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K Li

Publications and source records attributed to K Li.

At least 289 records · Page 16Linked to original sources
PubMed1996

Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.

DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.

Affinity Labels↗
PubMed1996

Cloning of the gene for human pemphigus vulgaris antigen (desmoglein 3), a desmosomal cadherin. Characterization of the promoter region and identification of a keratinocyte-specific cis-element.

Pemphigus vulgaris antigen is a cadherin-like desmosomal cell adhesion molecule expressed primarily in suprabasal keratinocytes within the epidermis. Previously characterized structural features have defined this molecule as a desmoglein, DSG3. In this study, we have cloned the human DSG3 gene and examined the transcriptional regulation of its expression. The total gene consisted of 15 exons and was estimated to span >23 kilobases. Comparison of exon-intron organization of DSG3 with bovine DSG1 and several classical cadherin genes revealed striking conservation of the structure. Up to 2.8 kilobases of the upstream genomic sequences were sequenced and found to contain several putative cis-regulatory elements. The promoter region was GC-rich and TATA-less, similar to previously characterized mammalian cadherin promoters. The putative promoter region was subcloned into a vector containing chloramphenicol acetyl transferase reporter gene. Transient transfections with a series of deletion clones indicated that the DSG3 promoter demonstrated keratinocyte-specific expression, as compared with dermal fibroblasts examined in parallel, and fine mapping identified a 30-base pair segment at -200 to -170 capable of conferring epidermal specific expression. The results provide evidence for the transcriptional regulation of the pemphigus vulgaris antigen gene, potentially critical for development of the epidermis and physiologic terminal differentiation of keratinocytes.

Amino Acid Sequence↗
PubMed1996

Point mutations abolish 11 beta-hydroxysteroid dehydrogenase type II activity in three families with the congenital syndrome of apparent mineralocorticoid excess.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol into mineralocorticoid receptor inactive cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME), although proof of mutant protein synthesis was not provided. In the present study we have examined the metabolism of cortisol in mammalian cells transfected with plasmids expressing the wild type and mutant enzymes from three additional families of patients with mutations in the HSD11B2 gene. These studies revealed that the mutants were enzymatically inactive in intact mammalian cells expressing significant levels of both full length and truncated proteins. This is the first study to definitively show that point mutations in the HSD11B2 gene abolish 11 beta HSD2 enzymatic activity in the syndrome of AME.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

The homeotic target gene centrosomin encodes an essential centrosomal component.

The centrosomin (cnn) gene encodes a protein associated with mitotic centrosomes in Drosophila melanogaster and is a target of homeotic gene regulation. Here we report that CNN is an essential component of the centrosome. Loss of zygotic cnn expression disrupts the development of the second midgut constriction, the gastric caeca, and the nervous system. Embryos that lack maternal as well as zygotic cnn expression display defects in nuclear division, chromosome alignment, and microtubule organization, while adult flies that are mosaic for cnn-cells exhibit defects indicative of a block in cell proliferation. We propose that cnn provides an example of homeotic genes directly regulating the accumulation of essential cellular proteins to carry out segment-specific morphogenetic functions.

Animals↗
PubMed1996

Cloning and kidney cell-specific activity of the promoter of the murine renal Na-K-C1 cotransporter gene.

The murine Nkcc2/Slcl2a1 gene encodes a bumetanide-sensitive Na-K-Cl cotransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle. Nuclear run-off assays demonstrated that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription. To begin study of the gene promoter, a genomic clone that contained 13.5 kilobases of the 5'-flanking region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The sequence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initiator. A (G-A)28.(C-T)28 microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins, were also identified. To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporter gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E)Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K((NH4)+)-Cl cotransport activity and endogenously expressed the 5.0-kilobase Nkcc2 transcript. Luciferase activity was 130-fold greater following transfection into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that promoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further deletion to -190 resulted in a 76% decrease in activity. We conclude that the Nkcc2 promoter exhibits kidney cell-specific activity. Regulatory elements required for maximal promoter activity are located in a 280-bp DNA segment that contains consensus binding sites for several transcription factors expressed in the kidney.

Amino Acid Sequence↗
PubMed1996

Magnetic resonance imaging detection of vascular occlusion of a pedicled muscle flap.

