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Biomedical subjects

H Kong

Publications and source records attributed to H Kong.

At least 37 records · Page 2Linked to original sources
PubMed1999

A comparison of the cytoplasmic domains of the Fas receptor and the p75 neurotrophin receptor.

The p75 neurotrophic receptor (p75) shares structural features with the Fas receptor (FasR). Both receptors contain extracellular cysteine-rich repeats, a single transmembrane domain, and intracellular death domains. However, it has not been clearly established whether their death domains are equivalent in their ability to mediate apoptosis. To understand better the role of p75 during apoptosis, we constructed chimeric receptors that contained the extracellular portion of the FasR and the intracellular portion of p75. These chimeric receptors, one containing the p75 transmembrane domain and the other containing the FasR transmembrane portion, as well as wild-type p75 and Fas receptors, were transiently transfected into human U373 glioma cells and human embryonic kidney 293 cells (293 cells), which are both responsive to Fas-mediated apoptosis. Whereas expression of FasR was sufficient to induce apoptosis in U373 and 293 cells, expression of p75 and the chimeric receptors induced only minimal levels of cell death compared to FasR. The results indicate that the magnitudes of FasR- and p75-induced killing are different and suggest that the death domain of p75 does not function in the same manner as the FasR death domain.

Animals↗
PubMed1999

Intercellular adhesion molecule-1 and accumulation of eosinophils in nasal polyp tissue.

OBJECTIVE: To investigate the relationship between intercellular adhesion molecule-1 (ICAM-1) and the accumulation of eosinophils in nasal polyp tissue to better understand the mechanism of airway eosinophilic inflammation. METHODS: The expression of ICAM-1 and its natural ligand, lymphocyte function-associated antigen-1 (LFA-1), in normal nasal mucosa from 6 controls and in nasal polyp tissue from 19 patients with nasal polyposis were determined with immunohistochemistry. With dual immunohistochemistry and May-Griünwald Giemsa stain (MGG), the expression of LFA-1 and infiltrating eosinophils in nasal polyp tissue was observed. RESULTS: The expression of ICAM-1 and LFA-1 was stronger in the nasal polyp tissue than in normal nasal mucosa. There was a positive relationship between the infiltration of eosinophils and the expression of LFA-1 on eosinophils. CONCLUSION: Accumulation of eosinophils in nasal polyp tissue is associated with the counter-effect between adhesion molecules and its ligand on eosinophils.

Adult↗
PubMed1999

[Cell infiltration in nasal polyps and it's pathological significance].

OBJECTIVE: To study the relation between inflammatory cells infiltration in nasal polyp tissues and it's pathogenesis. METHOD: Nasal mucosa from 10 normal individual and 4 patients of nasal polyps were studied with immunohistochemical technique and histopathologic examination. RESULT: The number of eosinophils, CD68 positive cells and monocytes was significantly higher (P < 0.05) in nasal polyps tissues than in the normal controls. CONCLUSION: Infiltration of inflammatory effector cells such as eosinphils and chronic inflammatory changes of the nasal mucosa might play a role in nasal polyps sprouting.

Antigens, CD↗
PubMed1998

Analyzing the functional organization of a novel restriction modification system, the BcgI system.

BcgI is a novel, multi-subunit, restriction-modification (R-M) system that differs from all the other types of R-M system in its genetic and functional organization. The holoenzyme contains two different subunits, BcgI A and BcgI B. Both are required for endonuclease and methyltransferase activities. Here, we show that the endonuclease activity is mediated by the N-terminal portion of the A subunit. We made this assignment by mutational analysis. The analytic strategy involved three steps. First, the methyltransferase activity was inactivated by site-directed mutagenesis of a conserved methyltransferase motif also found in the A subunit. One of the R+M- mutants could not methylate DNA but was still able to cleave it, therefore expression of this mutant gene was lethal to the host. This lethal phenotype allowed the selective isolation of cleavage-deficient (R-) mutations in a second round of random mutagenesis in this mutant background. The R- mutations were all located in the N-terminal portion of the A subunit. There are five potential endonuclease motifs within this region. Conserved acidic residues in each of these motifs were substituted with alanine by site-directed mutagenesis of the wild-type A gene. The results identified one motif, P52E53-(X)12-E66D67K68, as the probable endonuclease active-site. Further support for this assignment was obtained by another round of site-directed mutagenesis directed to residues surrounding this motif. The results showed that DNA cleavage activity was mediated by the predicted, conserved residues, and not any of the surrounding non-conserved residues. One mutant protein, BcgI-E53A, with a single amino acid substitution decreased the DNA cleavage activity at least 700-fold. Our present model for the functional organization of BcgI locates both endonuclease and methyltransferase domains in the A subunit, with the target recognition domain located in the B subunit.

