Biomedical subjects

I Lee

Publications and source records attributed to I Lee.

At least 55 records · Page 3Linked to original sources

PubMed2000

Human Ku70 interacts with heterochromatin protein 1alpha.

Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres. It consists of a heterodimer of 70- and 80-kDa subunits. Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein. Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin. HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells. The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro. The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha. This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5. Ku80 did not interact with HP1alpha directly. The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively. Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C. In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells. The implication of this interaction is discussed.

Antigens, Nuclear↗

PubMed2000

Two closely related cDNAs encoding actin-depolymerizing factors of petunia are mainly expressed in vegetative tissues.

Actin-depolymerizing factor (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. We have isolated two cDNA clones, PhADF1 and PhADF2, encoding ADF from cDNA libraries constructed from petal protoplast cultures and flowers of Petunia hybrida. PhADF1 and PhADF2 encode polypeptides of 139 and 143 amino acids with a calculated molecular mass of 16.04 and 16.51kDa, respectively. Co-sedimentation assay showed that the recombinant PhADF1 protein produced in Escherichia coli binds to F-actin at pH7. 0 and preferentially depolymerizes it at pH8.0. Gene tree analysis indicates that the plant ADF family can be grouped into four classes, and PhADFs are included in class I. Southern blot analyses revealed that one or two copies of PhADF genes are present in petunia genome, and several other related isoforms also exist. Northern blot analyses indicated that PhADF1 and PhADF2 are closely related and abundantly expressed in every plant organ except pollen. In addition, they are highly accumulated in mature vegetative tissue (petal, leaf, and stem). Our results indicate that the transcription of petunia ADF genes is differentially regulated by developmental signals.

Actin Depolymerizing Factors↗

PubMed2000

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region.

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.

Amino Acid Sequence↗

PubMed2000

Diagnosis and management of infections involving implantable electrophysiologic cardiac devices.

BACKGROUND: Optimal treatment of infections related to implantable electrophysiologic cardiac devices is poorly defined. OBJECTIVE: To describe the clinical presentation, treatment, and outcome of patients with such infections. DESIGN: Retrospective case series. SETTING: The Cleveland Clinic Foundation, Cleveland, Ohio. PATIENTS: 123 patients with infections involving implantable cardiac electrophysiologic devices. MEASUREMENTS: Demographic characteristics, clinical manifestations, time to diagnosis, management, and outcome. RESULTS: 87 patients with permanent pacemakers and 36 patients with implantable cardioverter defibrillators had infections. The most common signs and symptoms were pocket erythema and local pain. The most common pathogens were coagulase-negative staphylococci (68%) and Staphylococcus aureus (23%). In 117 patients (95%), all equipment was extracted and antibiotic therapy lasted a median of 28 days. Operative mortality was zero. Follow-up showed crude mortality and relapse rates of 8% and 3%, respectively. CONCLUSION: For infections related to implantable electrophysiologic devices, complete device removal and antimicrobial therapy allow timely, successful reimplantation at a remote anatomic site without substantial risk for operative mortality or recurrent infection.

Adolescent↗

PubMed2000

The AGAMOUS-LIKE 20 MADS domain protein integrates floral inductive pathways in Arabidopsis.

The very late-flowering behavior of Arabidopsis winter-annual ecotypes is conferred mainly by two genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). A MADS-domain gene, AGAMOUS-LIKE 20 (AGL20), was identified as a dominant FRI suppressor in activation tagging mutagenesis. Overexpression of AGL20 suppresses not only the late flowering of plants that have functional FRI and FLC alleles but also the delayed phase transitions during the vegetative stages of plant development. Interestingly, AGL20 expression is positively regulated not only by the redundant vernalization and autonomous pathways of flowering but also by the photoperiod pathway. Our results indicate that AGL20 is an important integrator of three pathways controlling flowering in Arabidopsis.

AGAMOUS Protein, Arabidopsis↗

PubMed2000

Interaction of human Ku70 with TRF2.

Ku, a heterodimer of 70- and 80-kDa subunits, plays a general role in the metabolism of DNA ends in eukaryotic cells, including double-strand DNA break repair, V(D)J recombination, and maintenance of telomeres. We have utilized the yeast two-hybrid system to identify Ku70-interacting proteins other than Ku80. Two reactive clones were found to encode the dimerization domain of TRF2, a mammalian telomeric protein that binds to duplex TTAGGG repeats at chromosome ends. This interaction was confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells overexpressing TRF2. The transfected TFR2 colocalized with Ku70.