Contrast-enhanced magnetic resonance imaging (MRI) can be a highly sensitive monitor of tissue blood perfusion. This technique has been used to assess blood flow through liver, kidney, and certain tumors, but has not been widely applied to the study of skeletal muscle circulation. In our study, we used a novel scanning software to obtain contrast-enhanced T2*-weighted gradient echo MRI images of pedicled quadriceps muscle flaps in rabbits in order to study images of arterial, venous, and arterio-venous occlusion. We administered an intravenous bolus of gadoteridol contrast agent at the initiation of scanning, which produces a decrease in T2*-signal and improves the sensitivity of measuring blood perfusion. Within 30 seconds of MRI scanning, control flaps with intact pedicles exhibited a rapid decrease in T2*-signal intensity, indicating adequate perfusion of blood through muscle tissue; however, occluded pedicled flaps showed no significant change in signal intensity, indicating lack of blood perfusion. Differences in signal intensities as measured by MRI between occluded and control flaps were statistically significant (P < 0.05). Selective vascular occlusion of either artery alone, or both artery and vein were detected within 15 minutes, whereas selective venous occlusion could be detected after 2 hours. We conclude that MRI has the ability to assess skeletal muscle perfusion, and is capable of noninvasively evaluating a cross-section of tissue in both superficial and buried flaps. MRI, therefore, may have the potential for evaluating perfusion in muscle flaps (including buried flaps), and other disorders of muscle circulation such as compartment syndrome.

Animals↗
PubMed1996

Endocardial endothelium and myocardial performance in rats: effects of changing extracellular calcium and phenylephrine.

It is known that endocardial endothelium (EE) modulates the performance of its subjacent myocardium. However, one species where these effects have been more difficult to describe is the rat. As the rat is a species which is used to evaluate the contractile effects of various pathologic conditions, a better appreciation of the contractile effects of EE and its modulatory role on the contractile effects of inotropic substances is important. In this study, the contractile effects of increasing extracellular calcium concentrations (0.7 mM to 3.25 mM) and phenylephrine (10(-7) to 10(-4) M) on rat papillary muscles with intact endocardial endothelium (EE on) and after endocardial endothelial removal (EE off) were assessed. At 0.7 mM extracellular calcium concentration, endocardial endothelial removal decreased all measured indices of myocardial performance (P < 0.05 EE on v EE off), except for maximum rate of unloaded muscle shortening (Vmax) which did not change, and decreased time to peak tension development (TTPT) as well as shortening of time to half tension decline from peak tension (RT1/2) shortening. Increasing extracellular calcium concentration from 0.7 mM to 3.25 mM caused all indices of myocardial performance and RT1/2 to increase more in muscles without EE, such that at 2.5 mM extracellular calcium all differences between EE on and EE off had disappeared. The only exception was TTPT which decreased with increasing extracellular calcium concentrations in muscles with EE on but increased in muscles with EE off. Again, at 2.5 mM extracellular calcium concentration differences in TTPT between EE on and EE off had disappeared. Except for minor differences on TTPT, increasing phenylephrine concentrations had similar contractile effects on muscles with EE on and EE off. These results indicate that EE modulates rat myocardial contraction and that these effects are best observed at lower extracellular calcium concentrations (0.7 mM). They also indicate that EE does not appear to significantly modulate the myocardial effects of alpha-adrenergic agonists in the rat.

Animals↗
PubMed1996

Vascular-derived myocardial contractile factor: positive myocardial inotropic substance released from medial layer of the canine aorta.

Interactions between the various cell types that make up the cardiovascular system are known to play an important role in maintaining homeostasis. One area about smooth muscle cells that has received little attention, despite the production of a wide variety of mediators by smooth muscle cells, is their effect on myocardial function. In this study, the myocardial contractile effects of four different types of dog aortic strips on rabbit papillary muscles were evaluated. Of these, medial vascular smooth muscle strips most consistently (65% of the time) produced a "vascular-derived contractile factor" (VDCF), which caused a 15% increase in isometric twitch tension and a 24% increase in isotonic twitch shortening with no change in twitch configuration. Endovascular strips with or without intact endothelium and complete aortic rings had less consistent effects. Vascular-derived contractile factor was stable after freezing at -80 degrees C, its activity was not modified by a broad spectrum peptidase, but it was heat-labile. The angiotensin II blocker, losartan, did not modify its effects. However, incubation with indomethacin did reduce, but did not eliminate, the contractile effects of vascular strips. The addition of alpha 1- and beta-blockers did not further modify the effects of VDCF. Endocardial endothelial removal increased the effects of VDCF. No correlation existed between endothelin levels and the contractile effects of vascular strips. It is concluded that VDCF is produced by the medial layer of large vessels but its exact cellular origin is uncertain. These findings expand the ever-increasing understanding of the inter-relationship between the structures that make up the cardiovascular system, and open the door to new studies evaluating the inter-relationship of vessels and myocardium.

