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A Hung

Publications and source records attributed to A Hung.

10 recordsLinked to original sources

Temporal and spatial expression of murine acid beta-glucosidase mRNA.

The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid beta-glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally.

Animals↗

A prospective randomized study to determine the optimal dose of intravenous vitamin K in reversal of over-warfarinization.

The optimal management of asymptomatic overanticoagulated patients remains unknown. We measured international normalized ratio (INR), activated partial thromboplastin time (APTT) and prothrombin fragment 1 + 2 (F1 + 2) over 7 d in 24 asymptomatic or mildly haemorrhagic patients on warfarin with prolonged INR of > 7.0 who were randomized to receive 0.5 mg, 1 mg or 2 mg intravenous vitamin K. Of six severely overanticoagulated patients (INR > 9.5 with APTT ratio > 2), five failed to achieve an INR < or = 4.0 on day 1, irrespective of vitamin K dose given. In the remaining 18 cases, an optimal response (INR 2-4 at day 1) was observed in 67% of those receiving 0.5 mg vitamin K, but only in 33% of those receiving 1 or 2 mg, the majority of whom developed an INR < 2.0. Our results support an optimal dose of 0.5 mg i.v. vitamin K for most overanticoagulated patients, with possibly a repeat dose in the small group of severely overanticoagulated patients.

Adult↗

Cloning of rat and human inducible penile nitric oxide synthase. Application for gene therapy of erectile dysfunction.

Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the aging rat model, erectile dysfunction is accompanied by a reduction of penile smooth muscle compliance and, in very old animals, by a decrease in penile nitric oxide synthase (NOS), which is responsible for the synthesis of the mediator of penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by local induction or gene therapy can mitigate erectile dysfunction in the aged rat. A mix of iNOS (inducible NOS) inducers was continuously delivered to the penises of 5- ("adult"), 20- ("old"), and 30- ("very old") mo-old rats for 3-6 days, and the erectile response to electrical field stimulation of the cavernosal nerve was measured. The erectile dysfunction observed in old and very old rats as compared to adult animals was ameliorated by treatment with iNOS inducers. Penile iNOS was detectable in the penis of these rats by Western blot, NADPH diaphorase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indicating that the increased NOS levels were under physiological control. The iNOS cDNA was cloned from induced RPSMC mRNA and generated by reverse transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions from both the rat (RPiNOS) and human (HPiNOS) penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissues. RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal tissue by PCR, and its expression by RT-PCR and Western blots. These results open the way for the possible use of NOS isoforms in the management of erectile dysfunction.

Aging↗

Isolation of two pyruvate kinase activities in the parasitic protozoan Leishmania mexicana amazonensis.

Using phosphocellulose affinity chromatography we were able to separate two pyruvate kinase (EC 2.7.1.40) activities in the parasitic protozoan Leishmania mexicana amazonesis. One activity (PYK1) showed hyperbolic kinetics and was decreased by fructose 2,6-bisphosphate, whereas the second activity (PYK2) showed sigmoidal kinetics for the substrate phosphoenolpyruvate and was activated by fructose 2,6-bisphosphate. Molecular sieve chromatography (Sephacryl S-400) of PYK1 produced a single peak of apparent molecular mass around 200,000, while PYK2 eluted at a position corresponding to M(r) 55,000.

Animals↗

Similarities between the regulatory sequences of the unrelated tetracycline genes of pBR322 and Tn10.

The regulatory regions of the tetracycline genes present in pBR322 (pSC101) and in the transposon Tn10 are compared. They show a low degree of nucleotide sequence similarity but a high level of structure similarity. Furthermore, analyses of RNAs transcribed in the opposite direction of the pBR322 tet gene show that there are two mRNA initiation sites separated by 29 nucleotides. This suggests the existence of two promoters for the tet repressor gene in Tn10. These features reveal a strong resemblance of the mode of regulation between the tet operons of Tn10 and pSC101.

Base Sequence↗

In vivo selected promoter and ribosome binding site up-mutations: demonstration that the Escherichia coli bla promoter and a Shine-Dalgarno region with low complementarity to the 16 S ribosomal RNA function in Bacillus subtilis.

