoxamine (DFO) 1.5 g twice weekly for aluminum bone disease developed fever and bilateral calf pain caused by myonecrosis with gas gangrene. She had a rapidly fatal outcome. The cultures of blood and aspirates from both calf muscles demonstrated Aeromonas hydrophila. No obvious entry point could be traced. The in vitro growth of the patient's strain was found to be stimulated by the deferoxamine-iron complex in an iron-deprived medium. It is suggested that high-dose DFO therapy in this patient was responsible for promoting a bacterial infection by this microorganism.

Aeromonas hydrophila↗
PubMed1996

Hypertrophy of oxytocinergic magnocellular neurons in the hypothalamic supraoptic nucleus from gestation to lactation.

In the present experiments, we examined the changes in cell size (profile area) of oxytocinergic magnocellular neurons during reproductive states and dehydration with quantitative immunohistochemistry. During lactation, hypertrophy was observed in oxytocinergic magnocellular neurons but not in vasopressinergic ones in the supraoptic nucleus. In virgin rats, chronic dehydration increased the cell size in both oxytocinergic and vasopressinergic neurons. After normal weaning time, the cell size decreased, returning to virgin level within 20 days. However, if the mothers were deprived of their litters immediately after parturition, the cell size rapidly returned to virgin level within 5 days. Furthermore, the increase in the cell size of the mothers was not affected by the size of their nursing litters.

Animals↗
PubMed1996

Infective endocarditis complicated with rapidly progressive glomerulonephritis: a case report.

A 26-year-old man who had suffered from intermittent chills and fever over a two month period was quite clear of heart or kidney involved developed acute deterioration of renal function. A new pansystolic murmur over the apex of the heart was heard on auscultation, and echocardiography clearly showed a vegetation about 0.7-0.9 cm in size on the atrial site of the mitral value. Laboratory investigation displayed normochromic anemia with negative Coombs' test. Immunological studies were positive for rheumatoid factor and circulating immune complex. High serum levels of erythrocyte sedimentation rate and C-reactive protein, nephritic sediment of urinalysis and negative blood cultures for bacteria, tuberculosis or fungus were also noted. Abdominal sonography showed normal kidney size, bilaterally. Renal biopsy revealed typical crescentic glomerulonephritis. After intravenous penicillin therapy for two weeks, the serum creatinine level recovered from 6.7 mg/dl to 2.0 mg/dl and circulating immune complex disappeared. In consideration of cardiac insufficiency and the potential risk for complications of the vegetation, the patient underwent mitral valve replacement. Four weeks after operation, all the abnormal data had resolved completely. These data suggested that infective endocarditis with rapidly progressive glomerulonephritis is curable by antibiotic therapy and surgical intervention.

Adult↗
PubMed1996

The liver biochemical tests and serological markers of hepatitis B virus in the very old-aged population in Taiwan.

BACKGROUND: Human liver alters with aging. Lower serum albumin with elevation in serum cholesterol and alkaline phosphatase (ALP) has been reported in the elders, but there was no further report on the very old people (i.e., >75 Y/O). We evaluated changes of liver biochemical tests and hepatitis B virus (HBV) serological markers in the very old-aged population. METHODS: From October 1993 to June 1994, 2,549 subjects attended our hospital for physical check-up. Of them 1,742 were healthy subjects (M/F = 1,058/648) and were enrolled for analysis of the normal range of liver biochemical tests in different age groups. They were divided into four groups; young-aged group (age between 18 and 40, n = 231), middle-aged group (age between 41 and 64, n = 991), old-aged group (age between 65 and 75, n = 424), and very old-aged group (age > or = 76, n = 96). The items of liver biochemical tests included albumin, total bilirubin, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALP and gamma-glutamyl transpeptidase (GGT). The whole population, totally 2,549 subjects, were used for the analysis of the prevalence of HBV serological markers in different age groups. RESULTS: Serum albumin was the only biochemical parameter that persisted to decline after the age of 75 (p <0.05). Cholesterol began to elevate in the middle-age but decreased after 75 Y/O. The old-aged and very old-aged subjects had higher levels of serum bilirubin, AST, ALP, and GGT levels than the other two groups (p <0.05), however, the changes of ALT levels among these groups were not so remarkable. The HBV carrier rates in the young-aged, middle-aged, old-aged and the very old-aged groups were 16.8%, 12.3%, 6.8% and 7.0%, respectively (p <0.001). Furthermore, the very old-aged group had a significantly higher prevalence of negativity in both HBsAg and anti-HBs (p <0.001). CONCLUSIONS: Serum albumin is the only biochemical parameter that persists to decline after the age of 75. In contrast, elevation of cholesterol tends to reverse after that age. All the aging-related changes in biochemical tests are clinically nonsignificant though some of them are significant in statistical analysis. In addition, a low HBV carrier rate and a high prevalence of negativity in both HBsAg and anti-HBs are noted in the very old-aged people, possibly due to the progressive loss of HBV markers in the elders.