Animals↗
PubMed1996

Altered responsiveness to endothelin-1 of myocardium from pacing-induced heart failure model in the dog.

In congestive heart failure, in addition to a compensatory increase in neurohumoral activation, there is an increase in the endothelial-derived vasoconstrictive and positive inotropic substance, endothelin. Whether downregulation of the cardiac inotropic effects of this endothelial-derived substance occurs, as has been shown to occur with neurohumoral beta- and alpha-adrenergic agonists, remains unknown. In this study we investigated the effects of endothelin-1 [dose-response curve (10(-11) to 10(-7) M)] on the contractile characteristics of isolated papillary muscles from normal dogs and from dogs with heart failure induced by pacing overdrive, with or without removing endocardial endothelium from the papillary muscles. Endothelin-1 caused a similar absolute increase in myocardial contractile indices in all four groups, except for shortening, which increased more in muscles with heart failure without endocardial endothelium. However, in muscles with an intact endocardial endothelium, the relative increase was greater in muscles from pacing overdrive dogs (failing) as compared with normal dogs (tension increase of 110% vs. 53%, p < 0.01 and shortening increase of 127% vs. 24%, p < 0.01). Also, failing muscles with intact endocardial endothelium began responding to endothelin-1 at lower endothelin-1 concentrations (10(-10) vs. 10(-9) M) than normal muscles with intact endocardial endothelium. Endocardial endothelial removal increased the contractile effects of endothelin-1, whether this was done in normal or failing myocardium. This study thus indicates that, in contrast to other positive inotropic substances, in this model of heart failure there is an increase in sensitivity and relative response to endothelin-1. It also indicates that although endocardial endothelial removal increases the relative effects of endothelin-1 in both normal and failing myocardium, the increased responsiveness of failing myocardium is not endocardial endothelial dependent.

Animals↗
PubMed1996

The 11 beta-hydroxysteroid dehydrogenase type II enzyme: biochemical consequences of the congenital R337C mutation.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol to cortisone, allowing the non-selective mineralocorticoid receptor to bind aldosterone. When the activity of this enzyme is compromised, as occurs in licorice intoxication or in the congenital syndrome of apparent mineralocorticoid excess (AME), there is marked sodium retention, hypokalemia, and hypertension. The first proof that this enzyme was defective in AME came from the identification of the R337C mutation in a number of siblings with the syndrome. Subsequent expression studies showed that the mutant had a Km one order of magnitude higher than the wild-type enzyme while in the cell-free system it was without detectable activity. In the present work we have extended our studies on this mutant and provide evidence that the mutant protein may also partially inhibit the wild-type enzyme in heterozygotes. Furthermore, experiments incorporating the protein synthesis inhibitor cycloheximide show that the mutant enzyme is less stable than the wild-type activity in intact cells. These results suggest that mutations in the 11 beta HSD2 enzyme may have multiple consequences for the mineralocorticoid target cell.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1996

An integrated YAC map of the human X chromosome.

The human X chromosome is associated with a large number of disease phenotypes, principally because of its unique mode of inheritance that tends to reveal all recessive disorders in males. With the longer term goal of identifying and characterizing most of these genes, we have adopted a chromosome-wide strategy to establish a YAC contig map. We have performed > 3250 inter Alu-PCR product hybridizations to identify overlaps between YAC clones. Positional information associated with many of these YAC clones has been derived from our Reference Library Database and a variety of other public sources. We have constructed a YAC contig map of the X chromosome covering 125 Mb of DNA in 25 contigs and containing 906 YAC clones. These contigs have been verified extensively by FISH and by gel and hybridization fingerprinting techniques. This independently derived map exceeds the coverage of recently reported X chromosome maps built as part of whole-genome YAC maps.

Chromosome Mapping↗
PubMed1996

Cloning of mouse type VII collagen reveals evolutionary conservation of functional protein domains and genomic organization.