Amino Acid Sequence↗
PubMed1998

Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and mRNA in extrahepatic tissues of largemouth bass (Micropterus salmoides).

Low levels of all of the enzymes required for urea synthesis via the urea cycle, including mitochondrial glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase III (CPSase III) and cytosolic glutamine synthetase, are known to be present in liver of the teleost fish largemouth bass (Micropterus salmoides). The levels of these enzymes are higher than those in most other teleosts, but they are significantly lower than the levels present in liver of ureoosmotic elasmobranchs. The purpose of this study was to assess the physiological role of CPSase III in the context of urea synthesis in adult bass. The results showed that urea-N accounts for about 30% of the total nitrogen (ammonia-N plus urea-N) excreted under control conditions. The rate of urea-N excretion did not increase in response to exposure to 1 mM NH4Cl (3 days) or 0.25 mM NH4Cl (12 days) in the external water, except for a transient increase after a day or two of exposure. CPSase III activity in liver also did not increase in response to exposure to ammonia. Adult largemouth bass, while apparently ureogenic, are primarily ammonotelic and remain so even in the presence of relatively high concentrations of ammonia in the external environment. The total units of CPSase III activity in liver are not sufficient to account for the quantity of urea that is excreted. However, CPSase III and ornithine carbamoyltransferase (OCTase) activities were found to be present in intestinal tissue and, unexpectedly, in muscle tissue. The total units of CPSase III and OCTase in muscle, intestine, and liver appear to be sufficient to account for the observed rate of urea excretion. The sequence of CPSase III cDNA was determined, which permitted the use of ribonuclease protection assays to demonstrate the presence of CPSase III mRNA in these tissues.

Amino Acid Sequence↗
PubMed1998

Neurotrophin receptors: mediators of life and death.

The mechanism of action of NGF has continued to provide a challenging and formidable problem in signal transduction. NGF can bind independently to two different receptors, the trkA tyrosine kinase receptor and the p75 neurotrophin receptor, which are involved in many different signaling events. In addition to promoting cell differentiation survival, NGF can paradoxically be an inducer of cell death. Several receptor mediated mechanisms are proposed to explain how NGF might act as a trophic factor and as a cell killer. The survival and cell death properties of the receptors are dependent upon the relative ratio of receptors and the persistent nature of the signaling events.

Animals↗
PubMed1998

Neurotrophins: the biological paradox of survival factors eliciting apoptosis.

Neurotrophins are target-derived soluble polypeptides required for neuronal survival. Binding of neurotrophins to Trk receptor tyrosine kinases initiate signaling cascades that promote cell survival and differentiation. All family members bind to another receptor (p75NTR), which belongs to the tumor necrosis factor superfamily. Hence, nerve growth factor (NGF) and related trophic factors are unique in that two separate receptor types are utilized. Although the biological function of p75NTR has been elusive, it has been suggested to mediate apoptosis of developing neurons in the absence of Trk receptors. This presents a tantalizing paradigm, in which life-death decisions of cells are dependent upon the expression and action of two different receptors with distinctive signaling mechanisms. In the presence of TrkA receptors, p75 can participate in the formation of high affinity binding sites and enhanced NGF responsiveness leading to a survival signal. In the absence of TrkA receptors, p75 can generate, in only specific cell populations, a death signal. Here we discuss the unique features and implications of this unusual signal transduction system.

Animals↗
PubMed1998

[Expression and significance of vascular permeability factor in nasal polyps].

