Biomedical subjects

L C Huang

Publications and source records attributed to L C Huang.

At least 19 recordsLinked to original sources

PubMed1999

A model phosphatase 2C --> phosphatase 1 activation cascade via dual control of inhibitor-1 (INH-1) and DARPP-32 dephosphorylation by two inositol glycan putative insulin mediators from beef liver.

Two inositol phosphoglycans (IPG) isolated from beef liver and designated as putative insulin mediators were demonstrated to reciprocally enhance the dephosphorylation of inhibitor-1 (INH-1) and DARPP-32, thus directly activating phosphatase 2C and disinhibiting phosphatase 1 in a potential protein phosphatase 2C --> phosphatase 1 cascade mechanism. One IPG termed pH 2.0, containing Dchiro-inositol and galactosamine, stimulated the dephosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. A second, termed pH 1.3, containing myo-inositol glucosamine and mannose acted reciprocally to inhibit the cAMP-dependent protein kinase phosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. These model experiments are discussed in terms of the observed dephosphorylation of INH-1 with insulin action documented in the literature and the activation of both phosphatase 1 and 2C described in intact cells and in vivo with insulin action.

Animals↗

PubMed1998

Phosphoinositol glycan derived mediators and insulin resistance. Prospects for diagnosis and therapy.

While much work remains, the evidence has become strong that IPG generation following insulin action is a significant signaling mechanism. A considerable body of data has established IPG release by insulin and other growth factors from cell membranes, cells and in human blood and muscle biopsies in vivo. Two separate IPG species containing D-chiro-inositol and myo-inositol have been separated by ion exchange. These IPGs have separate actions in vitro and are both active as insulin surrogates in vivo. A deficiency of the chiro-inositol system has been demonstrated in urine and tissues in humans and directly related to insulin resistance. Accordingly, D-chiro-inositol was administered to STZ diabetic rats and rhesus monkeys and shown to decrease hyperglycemia and enhance glucose disposal. Two trials in humans with impaired glucose tolerance and women with PCOS have now also proven successful. Thus, the pathophysiology in the chiro-inositol system related to insulin resistance and its reversal by chiro-inositol administration, in addition to the basic work, argues strongly for the physiological significance of this novel signaling system in the control of glucose metabolism.

Animals↗

PubMed1997

Human papillomavirus-16 E6/E7 transfected retinal cell line expresses the Müller cell phenotype.

The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Müller cells, the predominant glial cells in the mammalian retina, have been isolated using a variety of techniques, most of these cultures have limited capacity for cell division and are often contaminated by other cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which express Müller cell characteristics and exhibit unlimited growth in vitro. We now report the techniques involved in the propagation and characterization of these cultures. Mixed retinal cultures isolated from dystrophic rat retinas were infected with defective retroviruses coding for human papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled viral constructs also contained the neomycin gene allowing selection of the cultures using Geneticin, a neomycin analogue. Pure cultures were then obtained from Geneticin-selected populations by limiting end-dilution techniques. The expression of the HPV-16 E6/E7 genes in the transfected cell line was established using an HPV-16 E6/E7 PCR product to probe Northern blots. Cloned cells were found to be highly reactive for Müller cell markers including S-100, carbonic anhydrase-C, cellular retinaldehyde binding protein, and glial fibrillary acidic protein but not for glutamine synthetase. Ultrastructural studies showed stacks of cells with long elaborate processes, short microvilli, coated pits, cytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum, and smooth endoplasmic reticulum extending to the cell processes. Growth patterns of late passage cells (> 50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual confluence at day 5. Since the cultures have undergone more than 240 population doublings, they can be characterized as a continuous cell line with unlimited lifespan. The HPV-16 E6/E7 transfected Müller cell line may prove useful in studies requiring abundant and pure cultures of Müller cells.

Animals↗

PubMed1997

Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: the FLIRT system.