Adolescent↗
PubMed1995

Rapid inhibition of protein histidine phosphorylation by UV-irradiation in Xanthomonas oryzae pv. oryzae.

Exposure of Xanthomonas oryzae pv. oryzae cells to 254 nm UV radiation resulted in an alteration of protein phosphorylation. Labelling of the phosphohistidine-containing proteins with molecular masses of 81 and 32 kDa, named p81 and p32, was rapidly reduced following UV irradiation in the early exponential cells, but the decrease was not detected in mid-exponential cells. Mitomycin C, a DNA replication inhibitor, and rifampicin, a drug generally used to inhibit RNA synthesis and DNA replication, were also found to reduce the histidyl phosphorylation. However, this alteration of protein phosphorylation was not hindered by chloramphenicol treatment. A possible role for these histidyl phosphoproteins in sensing UV light is proposed.

Bacterial Proteins↗
PubMed1995

Structure and function of C-CAM1: effects of the cytoplasmic domain on cell aggregation.

C-CAMs are epithelial cell-adhesion molecules of the immunoglobulin supergene family with sequences highly homologous to carcinoembryonic antigen (CEA). C-CAMs and their human homologues, biliary glycoproteins, are unique among the CEA-family proteins in that they have cytoplasmic domains. Furthermore, alternative splicing generates C-CAM isoforms with different cytoplasmic domains, suggesting that the cytoplasmic domains of C-CAM may play important roles in regulating the function or functions of C-CAM. By using both sense and antisense approaches, we have shown that C-CAM1 is a tumour suppressor in prostate carcinogenesis. This observation raises the possibility that the cytoplasmic domain of C-CAM1 may be involved in signal transduction or interaction with cytoskeletal elements to elicit the tumour suppressor function. The cytoplasmic domain of C-CAM1 contains several potential phosphorylation sites, including putative consensus sequences for cyclic AMP-dependent kinase and tyrosine kinase. One of the potential tyrosine phosphorylation sites is located within the antigen-receptor homology (ARH) domain. The ARH domain of the membrane-bound IgM molecule is necessary for signal transduction in B-cells. These structural features suggest that the cytoplasmic domain of C-CAM1 may be important for signal transduction. To test this possibility, we generated several site-directed C-CAM1 mutants and tested their ability to support adhesion and their abilities to be phosphorylated in vivo. Results from these studies revealed that Tyr-488 is phosphorylated in vivo. However, replacing this tyrosine with phenylalanine did not significantly compromise its adhesion function. Similarly, Ser and Thr residues are phosphorylated in vivo, but deletion of the potential cyclic AMP-dependent kinase site did not significantly reduce the adhesion function. These results suggest that the kinase phosphorylation sites in the cytoplasmic domain of C-CAM1 are not required for the adhesion function. However, these phosphorylation sites are probably involved in the regulation of C-CAM-mediated signal transduction. Thus, there are probably distinct structural requirements for the adhesion and the signal transduction functions of C-CAM. Incidentally, a C-CAM1 deletion mutant containing a 10-amino-acid cytoplasmic domain was able to support adhesion activity. This is in contrast to our previous finding that a C-CAM isoform, C-CAM3, with a 6-amino-acid cytoplasmic domain could not support cell adhesion. This result indicates that the extra four amino acids, which are absent in C-CAM3 and contain a potential Ser/Thr phosphorylation site, are important for the adhesion function.

