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J Lin

Publications and source records attributed to J Lin.

At least 181 records · Page 10Linked to original sources
PubMed1996

The role of Glu 57 in the mechanism of the Escherichia coli MutT enzyme by mutagenesis and heteronuclear NMR.

The role of the conserved residue Glu-57 in the mechanism of the MutT enzyme from Escherichia coli was investigated by mutagenesis and heteronuclear NMR methods. The enzymatic activity of the E57Q mutant is at least 10(5)-fold lower than that of the wild type enzyme. The solution structure of the E57Q mutant, based on comparisons of 1H-15N NOESY HSQC spectra and 1H-15N HSQC spectra to those of the wild type enzyme, differs in a region near Glu-57. The dissociation constants (KD) of the E-Mg2+ and E-Mn2+ complexes increased 3.3- and 3.6-fold, respectively, in the E57Q mutant, while the KD of E-dGTP is unaltered from that of the wild type enzyme. The enhanced paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons is halved in the E57Q mutant indicating an altered metal-binding site. 1H-15N HSQC titrations of E57Q with MnCl2 show selective attenuation of the side chain NH signals of Gln-57 and the backbone NH signals of Gly-37, Gly-38, Lys-39, Glu-53, Glu-56, Gln-57, and Glu-98, indicating proximity of bound Nm2+ to these residues. The same resonances of the wild type and the E57Q mutant enzymes are attenuated by Mn2+, but significantly smaller paramagnetic effects (relative to the largest effect on Lys-39) are found on Gly-37, Gly-38, Val-58, and Glu-98 of the mutant, indicating an altered position of the bound divalent cation. Thus Glu-57 is probably a ligand to the enzyme-bound metal, and the profound loss of catalytic activity in the E57Q mutant results from structural and electronic changes at the site of the enzyme-bound divalent cation. 1H-15N HSQC titrations of the wild type enzyme with MgCl2 show changes in chemical shifts of 15N and NH resonances in regions closely overlapping those induced by the E57Q mutation itself, suggesting that the loss of the negative charge at Glu-57, either by mutation or by neutralization with Mg2+, induces a similar effect. In the E57Q mutant, the slow exchange of the side chain NH2 protons of Gln-57 and NOE's from the NH2 protons of Gln-57 to the beta and gamma protons of Glu-98 suggests hydrogen bonding of Gln-57 to Glu-98 in the free enzyme. 1H-15N HSQC titrations of both the wild type and mutant enzymes with dGTP show changes in 15N and NH chemicals shifts of residues in a cleft formed by beta-strands A, C, and D on one side and loop I, the end of loop IV, and the beginning of helix II on the other side, suggesting this cleft to be the nucleotide binding site. These changes in chemical shift were smaller or absent in titrations of the wild type or mutant enzymes with AMPCPP or Mg2+-AMPCPP, in accord with the strong preference of the MutT enzyme for guanine over adenine nucleotide substrates.

Amino Acid Sequence↗
PubMed1996

The cotranslational integration of membrane proteins into the phospholipid bilayer is a multistep process.

During the cotranslational integration of a nascent protein into the endoplasmic reticulum membrane, the transmembrane (TM) sequence moves out of an aqueous pore formed by Sec61alpha, TRAM, and other proteins and into the nonpolar lipid bilayer. Photocross-linking reveals that this movement involves the sequential passage of the TM domain through three different proteinaceous environments: one adjacent to Sec61alpha and TRAM and two adjacent to TRAM that place different restrictions on TM domain movement. In addition, the TM sequence is not allowed to diffuse into the bilayer from the final TRAM-proximal site until translation terminates. Cotranslational integration is therefore linked to translation and occurs via an ordered multistep pathway at an endoplasmic reticulum site that is multilayered both structurally and functionally.

Cross-Linking Reagents↗
PubMed1996

Expression of Rab3D in dispersed chief cells from guinea pig stomach.

Rab3 proteins are low molecular weight GTP-binding proteins that are expressed in neurons and other secretory cells. These proteins are localized to secretory vesicles and may play a role in regulated exocytosis. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have been identified. We examined the expression of Rab3 isoforms in dispersed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cytosolic and membrane fractions, but staining was more intense in membrane fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To determine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obtained by reverse transcription and subjected to PCR using degenerate primers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced amino acid sequence was identical to that of mouse Rab3D (amino acids 16-83). Moreover, Rab3D was the only isoform detected in chief cells by these methods. To identify rab3 transcripts, the guinea pig rab3D fragment obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in chief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These results indicate that Rab3D is expressed in gastric chief cells.

Amino Acid Sequence↗
PubMed1996

cDNA sequence analysis and expression of cardiotoxins from Taiwan Cobra.