Type VII collagen is the major component of anchoring fibrils, attachment structures necessary for stable association of the dermal-epidermal basement membrane to the underlying dermis. The critical role of the anchoring fibrils in providing integrity to the cutaneous basement membrane zone is attested to by demonstration of mutations in the type VII collagen gene (COL7A1) in patients with dystrophic epidermolysis bullosa. To gain insight into the evolutionary conservation of the type VII collagen gene, in this study we have cloned the entire mouse type VII collagen cDNA and elucidated the intron-exon organization of the corresponding gene, Col7a1. The coding region of the cDNA consists of 8832 nucleotides encoding a polypeptide of 2944 amino acids with a calculated molecular mass of approximately 295 kDa. Computer analysis predicted the presence of an 18-amino acid signal peptide. Comparison of the deduced mouse alpha1(VII) collagen polypeptide with the corresponding human sequence indicated 84.7% identity and 90.4% homology at the amino acid level. In addition, the domain organization, including imperfections and interruptions within the collagenous domain consisting of Gly-X-Y repeat sequences, was highly conserved. The unit of evolutionary period between the full-length human and mouse polypeptides was calculated to be 6.5 million years, however, suggesting relatively rapid evolutionary divergence in comparison to other collagen genes. Elucidation of the intron-exon organization of the mouse Col7a1 gene revealed 118 distinct exons, the same number as present in the human gene. These data indicate a high degree of structural conservation between the human and mouse type VII collagen, supporting the critical role of this collagen as the major component of the anchoring fibrils.

Amino Acid Sequence↗
PubMed1996

Gender influences the magnitude of the inflammatory response within embolic cerebral infarcts in young rats.

BACKGROUND AND PURPOSE: The inflammatory response within cerebral infarcts may have an influence on tissue damage. Since old animals with an impaired immune response have decreased inflammation after experimental cerebral infarction, we postulated that female animals with an increased immune response will have an increased inflammatory response after cerebral infarction. METHODS: Embolic cerebral infarcts were produced by photochemical irradiation of the right carotid artery in 12 female Fischer rats. The inflammatory response within 4-day-old infarcts was quantitated by histology with the use of computer-assisted image analysis and compared with that in 12 male rats from a previous series. RESULTS: Severe infarcts had the most pronounced inflammatory response. Female rats had an increased inflammatory response in infarcts of all severity, which was statistically significant in severe cerebral infarcts even after adjustment for infarct size. Severe infarcts in males were significantly larger than those in females. CONCLUSIONS: Gender influences the outcome of embolic cerebral infarcts after photochemical damage to the carotid artery, both in terms of the magnitude of the inflammatory response and infarct size. There are numerous gender-related differences in neurochemicals, cytokine production, and drug metabolism that may influence tissue damage after stroke and responsiveness to therapeutic intervention. The preponderance of male animals in stroke research may produce results not applicable to female stroke patients. The use of female animals will be required to provide adequate models for the study of stroke in women.

Animals↗
PubMed1996

Mineral dusts cause elastin and collagen breakdown in the rat lung: a potential mechanism of dust-induced emphysema.

It is now accepted that workers with exposure to mineral dusts can develop airflow obstruction. The basis of this process is uncertain, but carefully performed morphologic studies suggest that coal, silica, and perhaps other dusts may produce emphysema in humans. To investigate the mechanisms involved in this process, we administered crystalline silica (quartz) or titanium dioxide (rutile) to rats in a single intratracheal instillation. At varying times after instillation, the animals' lungs were lavaged, the lavageate from one lung was dried and hydrolyzed, and the amounts of desmosine (DES),as a measure of elastin breakdown, and hydroxyproline (HP), as a measure of collagen breakdown, were determined. The lavageate from the other lung was counted for inflammatory cells. Both silica and titanium dioxide caused a dose-dependent increase in DES and HP 24 h after instillation. When an equivalent dose (30 mg) of silica or rutile was administered and animals were sacrificed at various times up to 21 d, a sustained increase in lavage DES and HP was seen in the silica-treated animals, and this was accompanied by a sustained increase in polymorphonuclear leukocytes (PMN); in contrast, both lavage PMN and lavage DES/HP rapidly peaked and then declined in the titanium dioxide-treated animals. Numbers of macrophages remained elevated over the 21-d period of sacrifice with both types of treatment. These data show for the first time that mineral dusts can cause connective-tissue breakdown in the lung, with the release of matrix components into the alveolar spaces. The amount of connective-tissue breakdown appears to parallel the number of PMN but not the number of macrophages in the alveolar spaces, suggesting that PMN-derived proteolytic enzymes are responsible for the breakdown. This process probably plays a role in dust-induced emphysema.

Animals↗
PubMed1996

Relationships between type A behavior, narcissism, and maternal closeness for college students in Japan, the United States of America, and the People's Republic of China.

The authors examined the relationships between Type A behavior and narcissism based on scores of college students in Japan, the United States of America, and the People's Republic of China. The scores on narcissism and Type A behavior differed significantly across the groups, being highest among the Chinese. In all three groups, the Type A scores were significantly and positively correlated with the scores on narcissism, and the latter were significantly and negatively correlated with the scores of mother's care. We refer in this study to cross-cultural comparisons from viewpoints of sociocultural and psychological family structure.

Adolescent↗