We have created a system that utilizes the FLP recombinase of yeast to introduce exogenous cloned DNA reversibly at defined locations in the Escherichia coli chromosome. Recombination target (FRT) sites can be introduced permanently at random locations in the chromosome on a modified Tn5 transposon, now designed so that the inserted FRT can be detected and its location mapped with base pair resolution. FLP recombinase is provided as needed through the regulated expression of its gene on a plasmid. Exogenous DNA is introduced on a cloning vector that contains an FRT, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. High yields of targeted integrants are obtained, even in a recA background. This system permits rapid and precise excision of the introduced DNA when needed, without destroying the cells. The efficiency of targeting appears to be affected only modestly by transcription initiation upstream of the chromosomal FRT site. With rare exceptions, FRTs introduced to the bacterial chromosome are targeted with high efficiency regardless of their location. The system should facilitate studies of bacterial genome structure and function, simplify a wide range of chromosomal cloning applications, and generally enhance the utility of E. coli as an experimental organism in biotechnology.

Blotting, Southern↗

PubMed1997

Maintaining genetic stability through TP53 mediated checkpoint control.

TP53 serves as a key relay for signals elicited by cellular stresses arising from diverse environmental or therapeutic insults. This relay then activates a cell cycle arrest or cell death program, depending on the stimulus and cell type. The absence of TP53 function disables the cell death or arrest programmes, thereby allowing the emergence of variants with various types of genomic alterations. The data discussed focus on two different types of signals that trigger the TP53 relay system. Firstly, TP53 arrests cell cycle progression in response to the types of DNA damage most commonly detected in cells undergoing tumour progression. Secondly, TP53 is activated by specific depletion of ribonucleotide pools, which prevent cells from entering S phase under conditions that could lead to chromosome breakage. The contribution of both responses limits the emergence of genetic variants. The DNA damage induced arrest appears to be triggered by as few as one double strand break in normal human fibroblasts. Analysis of the arrest kinetics after ionizing radiation shows that TP53 activates a prolonged arrest response in cells with irreparable DNA damage and that high efficiency cell elimination is achieved by a process that can be activated over multiple cell cycles. These data indicate that the primary function of the TP53 arrest/apoptosis pathway in response to double strand break is to eliminate damaged cells from the proliferating population, not to allow additional time for lesion repair. However, it remains possible that repair of other types of damage may benefit from TP53 mediated arrest. Analyses in model genetic systems indicate that the absence of TP53 function allows, but does not ensure, a high intrinsic rate of genetic variation and that instability is increased substantially when cells proceed through S phase under inappropriate growth conditions. This implies that inactivation of TP53 function in combination with other genetic alterations, such as oncogene activation, could accelerate genomic instability and tumour progression.

DNA Damage↗

PubMed1996

p53-dependent cell cycle arrests are preserved in DNA-activated protein kinase-deficient mouse fibroblasts.

p53 is involved in at least three cell cycle checkpoints in normal cells: two in G1, activated by either DNA damage or by ribonucleotide pool depletion in the absence of damage, and one in metaphase/anaphase activated by an incomplete mitotic spindle. We tested whether any of these checkpoints require the DNA-activated protein kinase (DNAPK), since data indicate that it is activated by damaged DNA to modify p53 in cultured cells and in cell-free systems. Fibroblasts isolated from mice with severe combined immune deficiency (SCID) were used because the sole genetic defect underlying this syndrome lies within the DNAPK gene. This report shows that age-matched SCID and isogenic wild-type embryonic fibroblasts arrested in response to DNA damage, ribonucleoside triphosphate depletion, and spindle poisons, whereas p53-/- fibroblasts failed to do so. Therefore, DNAPK-deficient scid cells preserve normal p53-dependent cell cycle checkpoints. The data provide one explanation of why scid mice are not tumor prone though they are deficient in double-strand break repair.

Animals↗

PubMed1996

Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.

The tumor suppressor p53 contributes to maintaining genome stability by inducing a cell cycle arrest or apoptosis in response to conditions that generate DNA damage. Nuclear injection of linearized plasmid DNA, circular DNA with a large gap, or single-stranded circular phagemid is sufficient to induce a p53-dependent arrest. Supercoiled and nicked plasmid DNA, and circular DNA with a small gap were ineffective. Titration experiments indicate that the arrest mechanism in normal human fibroblasts can be activated by very few double strand breaks, and only one may be sufficient. Polymerase chain reaction assays showed that end-joining activity is low in serum-arrested human fibroblasts, and that higher joining activity occurs as cells proceed through G1 or into S phase. We propose that the exquisite sensitivity of the p53-dependent G1 arrest is partly due to inefficient repair of certain types of DNA damage in early G1.

