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D Kang

Publications and source records attributed to D Kang.

At least 109 records · Page 6Linked to original sources
PubMed1996

Analysis of a lysozyme gene from the malaria vector mosquito, Anopheles gambiae.

A genomic DNA sequence encoding a basic lysozyme was isolated from the malaria vector mosquito Anopheles gambiae by screening a library with a probe prepared by PCR of reverse transcribed adult RNA. The sequence consists of an upstream region of about 2 kb, a coding region containing three exons and two introns, and a short 3' untranslated region. The coding region indicates that this mosquito lysozyme consists of a signal peptide of 20 residues followed by an 120 aa mature protein which is very similar to other basic lysozymes. The two small introns, 67 and 76 bp, are located at evolutionarily conserved sites. RT-PCR indicated that this gene is expressed abundantly in sugar-fed adults, and at considerably lower levels when females have fed on blood. Although it remains to be seen whether this gene is induced by bacterial infection, the surrounding sequence contains six sequence motifs very similar to the consensus binding sites for a transcription factor similar to NF-kappa B that are found associated with most insect immune response genes. This lysozyme gene maps to division 27 on the left arm of polytene chromosome 2L. An ORF unrelated to any animal protein in current data bases was found at the 5' end of the clone.

Amino Acid Sequence↗
PubMed1996

Trichoplusia ni attacin A, a differentially displayed insect gene coding for an antibacterial protein.

The mRNA differential display method was used to isolate antibacterial defense genes from Trichoplusia ni. The mRNA population in last-instar T. ni larvae injected with bacteria was compared to that of untreated larvae. Using a PCR amplified probe corresponding to an induced mRNA, we were able to clone an attacin homolog from a lambda cDNA library from vaccinated larvae. The corresponding protein showed 63% identity to Hyalophora cecropia acidic attacin. The induction kinetics of T. ni attacin A gave optimal mRNA levels at 20 h post-infection. Genomic analysis showed this to be a single-copy gene with two introns.

Amino Acid Sequence↗
PubMed1996

PCR differential display of immune gene expression in Trichoplusia ni.

The immune state of insects is defined by a set of proteins that is absent in the naive state. To explore the immune system of Trichoplusia ni in more detail we have employed a PCR differential display technique to compare the mRNA population of untreated last instar larvae to that of immunized animals. In the primary display, more than one hundred bands seemed induced upon bacterial challenge. When they were used as probes in Northern blots, 35% of these probes detected inducible mRNA species. Such probes were used to screen a cDNA library from immunized larvae. We isolated clones for T. ni homologs of cecropin A, lysozyme and attacin. One differentially expressed band hybridized to clones for BJHSP1, a hemacy-anin-related protein which is hormonally up-regulated in last instar larvae; this induction is probably not related to the bacterial infection. Still other probes recognized inducible mRNAs of 1.6 and 1.0 kb. The corresponding cDNA clones did not show strong sequence homology to any known proteins. We have demonstrated the potential of this PCR technique to display both known and unknown genes specific for the immune state of whole insects against a background of genes involved in larval development.

Amino Acid Sequence↗
PubMed1996

Polycyclic aromatic hydrocarbon metabolites in urine as biomarkers of exposure and effect.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) from various occupational, environmental, medicinal, and dietary sources. PAH metabolites in human urine can be used as biomarkers of internal dose to assess recent exposure to PAHs. PAH metabolites that have been detected in human urine include 1-hydroxypyrene (1-OHP), 1-hydroxypyrene-O-glucuronide (1-OHP-gluc), 3-hydroxybenzo[a]pyrene, 7,8,9,10-tetrahydroxy-7,8,9, 10-tetrahydrobenzo[a]pyrene, and a number of other hydroxylated PAHs. The most widely used of these is 1-OHP-gluc, the major form of 1-OHP in human urine, by virtue of its relatively high concentration and prevalence in urine and its ease of measurement. This metabolite of pyrene can be measured as 1-OHP after deconjugation of the glucuronide with beta-glucuronidase or directly as 1-OHP-gluc without deconjugation. Elevated levels of 1-OHP or 1-OHP-gluc have been demonstrated in smokers (versus nonsmokers), in patients receiving coal tar treatment (versus pretreatment), after workshifts in road pavers (versus before shifts or versus controls), after shifts in coke oven workers (versus before shift), and in subjects ingesting charbroiled meat (versus preingestion). More importantly, this metabolite is found (at low levels) in most human urine, even in persons without apparent occupational or smoking exposure. Although measurement of these metabolites is useful in assessing recent exposure to PAHs, their value as predictive markers of biological effect or health outcomes has not been rigorously tested and at present can only be inferred by association.

Animals↗
PubMed1995

Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine.