Adenosine Triphosphatases↗
PubMed1995

An equivalent site mechanism for Na+ and K+ binding to sodium pump and control of the conformational change reported by fluorescein 5'-isothiocyanate modification.

We have previously shown that two K+ must bind to cause the conformational change in Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase reported by fluorescein 5'-isothiocyanate modification and that the ratio of the macroscopic K+ dissociation constants is 4 [Smirnova, I. N., & Faller, L. D. (1993) J. Biol. Chem. 268, 16120-16123]. Since 4 is the ratio expected for random binding to two identical and independent sites, in this paper we propose a two-equivalent-site mechanism for Na+ as well as K+ binding to sodium pump and control of the conformational change between E1 and E2. The equivalent site mechanism is tested by fitting algebraic equations for the empirical first-order rate constant and for the magnitude of the fluorescence change observed in stopped-flow experiments to an array of data. The estimated rate constants for the conformational change at 15 degrees C are kf = 150 +/- 19 s-1 and kr = 0.13 +/- 0.03 s-1, and the estimated dissociation constants for K+ and Na+ from the E1 conformation of the enzyme are KK = 4.8 +/- 1.2 mM and KNa = 0.38 +/- 0.12 mM, in good agreement with estimates from published binding measurements. Although the possibility of ordered and anticooperative binding cannot be excluded, the equivalent site interpretation is supported by quasi-independent estimates of the macroscopic Na+ dissociation constants that give the ratio 4 predicted for binding to identical, noninteracting sites and by equivalent site interpretations of published transport experiments. Six alternative mechanisms are tested. Noncompetitive binding and mutually exclusive binding of the transported ions can be ruled out. Comparable fits to the data are obtained by three-equivalent-site mechanisms in which enzyme with either two or three K+ bound can undergo the conformational change. Therefore, this study quantitatively supports the conformational hypothesis by showing that a conformational change in sodium pump has the right properties to explain transport.

Animals↗
PubMed1995

Application of a tumor suppressor (C-CAM1)-expressing recombinant adenovirus in androgen-independent human prostate cancer therapy: a preclinical study.

Recently, we demonstrated that an androgen-regulated cell adhesion molecule, C-CAM, acts as a tumor suppressor in prostate cancer development. In this study, we further explored the possibility of applying C-CAM as a potential agent for developing prostate cancer gene therapy using an adenoviral delivery system. We found that prostate cancer cells, in general, were sensitive to adenoviral infection. In vitro characterization indicated that C-CAM1 protein was detected only in C-CAM1 adenovirus-infected cells but not in antisense control virus-infected cells, and the levels of expression showed dose dependency. Because of the stability of the protein, C-CAM expression in viral-infected cells appeared to be a long-lasting event, indicating that C-CAM may be superior to many other known tumor suppressors that have a short protein half-life. Most importantly, the delivery of a single dose of C-CAM adenovirus was able to repress the growth of PC-3-induced tumors in nude mice for at least 3 weeks. Taken together, these data indicate that C-CAM is a potential candidate for human prostate cancer therapy.

Adenosine Triphosphatases↗
PubMed1995

Consistent expression of an epithelial cell adhesion molecule (C-CAM) during human prostate development and loss of expression in prostate cancer: implication as a tumor suppressor.