We have constructed a plasmid, pQS1, in which a mouse dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP:oxidoreductase; EC 1.5.1.3; DHFR) cDNA is inserted in the unique PstI site of a gram-positive/gram-negative shuttle vector derived from pBR322. The cDNA is expressed under the control of the bla promoter, which, like most gram-negative bacterial genes, is considered not to be expressed in Bacillus subtilis, and its coding sequence is translated from a polycistronic message. We have selected in vivo and studied, in Escherichia coli and B. subtilis, expression mutants with promoter and ribosome binding site sequence mutations. One promoter mutation changes the third nucleotide of the -35 region from a C to a G. As expected, this substitution results in increased transcriptional activity in E. coli. In B. subtilis, this mutation induces the accumulation not only of a low but significant amount of dhfr mRNA but also of DHFR, demonstrating that binding strengths with a free energy as low as -9.4 kcal/mol are sufficient to promote ribosome binding in B. subtilis. The association of the promoter mutation (C-G) with a mutation which creates a strong B. subtilis ribosome binding site (-21 kcal/mol) results in the accumulation of a large amount of dhfr mRNA. This demonstrates the importance of having an efficient ribosome binding site in the evaluation of promoter function: for example, with this strong ribosome binding site we can show that the wild-type bla promoter is recognized by the B. subtilis transcription machinery.

Animals↗

Expression of the mouse dihydrofolate reductase cDNA in B. subtilis: a system to select mutant cDNAs coding for methotrexate resistant enzymes.

With the aim to obtain a cDNA coding for a mammalian methotrexate resistant dihydrofolate reductase (Dhfr) a plasmid ( pQS1 ) harboring the mouse wild type Dhfr cDNA was constructed and used to transform a methotrexate sensitive bacteria: B. subtilis. A plasmid, pQS4 , expressing large amount of Dhfr in both E. coli and B. subtilis was isolated through a two steps selection with two substrate analogues, trimethoprim followed by methotrexate. This new plasmid has a 54 bp duplication including the beta-lactamase promoter and a deletion of 564 bp removing the 5' end of the beta-lactamase coding region. These changes create a new -35 region TTGAAA and a potentially stronger binding site for both E. coli and B. subtilis 16S ribosomal RNA. pQS4 transformed B. subtilis were then grown in the presence of high level of methotrexate and resistant mutants isolated. One of them, pQS6 , which codes for an enzyme about 50 times more resistant to methotrexate than the wild type Dhfr was sequenced. It shows that a point mutation replaces the glutamine residue at position 35 by a proline.

Animals↗

Expression of inducible nitric oxide synthase in smooth muscle cells from rat penile corpora cavernosa.

Nitric oxide (NO), the main mediator of penile erection, is assumed to be synthesized in the penis by the neuronal constitutive nitric oxide synthase (nNOS). However, nNOS has not been identified in the penile smooth muscle, the target of NO action. The other NOS isozymes, the inducible NOS (iNOS) and the endothelial NOS (eNOS) have not been reported in any penile tissue. The smooth muscle vascular and trabecular tissue from rat corpora cavernosa is represented in vitro by cell cultures designated RPSMC. To determine whether iNOS can be expressed in penile smooth muscle, RPSMC were treated with different lymphokines and/or bacterial lipopolysaccharide (LPS). The selected inducer, LPS/interferon, elicited at 48 hours up to a 50-fold increase in nitrites in the medium; the nitroarginine methyl ester (L-NAME), aminoguanidine, actinomycin D, cycloheximide, transforming growth factor-beta1 (TGF-beta1), and dexamethasone, but was resistant to nifedipine and platelet-derived growth factor AB (PDGF-AB). iNOS induction increased with cell passage. The [3H]L-arginine/citrulline measurement of NO synthesis with intact cells confirmed these results. Incubations of soluble and particulate fractions showed that the cytosol contained most of the activity (Km = 43 microM), which was partially inhibited by ethyleneglycal-bis-tetraacetic acid (EGTA). The 4.4-kb iNOS mRNA peaked at a late period (24-30 hours) and remained high for up to 72 hours. iNOS mRNA induction was strongly inhibited by actinomycin D and dexamethasone, partially inhibited by TGF-beta1, inhibited slightly by PDGF-AB, and unaffected by nifedipine. These results show that iNOS can be expressed in RPSMC in a cell passage-dependent fashion that has so far not been reported for other cell lines, and that the induction reaches much higher levels than in rat or human vascular smooth muscle cells. The expression pattern is also distinctive for the penile cells in time course of induction, Ca2+ dependence, response to certain agents, and mRNA stability.

Animals↗