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O2-) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O2-, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10(6) cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO2 in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10(6) cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.

Acids↗
PubMed1995

NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease amyloid, binds A beta and stimulates A beta aggregation.

NACP, a 140-amino acid presynaptic protein, is the precursor of NAC [the non-amyloid beta/A4 protein (A beta) component of Alzheimer disease (AD) amyloid], a peptide isolated from and immunologically localized to brain amyloid of patients afflicted with AD. NACP produced in Escherichia coli bound to A beta peptides, the major component of AD amyloid. NACP bound to A beta 1-38 and A beta 25-35 immobilized on nitrocellulose but did not bind to A beta 1-28 on the filter under the same conditions. NACP binding to A beta 1-38 was abolished by addition of A beta 25-35 but not by A beta 1-28, suggesting that the hydrophobic region of the A beta peptide is critical to this binding. NACP-112, a shorter splice variant of NACP containing the NAC sequence, bound to A beta, but NACP delta, a deletion mutant of NACP lacking the NAC domain, did not bind A beta 1-38. Furthermore, binding between NACP-112 and A beta 1-38 was decreased by addition of peptide Y, a peptide that covers the last 15 residues of NAC. In an aqueous solution, A beta 1-38 aggregation was observed when NACP was also present in an incubation mixture at a ratio of 1:125 (NACP/A beta), whereas A beta 1-38 alone or NACP alone did not aggregate under the same conditions, suggesting that the formation of a complex between A beta and NACP may promote aggregation of A beta. Thus, NACP can bind A beta peptides through the specific sequence and can promote A beta aggregation, raising the possibility that NACP may play a role in the development of AD amyloid.

Alzheimer Disease↗
PubMed1995

Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport.

In the accompanying paper we have shown that four different histidine residues are involved in the maintenance of mouse band 3 in a state in which it is able to execute its anion transport function. Here we focus on the functional significance of His 752 and demonstrate that this residue, together with Glu 699, plays a key role in the control of pH dependence of Cl- transport. Mouse band 3-encoding cRNA was expressed in Xenopus oocytes, and band 3-mediated Cl- transport was measured at zero membrane potential over the pH range 6.0-9.2. Transport decreased with increasing H+ concentration and was governed by a single pK of 5.8. After correction for temperature differences, this result agrees well with measurements in erythrocyte ghosts of Cl- flux by Funder and Wieth [Funder, J., & Wieth, J. O. (1976) J. Physiol. 262, 679-698] and our own determinations by 35Cl NMR spectroscopy of Cl- exchange between the substrate binding site and the medium. After mutation of either Glu 699 to Asp or of His 752 to Ser, the maximal rate of transport is reduced and the rate of anion exchange is now governed by a single pK of about 6.8-6.9. This suggests that the formation of a hydrogen bond between His 752 and Glu 699 is essential for the decrease of band 3-mediated Cl- transport at low pH. We suggest that in the wild type band 3 both the decrease of the chloride exchange between the medium and the substrate binding site and the inhibition of chloride translocation across the membrane are dominated by a common rate-limiting step and that this step involves hydrogen bond formation between Glu 699 and His 752.

Amino Acid Sequence↗
PubMed1995

Intracellular localization of 8-oxo-dGTPase in human cells, with special reference to the role of the enzyme in mitochondria.

We examined the intracellular distribution of 8-oxo-dGTPase (8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of 8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble fractions of Jurkat cells, a human T-cell leukemia line. Electron microscopic immunocytochemistry, using a specific antibody against MTH1 protein, showed localization of MTH1 protein in the mitochondrial matrix. Activity in the mitochondria accounted for about 4% of the total activity. The specific activity in the mitochondrial soluble fraction (8093 units/mg protein) was as high as that in the cytosolic fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in cytosolic and mitochondrial soluble fractions co-eluted with MTH1 protein by anion-exchange chromatography, and the molecular mass of the mitochondrial MTH1 protein was much the same as that of the cytosolic MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an increased cytoplasmic signal together with a weak signal in the nucleus in in situ immunostaining of MTH1 protein, and the overexpressed MTH1 protein was recovered from both cytosolic and mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondrial and cytosol.

Blotting, Western↗
PubMed1995

Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system.