Cell adhesion molecules have been suggested to function as tumor suppressor molecules. We have been studying one of the epithelial cell adhesion molecules (C-CAM), which belongs to the immunoglobulin gene superfamily. Transfection of a C-CAM cDNA expression vector into a highly tumorigenic human prostate cancer cell line (PC-3) suppresses tumor formation in nude mice. Alternatively, reducing C-CAM expression levels in the nontumorigenic rat prostate epithelial cell line NbE by the antisense expression vector markedly increases tumorigenicity of NbE cells in nude mice. These results suggest that C-CAM may be a tumor suppressor in prostate cancer. In this study, we examined the relationship between C-CAM expression during human prostate development and neoplastic progression by immunohistochemical staining of frozen sections. C-CAM predominantly localized on the plasma membrane of the basal cell layer in both the fetal and normal adult prostate gland. However, an overall decreased staining was seen in benign prostatic hyperplasia and high grade prostatic intraepithelial neoplasia. Furthermore, C-CAM was not detected in prostate carcinomas. Thus, a decrease in C-CAM expression may be an early event in hyperplastic/neoplastic transformation. These observations support the suggestion that C-CAM is a tumor suppressor in prostate cancer progression.

Antineoplastic Agents↗
PubMed1995

Anti-peptide sera against cell-CAM 105 determine high molecular-mass variants of the long isoform in rat hepatocytes.

The glycoprotein cell-CAM 105 is a member of the carcinoembryonic-antigen-(CEA)-gene family, involved in cell-cell adhesion of rat hepatocytes and expressed on the cell surface as a long (L) and a short (S) isoform with slightly differing molecular masses and isoelectric points. The cDNA of the L-isoform has been isolated and sequenced, as confirmed by the preparation of specific anti-peptide sera [Lin, S.-H., Culic, O., Flanagan, D. & Hixson, D. C. (1991) Biochem. J. 278, 155-161]. Recently, two additional cDNAs have been sequenced, which possess identical deduced primary structures, including short intracellular domains 10 amino acids in length, which differ from the cytoplasmic domain of the L-isoform specifically in the last four C-terminal amino acids. Here, we report on the production of the polyclonal antiserum [anti-(peptide 2)] by immunization with a synthetic hexapeptide (GGSGSF) corresponding to the unique intracellular C-terminal domain of these short cell-CAM 105 cDNA isoforms. This antiserum was specific in ELISA, immunoblot and immunoprecipitation assays for a protein with the same biochemical properties as the S-isoform of cell-CAM 105 expressed in rat liver. In addition, CNBr peptide maps of the S-isoform and the protein immunoprecipitated with anti-(peptide 2) serum were identical. Together, these results provide strong evidence that anti-(peptide 2) serum is specific for the S-isoform of rat liver cell-CAM 105. In immunoblot analysis on liver plasma membrane extracts prepared without collagenase perfusion, at least seven high molecular-mass proteins were observed which showed strong reactivity with mAbs against extracellular epitopes and L-isoform-specific antibodies but no reactivity with anti-(peptide 2) serum. Like the L-isoform, these proteins are expressed on the cell surface and might represent structural variants of cell-CAM 105.

Adenosine Triphosphatases↗
PubMed1995

Tumor suppressive role of an androgen-regulated epithelial cell adhesion molecule (C-CAM) in prostate carcinoma cell revealed by sense and antisense approaches.

We recently demonstrated that C-CAM, an epithelial-cell adhesion molecule of the immunoglobulin supergene family, could be regulated by androgen and might act as a growth repressor during differentiation of the prostatic epithelium. To define the role of C-CAM in prostatic tumorigenesis, a tumorigenic human prostatic cancer cell line, PC-3, was transfected with an expression plasmid containing C-CAM1 (a C-CAM isoform). Transfected clones showed significantly lower growth rates, reduced anchorage-independent growth, and less tumorigenicity in vivo than control cells. Furthermore, transfection of an antisense vector into a nontumorigenic prostatic epithelial cell line, NbE, resulted in tumor formation in nude mice. Sublines derived from these NbE-induced tumors had lower levels of C-CAM than did control cells. These data suggest that C-CAM1 can function as a tumor suppressor in prostate tumorigenesis.

Adenosine Triphosphatases↗
PubMed1995

Craniofacial injuries in unhelmeted riders of motorbikes.