Base Sequence↗

PubMed1996

Alcoholism, hepatitis B and C viral infections, and impaired liver function among Taiwanese aboriginal groups.

Viral hepatitis and alcoholism prevail in four major Taiwanese aboriginal groups. To study the relative importance of the acquisition of hepatitis B virus or hepatitis C virus infection and alcoholism to the presence of impaired liver function in these groups, the authors conducted a semistructured clinical interview for alcoholism and test for seromarkers for viral hepatitis among 993 cohort members enrolled in 1990-1992 in an ongoing prospective study (Taiwan Aboriginal Study Project). The subjects' blood specimens were tested for serum alanine aminotransferase/aspartate aminotransferase levels and for the presence of hepatitis B surface antigen and anti-hepatitis C virus antibody. The prevalence of a combination of an alanine aminotransferase level of > 35 IU/liter and an aspartate aminotransferase level of > 40 IU/liter, implying impaired liver function or advanced liver disease, was 4.3% overall. Univariate and multiple logistic regression analysis showed that, rather than chronic hepatitis B virus infection, hepatitis C virus infection and alcoholism were the two dominant risk factors that signalled the risk of liver damage among these Taiwanese aborigines. In addition, these two contributing factors were able to act synergistically to cause impaired liver function.

Alcoholism↗

PubMed1996

Infusion of pH 2.0 D-chiro-inositol glycan insulin putative mediator normalizes plasma glucose in streptozotocin diabetic rats at a dose equivalent to insulin without inducing hypoglycaemia.

We compared the effects of infusing a chemically defined chiro-inositol glycan putative insulin mediator with an equivalent dose of insulin in low-dose (45 mg/kg) streptozotocin diabetic rats. Insulin decreased plasma glucose levels from 17.32 +/- 0.17 to 3.96 +/- 0.064 mmol/l (p < 0.0002) in 120 min, a decrease of 77.13%, while the putative mediator promoted a decrease in plasma glucose from 14.85 +/- 0.084 to 7.22 +/- 0.13 mmol/l (p < 0.007) in 60 min. The putative mediator maintained euglycaemia over the ensuing 60 min with a plasma glucose level of 7.01 +/- 0.10 mmol/l at 120 min. Thus, insulin further reduced the plasma glucose from euglycaemia at 60 min to produce hypoglycaemia at 120 min. The lack of production of hypoglycaemia by the putative mediator can be explained by its inhibition of glucose-stimulated insulin secretion by the islet beta cells, thus providing a potential negative feedback regulatory mechanism; or by its selective action on muscle to increase glycogen synthesis. The significance of these results in terms of future directions in drug design is herein considered.

Animals↗

PubMed1995

Genetic instability as a consequence of inappropriate entry into and progression through S-phase.

The stability of the mammalian genome depends on the proper function of G1 and G2 cell cycle control mechanisms. Two tumor suppressors, p53 and retinoblastoma (Rb), play key roles in progression from G1 into S-phase. We address the mechanisms by which these proteins mediate a G1 arrest in response to DNA damage and limiting metabolic conditions. Gamma-irradiation induced a prolonged, p53-dependent G1 arrest associated with a long-term increase in the levels of the cdk-inhibitor p21WAFl/Cipl (p21). Microinjection of linear plasmid DNA also caused a G1 arrest. The p53-dependent arrest induced by inhibitors of UMP biosynthesis was reversible and occurred in the absence of detectable DNA damage. Both arrest mechanisms contribute to limiting the formation and propagation of damaged genomes. Cells containing mutant p53 but wild-type Rb do not generate methotrexate (Mtx) resistant variants. However, pre-treatment with DNA damaging agents prior to drug selection resulted in resistant clones containing amplified dihydrofolate reductase (DHFR) genes, suggesting that DNA breakage is a rate limiting step for gene amplification. The Mtx-induced arrest did not occur in cells with non-functional Rb. Rb acts as a negative regulator of the E2F transcription factors, and Rb-deficient primary mouse embryo fibroblasts (MEFs) produced elevated levels of mRNA and protein for key E2F target genes. Failure to prevent entry into S-phase in Rb-/- MEFs exposed to DNA-damaging or nutrient limiting conditions caused apoptosis and correlated with p53 induction. Taken together, these findings indicate a link between p53 and Rb function and suggest that their coordination insures correct entry into S-phase, minimizing the emergence of genetic variants.

