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Biomedical subjects

G Wang

Publications and source records attributed to G Wang.

At least 757 records · Page 42Linked to original sources
PubMed1993

pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo.

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.

Base Sequence↗
PubMed1993

Polymerase chain reaction-based restriction fragment length polymorphism analysis of the short arm of chromosome 3 in primary head and neck squamous carcinoma.

BACKGROUND: Deletion or loss of heterozygosity (LOH) at the polymorphic loci on the short arm of chromosome 3 has been reported in a large number of renal cell, small cell lung, non-small cell lung, and cervical carcinomas, suggesting the presence of one or more putative tumor suppressor genes at chromosome 3p. Similar studies in primary head and neck carcinoma are lacking. METHODS: To investigate the possibility of chromosome 3p deletions, the authors applied a polymerase chain reaction (PCR)-based, restriction fragment length polymorphism analysis, in conjunction with conventional Southern blot techniques, to DNA samples of matched normal mucosa and head and neck squamous cell carcinomas from 18 patients. The authors also assessed the merit of the PCR-based assay as a rapid screening tool, particularly in assaying limited tissue samples. RESULTS: Constitutional heterozygosity at the polymorphic loci varied in the 18 normal samples that the authors studied: 12 at the D3F15S2 locus (on telomeric 3p21), 7 at the D3S32 locus (on centromeric 3p21), and 9 at the THRB locus (on 3p24). In 18 matched carcinoma specimens, LOH (deletion) was observed at D3S32 in 0 of 7, at D3F15S2 in 9 of 12 (75%), and at THRB in 3 of 9 cases (33%). CONCLUSIONS: The results of the PCR-based assay and Southern blotting were completely concordant in all specimens the authors studied. This study indicates that deletion at 3p is a frequent abnormality in primary head and neck carcinoma and that the most common deletion region is telomeric to D3S32. The authors also observed an apparent correlation among poor histologic differentiation, DNA aneuploidy, and 3p deletions. Most poorly and moderately differentiated and aneuploid carcinomas manifested the 3p deletion. Therefore, the authors suggest an association between deletion at 3p and aggressive biologic behavior.

Aged↗
PubMed1993

A clinical and immunologic evaluation of women with silicone breast implants and symptoms of rheumatic disease.

OBJECTIVE: To describe the clinical and serologic features of women with silicone breast implants who were referred for symptoms of rheumatic disease. DESIGN: A case series. SETTING: University and private rheumatology practices. PATIENTS: A total of 156 women with silicone breast implants and rheumatic disease complaints. Controls for the serologic studies included women with silicone implants and no rheumatic symptoms (n = 12) and women with fibromyalgia without silicone implants (n = 174). MEASUREMENTS: Complete physical examination and testing for immunoglobulins; complement; C-reactive protein; rheumatoid factor; and autoantibodies by indirect immunofluorescence, immunodiffusion, and Western blot. RESULTS: Three subgroups of patients were defined based on clinical and laboratory findings: joint and muscle pain (n = 95), joint swelling (n = 32), and connective tissue disease (n = 29). Most women had normal immunologic studies. The patients with joint swelling had mild, asymmetric, rheumatoid-factor-negative synovitis that did not meet American College of Rheumatology criteria for rheumatoid arthritis. Fourteen patients had a scleroderma-like illness and anti-centromere or anti-PM-Sci antibodies by Western blot. Ten patients had a positive Western blot for BB' polypeptide, a small nuclear ribonucleoprotein (snRNP), but did not meet criteria for systemic lupus erythematosus. No autoantibodies to known disease-related polypeptides were detected on Western blot in the control groups. CONCLUSION: Most women with silicone implants and rheumatic complaints had normal results of serologic tests and nonspecific symptoms, suggesting no serious connective tissue disease. However, a subset of women had clinical signs and serologic tests that were unusual even for referred patients. These observations suggest, but cannot establish, that some women with silicone breast implants may develop atypical immunologic reactions.

Adult↗
PubMed1993

Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants.

An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.

Alleles↗
PubMed1993

Thermodynamic elucidation of structural stability of deuterated biological molecules: deuterated phospholipid vesicles in H2O.

Thermodynamic studies of deuterated phospholipids in H2O were conducted using deuterated lipid bilayer vesicles prepared from aqueous dispersions of 1,2-dimyristoyl-d54-, 1,2-dipalmitoyl-d62-, and 1,2-distearoyl-d70-phosphatidylcholines. The results obtained in combination with those previously reported in D2O made possible the evaluation of deuterium solvent and deuterium alkyl-chain substitution effects in deuterated phospholipids. These effects included the temperature (delta tm), the enthalpy (delta delta H), the free energy (delta delta G), the entropy (delta delta S), and the cooperative melting unit (delta n) of the main lipid phase transition from the gel to the liquid-crystalline phase. To supplement thermodynamic observations, fluorescence steady-state polarization measurements were also performed. The results were examined to elucidate the nature of interactions in deuterated compounds. The deuterium solvent effect increases the structural stability of deuterated lipid vesicles, due to an enhancement of hydrophobic interaction in D2O. This observation is contrary to that previously reported for deuterated proteins, phycocyanins, where structural stability is reduced in D2O solvent. These findings therefore support the proposal that an alteration in hydrophobic interactions in deuterated proteins due to deuterium substitution is not likely to play a significant role in contributing to the structural instability of proteins.