PURPOSE: A noncontact holmium:yttrium scandium gallium garnet (Ho:YSGG) laser system has been designed and tested for the micromanipulation of mammalian embryos. The purpose of this preliminary investigation was to determine the effectiveness of this laser for assisted hatching and evaluate its impact on embryo viability. The Ho:YSGG system, utilizing 250-microsecond pulses at a wavelength of 2.1 microns and 4 Hz, was used to remove a portion of the zona pellucida (ZP) of two- to four-cell FVB mouse embryos. RESULTS: In the first experiment there was no difference in blastocyst production or hatching rates following laser or conventional assisted hatching (LAH or AH, respectively) in contrast to control embryos cultured in a 5% CO2 humidified air incubator at 37 degrees C. In the second experiment a blastocyst antihatching culture model was employed and LAH-treated embryos were cultured in a serum-free HTF medium (HTF-o). Blastocyst formation was not influenced by LAH treatment and hatching was increased (P < 0.01) from 4 to 60% compared to HTF-o control group. CONCLUSIONS: These preliminary data demonstrate the utility and nontoxic properties of the Ho:YSGG laser system for quick and precise ZP drilling.

Animals↗
PubMed1995

A structural study of the carboxyl terminal region of the human erythrocyte band 3 protein.

Two peptides derived from the carboxyl terminal region of the human erythrocyte band 3 protein were identified as fragments releasable from cell membranes on trypsin digestion. These peptides, Asn-880-Lys-892 and Ala-893-Val-911-COOH, however, were resistant to trypsin, unless the cell membranes had been treated with high concentrations of NaOH. This suggests that the carboxyl terminal region is located in situ within the native band 3 molecule. Unlike in the cases of other portions of the band 3 protein, such as Gly-647-Arg-656, Ser-731-Lys-743, and Tyr-818-Lys-826, the release of the carboxyl terminal region was not inhibited by pretreatment of erythrocytes with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), indicating that there is no major structural difference in the carboxyl terminal portion between the outward and inward facing forms. The carboxyl terminal region of the band 3 protein has a negative charge cluster. In the middle of the negative charge cluster, consensus sequences, Val-Asp-X-X-X-Leu-Asp-Ala-Asp-Asp and Thr-Phe-Asp-Glu (TFDE), were found in the carboxyl terminal regions of aquaporin CHIP and glucose transporter 1, respectively. The sequence, TFDE, exists in the highly amphipathic 11-residue sequence of glucose transporter 1, and this amphipathic sequence has been suggested to promote normal membrane insertion of polytopic membrane proteins such as glucose transporter 1 and serine chemoreceptor. The role of the carboxyl terminal region of the band 3 protein is discussed.

Alanine↗
PubMed1995

Association of exposure to polycyclic aromatic hydrocarbons (estimated from job category) with concentration of 1-hydroxypyrene glucuronide in urine from workers at a steel plant.

OBJECTIVES: Increased risk of lung cancer has been associated with employment in the steel industry. This association is thought to be due in part to increased concentrations of polycyclic aromatic hydrocarbons (PAHs) in air found in this work environment. Measurement of PAH metabolites in human urine provides a means of assessing individual internal dose of PAHs. This study examined the relative contribution of occupation and smoking to urinary concentration of 1-hydroxypyrene glucuronide (1-OHPG) among a group of workers at a steel plant. METHODS: Concentrations of 1-OHPG in urine from 44 workers with jobs associated with increased air concentrations of PAHs and 40 workers with jobs with low or no exposure to PAHs were measured. 20 workers in each group were not current smokers. Urinary 1-OHPG was measured by synchronous fluorescence spectroscopy after immunoaffinity chromatography specific for PAH metabolites. RESULTS: Mean (SEM) urinary 1-OHPG concentration was 2.16 (0.42) pmol/ml urine among the 44 occupationally exposed workers compared with 0.38 (0.05) among the 40 workers with no or low exposure (P < 0.0001). Mean urinary 1-OHPG concentration was 1.82 (0.41) pmol/ml urine among the 44 current smokers compared with 0.75 (0.20) among the 40 non-smokers (P < 0.005). Mean 1-OHPG concentrations in non-smokers were 0.26 (n = 20), 0.70 (n = 15), and 2.84 pmol/ml urine (n = 5) for strata of exposure to PAHs (no or low, mid, and high) based on job category; the corresponding values in smokers were 0.55 (n = 20), 0.94 (n = 12), and 4.91 pmol/ml (n = 12), respectively. Multiple linear regression showed significant differences between subjects in different PAH exposure with increased concentrations of 1-OHPG in urine. Amounts of foods containing PAHs ingested by this group of workers were relatively low and did not contribute significantly to urinary 1-OHPG concentrations. CONCLUSIONS: These results indicate that 1-OHPG is a common urinary metabolite in people with recent occupational exposure to PAHs and is associated with both job category and estimated stratum of PAH exposure.

Adult↗
PubMed1995

1-Methyl-4-phenylpyridinium (MPP+) inhibits mitochondrial oxygen consumption mediated by succinate as well as malate in rat pheochromocytoma PC12 cells.