We studied 1160 consecutive craniofacial injuries sustained by unhelmeted motorbike riders in Taipei, Taiwan, between 1990 and 1993, in order to investigate the distribution, type and severity of these injuries. The average age of the victims was 31 years (SD 13.2), with 84 per cent of them being between ages 16 and 45. The facial and cranial areas were defined as being separated by the line between eyebrows and ears. The incidence of facial injuries was the same as that of cranial injuries (both 68 per cent). While facial injuries occurred most often in the cheek and chin, most cranial injuries occurred in the forehead and parietal region. Although the majority of facial injuries resulted in mild brain injuries, they may also cause serious cosmetic problems, and some were associated with serious brain damage. Motorbike riders need good face protection. Since most motorbikes in Taipei travel relatively slowly, these results may also apply to bicyclists; in other words, cyclists may also need good face protection.

Accidents, Traffic↗
PubMed1995

Fos expression in the hypothalamic magnocellular neurons of rats during pregnancy, parturition and lactation.

Changes in Fos expression of magnocellular neurons in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the rat hypothalamus were investigated using immunohistochemistry during pregnancy, parturition and lactation. Quantitative morphometrical analysis revealed that Fos-positive cells in the hypothalamic magnocellular neurons were rarely seen in days 10 and 20 pregnant rats, however, significantly numerous Fos-positive cells were found in parturient rats. The number of Fos-positive cells was drastically decreased within a few days after parturition. Moreover, it was found using dual immunohistochemistry that the percentage of Fos-positive cells in vasopressin (AVP) magnocellular neurons of the SON was higher than that of the PVN in parturient rats, although oxytocin (OXT) magnocellular neurons showed the same percentage of Fos-positive cells between the SON and PVN. These results demonstrate that the hypothalamic magnocellular neurons express Fos during a limited period after parturition, and Fos expression in AVP magnocellular neurons is heterogeneous between the SON and PVN.

Animals↗
PubMed1995

Temporal changes of c-fos expression in oxytocinergic magnocellular neuroendocrine cells of the rat hypothalamus with restraint stress.

The present experiments were undertaken to examine c-fos expression in magnocellular neuroendocrine cells (MNCs) of the rat hypothalamus with restraint stress using dual immunohistochemistry for c-fos and oxytocin. Restraint stress induced c-fos expression in oxytocinergic MNCs in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Quantitative immunohistochemical analysis revealed that percentages of c-fos-positive cells to oxytocin-immunoreactive MNCs in the SON and PVN maximally increased at 2 h after restraint stress had started, and began to decline in spite of the fact that the restraint of animals were continued. Similar results were obtained from time course of c-fos expression in parvocellular neurons of the PVN. When animals were released to move freely in their home cages following the 3-h restraint, the plasma levels of oxytocin declined to reach basal levels within 30 min and c-fos immunoreactivity in the hypothalamic MNCs and parvocellular neurons disappeared faster than those of the continually restrained. These results demonstrate that restraint stress induces c-fos expression in oxytocinergic MNCs in the SON and PVN, and that time course of c-fos expression is transient even in the continuation of restraint stress.

Animals↗
PubMed1995

Maintenance of ultrastructural plasticity of the hypothalamic supraoptic nucleus in the ovariectomized rat.

In the present experiments, we examined the effect of ovariectomy on the increases in litter weight and structural plasticity of MNCs in the supraoptic nucleus (SON) during lactation. Female rats were ovariectomized 2 days after parturition, and the increases in litter weight were measured as the index of milk let-down from dams during lactation. The lactation period was elongated up to 6 weeks by providing new litter to obtain more apparent effects of the ovariectomy. There was no significant difference in the increases in litter weight between non-operated and ovariectomized females. After lactation for 6 weeks, the ultrastructures such as juxtaposition (surface membrane apposition) and multiple synapses (terminals contacting with two or more postsynaptic elements) of MNCs in the SON in nonoperated and ovariectomized females were examined to compare with those of virgins. The percentage of juxtaposition and the number of multiple synapses significantly increased in nonoperated lactating females as compared with those of virgins. Ovariectomized rats showed similar structural changes to those of nonoperated females during lactation. Therefore, we conclude that ultrastructural plasticity of MNCs in the SON is maintained even in the absence of an ovary, and direct or indirect actions of suckling stimulation may be important in maintaining the plasticity during lactation.

Animals↗