Calorimetry, Differential Scanning↗
PubMed1993

Modulation of SPARC expression during butyrate-induced terminal differentiation of cultured human keratinocytes: regulation via a TGF-beta-dependent pathway.

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC+ cells increased with time and peaked immediately postconfluence (31.3 +/- 6.3% SPARC+). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G2/M and G1 phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-beta (TGF-beta) production since incubation in the presence of NaB+neutralizing antibodies to TGF-beta inhibited both the expression of SPARC by 72% and development of mature CEs.

Antibodies↗
PubMed1993

Reference values for blood toluene in the occupationally nonexposed general population.

Blood toluene was measured by gas chromatography--mass spectrometry in 232 occupationally nonexposed subjects, consisting of 126 rural and 106 urban workers, and 37 chemical workers. Mean blood toluene was significantly lower in rural (698 ng/l) and urban workers (984 ng/l) than in chemical workers (2789 ng/l). Blood toluene was not significantly different between the rural and urban workers or among the urban workers with different jobs. Smokers had significantly higher levels (median 606 ng/l) than nonsmokers (median 424 ng/l). Subjects who had smoked at least one cigarette in the last 2 h before blood sampling had significantly higher blood toluene (median 1170 ng/l) than those who had not smoked during this time (median 693 ng/l), for whom the level was not significantly different from that in nonsmokers. Blood toluene in the total population was less than 2863 ng/l in 95% cases.

Adult↗
PubMed1993

Functions and proteins of herpes simplex virus type-1 that are involved in raising the mutation frequency of infected cells.

When cells are infected by herpes simplex virus type-1 (HSV-1) the mutation frequency is increased. To find which functions of the virus are responsible for this, a variety of viral strains and one fragment of viral DNA were tested in a mutagenesis assay. Mutagenesis was dependent on the binding of the virus to the cell surface and disassembly of the virus particle, but expression of virus genes was not necessary. Since this implied that mutagenesis was a result of the exposure of the interior of the cell to an internal structural component of the virus, the role of two likely components was examined. The host-shutoff function of the virus was not required for mutagenesis. However, a fragment of DNA from within the minimum transforming region of HSV-1 that encodes a possible virion protein was mutagenic when expressed from a eukaryotic expression vector. The encoded product of this DNA fragment is therefore a candidate for a transforming protein of HSV-1, and is the only protein currently suggested to be responsible for that function.

Animals↗
PubMed1993

Effects of diethyl spermine analogues in human bladder cancer cell lines in culture.

The biochemical and antiproliferative effects of two recently developed N-alkylated analogues of naturally-occurring polyamines, N1,N14-diethylhomospermine (DEHSPM) and N1,N11-diethylnorspermine (DENSPM), were investigated in two human transitional cell carcinoma (TCC) lines, T24 and J82. Parallel studies with the ornithine decarboxylase enzyme inhibitor alpha-difluoromethylornithine (DFMO) were included for comparison. DENSPM displayed greater antiproliferative activity than DEHSPM in both TCC cell lines. Both analogues were strikingly more potent than DFMO. DEHSPM and DENSPM suppressed the activity of the major biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase. However, differences in the resulting polyamine depletion suggest that the substantial antiproliferative activity of these analogues may result from mechanisms other than polyamine depletion. The greater polyamine depletion seen with DENSPM is thought to result from its striking induction of spermidine/spermine N1-acetyltransferase. DENSPM is an attractive agent for further preclinical and clinical development, possibly as a chemopreventive agent, in TCC of the bladder.

Carcinoma, Transitional Cell↗
PubMed1993

Modified longitudinal preputial island flap urethroplasty for repair of hypospadias: results in 60 patients.

We report the results of hypospadias repair in 60 children requiring urethral reconstruction using a longitudinal or oblique preputial island flap technique that includes the inner and outer surfaces of the prepuce to construct the neourethra. Mean patient age was 3.2 years and mean length of the repair was 3.1 cm. The cosmetic and functional results were satisfactory. The incidence of urethrocutaneous fistula was 8.3% and that of meatal stenosis was 5%. Thus, this modification offers good results with a low complication rate. The range of applications can be extended by combining the technique with a tube of midline scrotal skin in cases of scrotal or perineal hypospadias. The surgical technique is described in detail.

Child, Preschool↗
PubMed1993

Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