Animals↗

PubMed1994

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Adipocytes↗

PubMed1993

Insulin stimulates the biosynthesis of chiro-inositol-containing phospholipids in a rat fibroblast line expressing the human insulin receptor.

HIRc-B cells (rat fibroblasts expressing the human insulin receptor) were incubated with myo-[3H]inositol for 48 hr, and the biosynthesis of chiro-[3H]inositol was investigated in the absence and presence of insulin following a time course up to 60 min. After phase separation, treatment with insulin for 15 min caused a 2.2-fold increase in the specific radioactivity of chiro-[3H]inositol-containing phospholipids in contrast to a 1.2-fold increase in the specific radioactivity of myo-[3H]inositol-containing phospholipids. No insulin-mediated change in the specific radioactivity was observed in the inositol phosphates or free inositols. Further detailed analysis of individual [3H]inositol-containing phospholipids demonstrated marked increases in specific activity of the chiro-[3H]inositol phospholipids after 15 min of incubation with insulin: phosphatidylinositol 4-phosphate and 4,5-bisphosphate, 4.2-fold; lysophosphatidylinositol, 1.5-fold; phosphatidylinositol, 3.2-fold. In contrast, myo-[3H]inositol-containing phospholipids demonstrated relatively small increases (1.1- to 1.4-fold) after 5 min of incubation with insulin. These findings indicate that insulin stimulates de novo synthesis of chiro-inositol-containing phospholipids at the inositol phospholipid level.

Animals↗

PubMed1993

Chiroinositol deficiency and insulin resistance. II. Acute effects of D-chiroinositol administration in streptozotocin-diabetic rats, normal rats given a glucose load, and spontaneously insulin-resistant rhesus monkeys.

The acute effects of administration of D-chiroinositol (D-CI), a component of a putative mediator of insulin action, on plasma glucose were examined in low dose streptozotocin-treated rats and normal rats given a glucose load and the effects on plasma glucose and insulin were determined in five obese rhesus monkeys with varying degrees of spontaneous insulin resistance. Single dose intragastric D-CI (10 mg/kg) administered to streptozotocin-treated rats produced a 30-40% decrease in plasma glucose (P < 0.05) at 30-120 min. Single dose intragastric D-CI (2-15 mg/kg) administered to normal rats 2 h before ip glucose produced a 30-50% decrease (P < 0.05) in plasma glucose. D-CI (10 mg/kg) caused a 50% increase (P < 0.05) in glucose disappearance rates in these rats. Myoinositol (10 mg/kg) was without effect. Intravenously administered single dose D-CI (100 mg/kg) increased both the glucose and insulin disappearance rates by 129 +/- 41% (mean +/- SE; P < 0.06) and 89 +/- 39% (P = 0.01), respectively, in all monkeys between 0-30 min compared to control values. D-CI administration, therefore, lowered elevated plasma glucose in streptozotocin-treated hyperglycemic rats, normal rats given a glucose load, and spontaneously insulin-resistant monkeys with or without noninsulin-dependent diabetes mellitus. Intravenous D-CI also lowered plasma insulin in these monkeys.

Animals↗

PubMed1993

Chiroinositol deficiency and insulin resistance. III. Acute glycogenic and hypoglycemic effects of two inositol phosphoglycan insulin mediators in normal and streptozotocin-diabetic rats in vivo.

Two insulin mediators, inositol phosphoglycans, were isolated from bovine liver by methods previously developed for rat liver, i.e. chromatography on an AG 1 x 8 ion exchange column and selective elution with HCl at pH 2.0 and 1.3. The pH 2.0 mediator containing D-chiroinositol stimulated pyruvate dehydrogenase phosphatase, whereas the pH 1.3 mediator containing myo-inositol inhibited cAMP-dependent protein kinase. Each mediator was further purified by thin layer and Bio-Gel P4 column chromatography and injected ip into normal fed rats together with [U-14C]glucose. After 2.5 h, diaphragms were removed, and glycogen isolated. Insulin mediators, like insulin, stimulated [U-14C]glucose incorporation into glycogen by 150-160% in a dose-dependent manner in the nanomolar range. Mediators injected iv in the nanomolar range into low dose streptozotocin-diabetic rats decreased plasma glucose 30-45% in 30-60 min, with a return to basal concentrations after 150-180 min. These in vivo insulin-like effects of mediator were observed without changes in serum insulin concentrations. The pH 2.0 mediator was 50-100 times more active (per nmol organic phosphate) than the pH 1.3 mediator in the ip diaphragm glycogenesis assay. Mediator effects on diaphragm were completely blocked by preincubation with an immunopurified inositol phosphoglycan antibody. Both mediators were equally active iv in lowering plasma glucose (per nmol inositol) at concentrations comparable to those of insulin.