When rat pheochromocytoma PC12 cells are cultured with 1 mM 1-methyl-4-phenylpyridinium (MPP+), the number of viable cells decreases to one third in 4 days while the number increases ten-fold without MPP+. Oxygen consumption by mitochondria in the presence of malate is inhibited about 80% by the treatment of the cells with MPP+ for 4 days. Unexpectedly, succinate-dependent oxygen consumption is also inhibited to essentially the same extent as malate-dependent one. These results suggest that the impairment of the respiration mediated by succinate as well as malate is important as a mechanism of MPP(+)-induced cell death.

1-Methyl-4-phenylpyridinium↗
PubMed1995

Metastatic pleural cancer in radionuclide angiocardiography. A pulmonary time-activity curve mimicking left-to-right cardiac shunt.

A 55-year-old man, with a history of nephrectomy for renal cell carcinoma, was evaluated using radionuclide angiocardiography to exclude cardiac shunts as the cause of vascular bruits heard at auscultation. A pulmonary time-activity curve derived from the right lung showed a pattern typical of a large left-to-right shunt, whereas that from the left lung was normal. Increased systemic arterial flow to the lateral right chest was noted in early dynamic images. Subsequent studies revealed that this flow and the abnormal time-activity curve of the right lung were due to systemic arterial supply to metastatic pleural cancer, originating from the kidney. The authors conclude that special caution should be undertaken in the interpretation of the radionuclide angiocardiography for determining left-to-right cardiac shunts, especially in those showing a asymmetric pulmonary time-activity curve.

Carcinoma, Renal Cell↗
PubMed1994

Anion transport function of mouse erythroid band 3 protein (AE1) does not require acylation of cysteine residue 861.

Cys-861 of mouse band 3 is equivalent to Cys-843 of human band 3, the only acylated cysteine residue in the anion exchanger AE1 of the red blood cell (Hamasaki et al. (1992) Progress Cell Res. 2, 65-71). Mutation of Cys-861 to serine or methionine caused no significant changes of band 3-mediated anion exchange as measured after expression of the appropriate cRNAs in Xenopus oocytes. Susceptibility to inhibition of transport by 4,4'-dinitrostilbene-2,2'-disulfonate and PCMBS was not affected. We conclude that palmitoylation is not an absolute requirement for the successful execution the anion transport function by the hydrophobic domain of band 3 in the plasma membrane.

Acylation↗
PubMed1994

Preferential heme transport through endoplasmic reticulum associated with mitochondria in rat liver.

The transport of de novo synthesized protoheme into the conventional microsomal fraction and endoplasmic reticulum associated with mitochondria (MAER) was studied by injecting amino[14C]levulinic acid (ALA) into phenobarbital-treated rats to evaluate the role of MAER in the trafficking of heme between mitochondria and endoplasmic reticulum. In mitochondria, the specific radioactivity of the radiolabeled heme reached a maximum level at 4 min after the injection of 14C-ALA. The specific radioactivity in cytosol was about 2-fold lower than that in microsomes, suggesting that the cytosolic pathway of the heme transport from mitochondria to endoplasmic reticulum is not predominant, because the specific radioactivity of heme in cytosol should be higher than that in microsomes if heme is transported mainly through cytosol. MAER showed higher specific radioactivity than the conventional microsomal fraction up to 4 min and thereafter the specific radioactivities in MAER and the conventional microsomal fraction became nearly the same. The extents of decrease in cytochrome P-450 and the radioactivity in microsomes by the treatment with allylisopropylacetamide which destroyed cytochrome P-450 but not cytochrome b5, were essentially the same, suggesting that most of the radiolabeled heme in microsomes was incorporated into cytochrome P-450. These results suggest that MAER is a preferential site for the protoheme transport from mitochondria to endoplasmic reticulum.

Animals↗
PubMed1994

Red blood cell band 3. Lysine 539 and lysine 851 react with the same H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid) molecule.

The band 3 protein of the red blood cell membrane catalyzes anion exchange that is inhibited by the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid). There is one H2DIDS binding site per 95,000-Da band 3 polypeptide. The single bound H2DIDS molecule can react covalently with 2 different lysine residues. The 2 lysines that react covalently with H2DIDS have been localized directly by sequencing fragments of human band 3 from cells labeled with [3H]H2DIDS. The most rapid covalent reaction is with Lys-539, in agreement with site-directed mutagenesis studies. The slower reaction is with Lys-851, which is known to be the primary site of binding of another anion transport inhibitor, pyridoxal phosphate (Kawano et al., 1988). These results indicate that the protein is folded to bring these 2 residues into close enough proximity to react covalently with the same H2DIDS molecule. In addition to defining the residues that react with H2DIDS, these studies have also defined new in situ proteolytic cleavage sites in band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