Animals↗

PubMed2000

Cloning, characterization and chromosomal assignment of NBC4, a new member of the sodium bicarbonate cotransporter family.

We report the cloning, characterization and chromosomal assignment of a new member of the sodium bicarbonate cotransporter (NBC) family, NBC4, from human heart. NBC4 maps to chromosome 2p13 and is a new candidate gene for Alstrom syndrome. NBC4 encodes a 1074-residue polypeptide with 12 putative membrane-spanning domains. Unlike other members of the NBC family, NBC4 has a unique glycine-rich region (amino acids 438-485). In addition, NBC4 lacks the lysine-rich C-terminus of NBC1 with which it is most homologous. The first of two putative stilbene binding motifs (K(M/L)(X)K) is lacking in NBC4 (amino acids 655-658). The approximately 6 kb NBC4 transcript is moderately expressed in heart, with the highest expression in liver, testes and spleen.

Amino Acid Sequence↗

PubMed2000

Kinetics and mechanism of addition of benzylamines to benzylidene-1,3-indandiones in acetonitrile

Nucleophilic addition reactions of benzylamines (BA; XC6H4CH2NH2) to benzylidene-1,3-indandiones (BID; YC6H4CH=C(C=O)2C6H4) have been studied in acetonitrile at 25.0 degrees C. The rate is first-order with respect to BA and BID, and no base catalysis is observed. The structure-reactivity behaviors (k2, rhoX, betaX, and betaY) are intermediate between the two series of addition reactions of BA to beta-nitrostyrene (NS) and benzylidenemalononitrile (BMN) in acetonitrile. The normal kinetic isotope effects, kH/kD > 1.0, involving deuterated BAs (XC6H4CH2ND2) are smaller than those for the reactions of NS and BMN suggesting a somewhat looser bond formation in the transition state. The reaction is predicted to proceed in a single step with concurrent C(alpha)-N bond formation and proton transfer to C(beta). A hydrogen-bonded, four-center type cyclic transition state is proposed.

Journal Article↗

PubMed2000

Kinetics and mechanism of the pyridinolysis of phenacyl bromides in acetonitrile

Kinetic studies of the reactions of substituted phenacyl bromides (YC6H4COCH2Br) with pyridines (XC5H4N) are carried out in acetonitrile at 45.0 degrees C. A biphasic Bronsted plot is obtained with a change in slope from a large (betaX approximately equals 0.65-0.80) to a small (betaX approximately 0.36-0.40) value at pKa = 3.2-3.6, which can be attributed to a change in the rate-determining step from breakdown to formation of a tetrahedral intermediate in the reaction path as the basicity of the pyridine nucleophile increases. This mechanism is supported by the faster rates with pyridines than with anilines and the change of cross-interaction constant rhoXY from a large positive (rhoXY = +1.4) to a small positive (rhoXY approximately +0.1) value. The large magnitude of Hammett rhoX (= -5.5 to -6.9) values for the pyridines with electron-withdrawing substituents and positive deviations of the pi-acceptors, p-CH3CO and p-CN, are quite similar to those for the pyridinium ion formation equilibria. The activation parameters are also in line with the proposed mechanism.

Journal Article↗

PubMed2000

Theoretical studies of hetero-diels-alder reactions involving N-sulfinyl dienophiles

The gas-phase hetero-Diels-Alder reactions between butadiene and X-substituted sulfinyl dienophiles, O(-)-S(+)=N-X, are investigated theoretically at the B3LYP/6-31G level. The Z-forms of the dienophiles are found to be more stable (by 5-7 kcal mol(-)(1)) than the E-forms. Four modes of cycloadducts are considered: Z-endo; Z-exo; E(X)(-)(endo)(); E(X)(-)(exo)(). Five factors are responsible for the decreasing energetic preferences of the adducts in the order E(X)(-)(endo)() > E(X)(-)(exo)() > Z-endo > Z-exo: (i) The sigma-sigma proximate charge-transfer interactions in the TS; (ii) the relative sizes of the LUMO AO coefficients on S and N atoms; (iii) steric hindrance in the TS; (iv) the levels of the ground state and the LUMOs of the dienophile; (v) bond energies of the C-S and C-N bonds that are formed in the TS. All the reactions proceed concertedly, but the adduct formation is asynchronous. The endo-additions are favored over the exo-additions kinetically (lower DeltaG()) as well as thermodynamically (lower DeltaG degrees ). The major secondary orbital interaction determining the endo preference is that between the lone pair on N (n(N)) and the d(3) (C(3)-C(4)) sigma orbital (n(N)-sigma(d3)) interactions, whereas the larger AO lobe (LUMO) sizes on S favor a greater degree of d(5) (C-S) bond formation than d(6) (C-N) bond. The solvent, C(6)H(6), uniformly lowers the activation barriers so that the energetic preferences in the gas phase between various modes are maintained in solution.