Animals↗

PubMed1993

Vulvar neoplasia associated with other primary malignancies.

It is recognized that a significant number of patients with vulvar neoplasia have had previous, concurrent, or subsequent genital tract neoplasia. This has resulted in speculation that there may be a common etiology and, in particular, of the possibility of an infection element. Among 59 cases of vulvar cancer diagnosed at the National Taiwan University Hospital from 1976 to 1991, there were seven cases (12%) associated with other primary malignancies. Carcinoma of the cervix was the most frequent other primary cancer (6/7, 86%). The possible role of human papilloma virus infection in genital neoplasia is discussed.

Adult↗

PubMed1992

In vivo conversion of [3H]myoinositol to [3H]chiroinositol in rat tissues.

We report here the in vivo conversion of [3H]myoinositol to [3H]chiroinositol. After labeling intraperitoneally with [3H]myoinositol for 3 days to reach radioisotope equilibrium in urine, [3H]chiroinositol was isolated from tissues and purified after 6 N HCl hydrolysis by two sequential paper chromatographies and high performance liquid chromatography (HPLC). Percent conversion of [3H]myoinositol to [3H]chiroinositol was highest in urine (36%), liver (8.8%), muscle (8.8%), and blood (7.6%) with intestine, brain, kidney, spleen, and heart decreasing in percentage from 2.8 to 0.7%. Labeling of other inositol isomers including scyllo-, neo-, and epi-, and mucoinositol was minimal, approximately 0.06% of [3H]myoinositol. Glucose was unlabeled, but glucuronate, the product of myoinositol oxidation, was labeled up to 1.5% of the [3H] myoinositol. Acid hydrolysates of combined inositol-containing phospholipids contain significant labeled chiroinositol. [3H]Phosphatidylinositols and [3H]glycosylphosphatidylinositols were extracted from liver, muscle, and blood, isolated by thin layer chromatography, and inositols purified by HPLC after acid hydrolysis. Percent conversion of [3H]myoinositol to [3H] chiroinositol was highest in blood (60.4%) followed by muscle (7.7%) and liver (2.2%).

Animals↗

PubMed1992

Age trends in lung cancer.

To investigate the relationship between the stages of cancer development and the age trends in histologic type found at the time of disease diagnosis, we studied 1,669 patients with histologically proven lung cancer. These patients were examined at the National Taiwan University Hospital using Mantel-Haenszel Chi-square testing to determine a linear trend. These patients were divided into three age groups: group 1 (< 45 years), 141 patients (8%); group 2 (45-64 years), 946 patients (57%); group 3 (65 years or more), 582 patients (35%). The ratio of men to women was 1.4 in group 1, 2.5 in group 2 and 2.3 in group 3. In men, there was a significant trend for the proportion of squamous cell carcinoma to increase from 26% to 42% and that of adenocarcinoma to decrease from 52% to 34% as age increased. In women, an age trend for histologic type was not observed. There was also a significant trend for local-stage squamous cell carcinoma in men to increase from 18% in group 1 to 35% in group 2 and to 42% in group 3. In men, but not in women, local-stage large cell carcinoma increased from 20% in group 1 to 31% in group 2 and to 64% in group 3. However, the age-stage trend for other cell types was not significant in men.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1991

A bacterial model system for chromosomal targeting.

A system that permits efficient site-specific chromosomal targeting of foreign DNA on the Escherichia coli chromosome has been developed, using the FLP site-specific recombination system derived from the yeast 2 mu plasmid. The system demonstrates the feasibility of using site-specific recombination for this purpose, and provides a means to gather information on parameters that may affect chromosomal targeting to guide efforts to establish similar systems in higher eukaryotes. In this model system, the efficiency of integration of foreign DNA is affected by the location of the target site in the chromosome, and the structure of the recombination sites.

Base Sequence↗