OBJECTIVE: To study the expression and significance of vascular permeability factor(VPF) in nasal polyps. METHODS: Expression of VPF and its receptor flk-1 in nasal polyps from 9 patients and inferior turbinates from 8 patients with chronic rhinitis were prospectively studied with immunohistochemistry. Each tissue section was observed under optical microscope. RESULTS: The VPF positivity in vascular endothelium and in gland cell was significantly higher in nasal polyps than in inferior turbinates (P < 0.01 and P < 0.05, respectively). The flk-1 labeling index of vascular endothelium was significantly higher in nasal polyps than that in inferior turbinates (P < 0.01). CONCLUSION: VPF may play a key role in the formation of heavy edema of nasal polyps.

Adult↗
PubMed1998

[Expression of intercellular adhesion molecules in nasal polyps tissue and its significance].

OBJECTIVE: To study the expression of intercellular adhesion molecule-1/lymphocyte function-associated antigen-1 in nasal polyps tissue and its significance. METHODS: Frozen sections of 9 normal nasal mucosa and 19 nasal polyp samples were studied with immunohistochemical technique and immunohistochemical and MGG dual staining method with ICAM-1 and LFA-1 monoclone antibodies. RESULTS: Expression of ICAM-1 and LFA-1 were stronger in nasal polyps tissue than in the normal nasal mucosa, and there is positive relationship between the expression of ICAM-1 and the expression of LFA-1 on eosinophils. CONCLUSION: High expression of ICAM-1 and LFA-1 in nasal polyps tissue showed that interaction of ICAM-1/LFA-1 may participate in the inflammatory reaction and prompt adhesion and migration of eosinophils. High expression of LFA-1+ on eosinophils may play an important role in the pathogens of nasal polyps.

Adult↗
PubMed1997

Substrate DNA and cofactor regulate the activities of a multi-functional restriction-modification enzyme, BcgI.

The BcgI restriction-modification system consists of two subunits, A and B. It is a bifunctional protein complex which can cleave or methylate DNA. The regulation of these competing activities is determined by the DNA substrates and cofactors. BcgI is an active endonuclease and a poor methyltransferase on unmodified DNA substrates. In contrast, BcgI is an active methyltransferase and an inactive endonuclease on hemimethylated DNA substrates. The cleavage and methylation reactions share cofactors. While BcgI requires Mg2+and S -adenosyl methionine (AdoMet) for DNA cleavage, its methylation reaction requires only AdoMet and yet is significantly stimulated by Mg2+. Site-directed mutagenesis was carried out to investigate the relationship between AdoMet binding and BcgI DNA cleavage/methylation activities. Most substitutions of conserved residues forming the AdoMet binding pocket in the A subunit abolished both methylation and cleavage activities, indicating that AdoMet binding is an early common step required for both cleavage and methylation. However, one mutation (Y439A) abolished only the methylation activity, not the DNA cleavage activity. This mutant protein was purified and its methylation, cleavage and AdoMet binding activities were tested in vitro . BcgI-Y439A had no detectable methylation activity, but it retained 40% of the AdoMet binding and DNA cleavage activities.

Base Sequence↗
PubMed1997

[Ultrastructural study of tumor infiltrating lymphocyte in tissues of laryngeal squamous cell carcinoma].

By transmission electron microscopy tumor infiltrating lymphocyte (TIL) in laryngeal squamous cell carcinoma tissues of 14 cases was studied. The results showed that the infiltrating lymphocyte in varying degrees eristed in laryngeal squamous cell carcinoma tissues. Most of them were present in a motionless state, and the others showed the feature of activated metrocyte and closely contracted with cancer cells and resulted in distinct morphological changes both the cancer cells and themselves. Therefore, this study provided an important morphological evidence for TIL as killer and inhibiter to the growth of cancer cells.

Carcinoma, Squamous Cell↗
PubMed1996

BaeI, another unusual BcgI-like restriction endonuclease.