Journal Article↗

PubMed2000

Structural organization of the human NBC1 gene: kNBC1 is transcribed from an alternative promoter in intron 3.

Several electrogenic sodium bicarbonate cotransporters have been cloned from different human organs. In the renal proximal tubule, the electrogenic sodium bicarbonate cotransporter kNBC1 (1035aa) mediates the majority of basolateral sodium bicarbonate absorption. In pancreatic ducts, the electrogenic sodium bicarbonate cotransporter pNBC1 (1079aa) mediates basolateral sodium bicarbonate influx. hNBC1 (hhNBC), cloned from human heart, is identical to pNBC1 at the amino acid level. We have demonstrated that kNBC1 and pNBC1 are highly homologous proteins that have different N-termini. In kNBC1, 41 amino acids replace the initial 85 amino acids of pNBC1. Whether these proteins are coded by one or more genes is unknown. In order to determine the genetic basis for these transcripts, we first characterized the genomic organization of the NBC1 gene (SLC4A4). NBC1 spans approximately 450 kilobases containing 26 exons that are flanked by typical splice donor and acceptor sequences at the intron-exon boundaries. Exon 1 is specific for the pNBC1 transcript. The first alternative exon of the hNBC1 transcript, containing the 5'-untranslated region, is derived from the last 43 nucleotides of intron 1 in the NBC1 gene coupled to exon 2. kNBC1 is transcribed from an alternative promoter in intron 3. In the first alternative exon of kNBC1, the last 313 nucleotides of intron 3 are coupled to exon 4, which is common to pNBC1 and hNBC1. The major transcription initiation site in kNBC1 is located 192 nucleotides upstream from the translation initiation codon. A minor start site is located 182 nucleotides upstream from the translation initiation codon. Structural analysis of the proximal kNBC1 promoter revealed an atypical TATA sequence (-33) and several potentially important transcription factor binding sites. Functional studies showed that the 5'-flanking region of the alternative kNBC1 promoter (-159 to+43) is sufficient for promoter activity. This work is the first demonstration that the three N-terminal transcripts of the human electrogenic sodium bicarbonate cotransporter NBC1 are encoded by the SLC4A4 gene. Furthermore, knowledge of the genomic organization and alternative promoter usage in the NBC1 gene provides a molecular basis for understanding disorders involving electrogenic sodium bicarbonate cotransporters and facilitates the elucidation of transcriptional control of NBC1 expression.

Amino Acid Sequence↗

PubMed2000

Kinetics and mechanism of the addition of benzylamines to benzylidenemalononitriles in acetonitrile

Nucleophilic addition reactions of benzylamines (BA) to benzylidenemalononitrile (BMN) have been studied in acetonitrile at 15.0 degrees C. The rate is first-order with respect to both BA and BMN and no base catalysis is observed. The rate decreases as the electron-withdrawing power of the substituent (Y) in the substrate increases (rho Y < 0). This is in contrast to the similar reactions of beta-nitrostyrenes (NS) with BAs in acetonitrile and the addition reactions of NS and BMN in aqueous solution (rho Y > 0). This sign change of rho Y is considered to result from the strong electron-withdrawing power of the (CN)2 group in BMN, which leads to polarization of C alpha delta+C delta- (CN)2 in the transition state. The mechanism of amine addition to BMN in acetonitrile is radically different from that in water. The reaction is predicted to proceed concertedly in a single step with a hydrogen-bonded, four-center cyclic transition state.

Journal Article↗

PubMed2000

Type of feeding during infancy and later development of schizophrenia.

Nutrition may be critical for neurodevelopment and can affect the later development of schizophrenia. Recently, a marked reduction in breast-feeding was reported in infants that developed schizophrenia in later life (McCreadie, R.G., 1997. The Nithsdale Schizophrenia Surveys. 16. Breast-feeding and schizophrenia: preliminary results and hypothesis. Br. J. Psychiatr. 170, 334-337). In the present study, we investigated feeding patterns during the infancy of 100 schizophrenia patients, 37 of their siblings and 200 age-matched healthy controls using a structured written questionnaire. Having been breast-fed was not negatively associated with schizophrenia.

Adult↗

PubMed2000

Nuclear matrix of calreticulin in hepatocellular carcinoma.

Nuclear matrix protein profiles of malignant cells vary from their normal counterparts. By two-dimensional gel electrophoresis, we analyzed nuclear matrix proteins in 11 hepatocellular carcinomas and compared them with corresponding non-neoplastic liver tissue. Although the compositions were mostly similar, several peptides were noted predominantly in the former. The most prominent one was an acidic protein of apparent Mr 62,000, which was identified to be calreticulin upon NH2-terminal amino acid sequencing. By immunoblotting, calreticulin was confirmed to be present abundantly in the nuclear matrix fraction of carcinomas but not in that of the nonmalignant liver tissue. Interestingly, the total content of calreticulin was similar between them. By immunofluorescence microscopy, evident nuclear immunostaining was detected in carcinomas. Calreticulin was also found to be in the nuclear matrices of various carcinoma cell lines. We conclude that calreticulin is a component of the nuclear matrix. The formation and/or expansion of the calreticulin-nuclear matrix may be related to the activated cell growth.

Adult↗

PubMed2000

Pyridinolysis of phenyl-substituted phenyl chlorophosphates in acetonitrile

The kinetics and mechanism of the reactions of phenyl-substituted phenyl chlorophosphates, I, with pyridines in acetonitrile are investigated at 25.0 degrees C. The rates are much faster and the transition state is much earlier on the reaction coordinate with smaller beta(X) (0.16-0.18) and smaller negative beta(XY) (-0.011) values than those for the corresponding reactions with anilines. The vertical approach of the pyridine ring to an apical position of the trigonal bipyramidal transition state enables the p(pi)-d(pi) overlap between the pi orbital of pyridine and an empty d-orbital of phosphorus with less steric hindrance in a concerted process. The activation parameters are in line with the proposed mechanism.

Journal Article↗

PubMed2000

Tumoricidal effects of onconase on various tumors.

BACKGROUND AND OBJECTIVES: The effects of Onconase (Onc) on the tumor growth in vitro and in vivo were examined. Because elevated tumor interstitial fluid pressure (TIFP) is one of the major causes of inadequate drug delivery into solid tumors, we tested if Onc could lower TIFP in solid tumors. METHODS: We used several assays including a clonogenic assay and a growth delay assay for the determination of anti-tumoricidal effects of Onc. We also measured Onc-induced changes in several tumor physiological parameters. RESULTS: Onc demonstrated cytotoxic effects in all eight exponentially growing cell lines in vitro. It effectively inhibited the growth of all four transplanted tumors in vivo, and significantly reduced TIFP in all four tumors. Onc also induced increases in tumor blood flow (TBF) as well as increases in median tumor oxygen partial pressure (pO(2)) in solid tumors. CONCLUSIONS: Onc showed anti-tumoral effects on various tumor cells in vitro as well as in vivo. We also gained some insight regarding the potential physiological benefit of Onc as a new therapeutic agent in cancer treatment. Due to increases in both TBF and tumor pO(2), Onc could be a potential candidate as a novel radiation enhancer; therefore, the study of the radiation response in vivo is warranted.

Animals↗

PubMed2000

The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy.

Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented.

Cellulase↗

PubMed2000

"Malgun" (clear) cell change of gastric epithelium in chronic Helicobacter pylori gastritis.

To characterize the Helicobacter pylori gastritis-associated epithelial change, we analyzed 251 randomly selected gastric biopsies. The "malgun" (clear) cell change of the gastric epithelium was noted in 229 biopsies (91.2%). Malgun cells were characterized by large, pale nuclei with a euchromatin pattern, enlarged nucleoli, and clear cytoplasm. In the proliferative zone, individual malgun cells and small clusters were often in close contact with infiltrating neutrophils, suggesting that they had developed individually in the background of acute foveolitis. Mitotic figures of malgun cells were not infrequent, including atypical ones. In the surface epithelium, most malgun cells were in clusters that were often large enough to occupy wide epithelial segments. With Warthin-Starry triple staining, they were distinguished by the absence of silver impregnation, while other cells showed staining of the heterochromatin. They displayed prominent immunostaining for low molecular weight cytokeratin (No. 8). Most malgun cells were PCNA-positive in both surface and proliferative zones, whereas Ki67-positive cells were found only in the proliferative zone. It was suggested that a population of malgun cells, which were positive for PCNA only, were in the process of active DNA repair. The malgun cell change may represent a "cellular pattern of activation" in a population which had significant DNA damage, but somehow escaped the detection by the apoptosis system. The notion of "damage at the genetic level" was supported by the observation that these cells remained at least for 8 weeks after eradication of the H. pylori infection.

Adult↗