BcgI and BcgI-like restriction endonucleases have a very distinct characteristic which causes them to differ from the other classified restriction enzymes; they all cleave double-stranded DNA specifically on both sides of the recognition sequence to excise a short DNA fragment including the recognition sites. Here we report a new BcgI-like restriction endonuclease, BaeI, isolated from Bacillus sphaericus. Like BcgI, BaeI also cleaves double-stranded DNA on both strands upstream and downstream of its recognition sequence (10/15)ACNNNNGTAYC(12/7). There are two dominant polypeptides in the final preparation of BaeI with molecular masses of approximately 80 and 55 kDa. Both are slightly larger than the two BcgI subunits. BaeI requires both Mg2+ and AdoMet to cleave DNA. Accompanying bilateral cleavage activity, the heteromeric BaeI also has an N6-adenine methyltransferase activity which modifies the symmetrically located adenines within its recognition sequence.

Bacillus↗
PubMed1996

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Amino Acid Sequence↗
PubMed1996

Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity.

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.

Amino Acid Sequence↗
PubMed1995

Interaction of somatostatin receptors with G proteins and cellular effector systems.

Somatostatin induces its multiple biological actions by interacting with a family of receptors, referred to as sstr1-sstr5. To determine the molecular mechanisms of action of somatostatin, we have investigated the interaction of the different cloned receptors with G proteins and cellular effector systems. sstr2, sstr3 and sstr5 associate with pertussis toxin-sensitive G proteins and are able to mediate the inhibition of adenylyl cyclase activity by somatostatin. Two forms of sstr2, sstr2A and sstr2B, are generated by alternative splicing and differ in their C-terminal amino acid sequence. sstr2B couples to adenylyl cyclase whereas sstr2A does not. To investigate the basis for the differential coupling to adenylyl cyclase, we truncated sstr2B to the point of amino acid sequence divergence from sstr2A. The truncated sstr2B mediated the inhibition of cAMP formation by somatostatin, indicating that the C-terminus is not needed for coupling sstr2 to adenylyl cyclase. It is likely that the C-terminus of sstr2A hinders coupling to adenylyl cyclase. sstr2A associates with Gi alpha 3 and G(o) alpha but does not effectively interact with Gi alpha 1, a G protein that is necessary for coupling somatostatin receptors to adenylyl cyclase. The differential association of the splice variants with Gi alpha 1 may explain their contrasting effects on adenylyl cyclase activity. sstr3 also couples to adenylyl cyclase. Gi alpha 1 links sstr3 to adenylyl cyclase and mutagenesis studies have shown that the C-terminus of Gi alpha 1 is necessary for this coupling. The C-terminus of the Gi alpha proteins differ by only a few amino acid residues and only Gi alpha 1 couples sstr3 to adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Molecular biology of somatostatin receptors.

The diverse physiological effects of somatostatin are mediated by a family of cell surface receptors that bind somatostatin selectively and with high affinity. The somatostatin receptors are members of the seven transmembrane segment receptor superfamily and molecular cloning studies have identified five types, designated sstr1-5. The human somatostatin receptors vary in size from 364 (sstr5) to 418 (sstr3) amino acids with 46-61% amino acid identity between receptors, and 105 amino acids are invariant. The sequences of the seven putative alpha-helical membrane-spanning domains are more highly conserved than those of the extracellular N- and intracellular C-terminal domains. Two forms of sstr2 have been identified in the mouse, sstr2A and sstr2B, which differ in size and sequence of the intracellular C-terminal domain. These two forms of sstr2 are products of a common gene and are generated by alternative splicing with sstr2A and sstr2B being the products of the unspliced and spliced forms, respectively, of sstr2 mRNA. Thus, functional diversity within the somatostatin receptor family may result from the expression of multiple types as well as from alternative splicing. The five somatostatin receptors have distinct patterns of expression in the central nervous system and peripheral tissues. They have also been expressed in vitro and shown to have different pharmacological properties. Somatostatin analogues selective for sstr2, sstr3 and sstr5 have been identified which will facilitate in vivo studies of the functions of these somatostatin receptors. Such studies to date suggest that sstr2 mediates inhibition of growth hormone secretion and sstr5 mediates inhibition of insulin secretion. The molecular cloning and functional characterization of the somatostatin receptor family is a first step in elucidating the diverse effects of somatostatin on cellular functions.

Amino Acid Sequence↗