The cDNAs encoding cardiotoxins I, III and N were constructed from the cellular RNA isolated from the venom glands of Naja naja atra by reverse transcription-polymerase chain reaction. A high degree of sequence homology was observed with the three cardiotoxins. The cardiotoxins were subcloned in the expression vector pET 20b(+) and transformed in BL 21(DE3) E. coli strain. The expressed protein was isolated from the inclusion bodies of E. coli and purified by reversed phase high performance liquid chromatography. The purified recombinant cardiotoxin III showed an immunoreactivity with anti-cardiotoxin III antibodies as revealed by immunoblot analysis.

Amino Acid Sequence↗
PubMed1996

Anti-CD2 monoclonal antibody-induced receptor changes. II. Interaction of CD2 and CD3.

Anti-CD3 monoclonal antibodies (mAbs) and anti-CD2 mAbs each prolong allograft survival and cause transient downmodulation of homologous receptor expression. Anti-CD2 mAbs also act synergistically with anti-CD3 mAbs to prolong allograft survival and induce tolerance. The effect of combined anti-CD2 and anti-CD3 mAb treatment on receptor expression was further analyzed with an in vitro model. The anti-CD2 mAb 12-15 caused CD2 expression on purified splenic T cells to decrease from 72.6% [mean channel fluorescence (MCF) 0.68] to 41.5% (0.45) total positive cells while CD3 expression remained unchanged [69.1% (3.47) to 76.4% (4.04)]. The anti-CD3 mAb 2C11 caused CD2 expression to increase from 72.6% (0.68) to 93.0% (1.74) while CD3 expression decreased from 69.1% (3.47) to 62.6% (2.15). The combination of anti-CD2 plus anti-CD3 preserved CD2 expression (72.6 to 71.1%) while still decreasing CD3 expression [69.1% (3.47) to 69.9% (2.37)]. Modulation of CD2 and CD3 expression was similar on mixed splenic T lymphocytes and isolated CD4 and CD8 subsets. Modulation did not change with the addition of the cytokines IL-1, IL-2, IL-4, IL-6, IL-10, TNF alpha, or TGF beta. Kinetic studies showed that modulation of CD2 was rapid, persistent, and of the same magnitude from Day 1 to Day 7 of culture while CD3 downmodulation was transient. The results of transcriptional analysis and receptor distribution suggested that downmodulation was due to receptor internalization while upmodulation was due to increased transcription. Analysis of expression of other adhesion molecules demonstrated that CD11a, CD18, CD44, CD45, CD48, CD54, and CD62L were significantly increased by either anti-CD2 or anti-CD3 mAbs while the combination was not synergistic. However, anti-CD3 significantly decreased VLA-4 alpha (CD49d) expression and anti-CD2 enhanced this decrease. Conversely anti-CD3 significantly increased IL-2R (CD25) expression and anti-CD2 profoundly inhibited the increase. These results show that anti-CD2 and anti-CD3 mAbs significantly modulate CD2 and CD3 expression on T cells and modulation is accompanied by changes in the array of other T cell surface receptors. Changes in cell surface receptor display may provide an additional explanation for the synergistic effect of anti-CD2 plus anti-CD3 in prolonging allograft survival.

Animals↗
PubMed1996

Human pharmacokinetics and tolerability of L-365,260, a novel cholecystokinin-B antagonist.

A study was conducted to examine the tolerability and pharmacokinetics of single and multiple oral doses of L-365,260, a novel antagonist for type B cholecystokinin (CCK) receptors and to quantify effects of selective blockade of type B CCK receptors through treatment with L-365,260 on measures of anxiety, hunger, and cognitive performance. Healthy volunteers were given single oral doses of up to 50 mg of L-365,260 and multiple oral doses of up to 25 mg every 6 hours for 10 days. Plasma concentrations of L-365,260 were quantified by means of high-performance liquid chromatography. Anxiety and hunger were assessed by visual analog scale and the Spielberger State Anxiety Index. Cognitive testing was used to evaluate attention level and short-term memory. L-365,260 was rapidly absorbed and a biphasic pattern of elimination was demonstrated with a terminal half-life (t1/2) of 8 to 12 hours. The mean (n = 6) values for peak plasma concentration (C(max)) and time to peak concentration (t(max)) of L-365,260 were 503 ng/mL and 1.25 hours, respectively, after a single 50-mg oral dose. Accumulation of L-365,260 plasma concentrations was seen after the prescribed multiple-dose regimens. Steady state was achieved after 3 days of oral administration. L-365,260 had an acceptable tolerability profile after oral administration. No changes in measures of anxiety, hunger, or short-term memory were observed at doses of L-365,260 shown to have antagonist activity at the CCK-B receptor.

Administration, Oral↗
PubMed1996

Extraskeletal mesenchymal chondrosarcoma of the labium majus.

Extraskeletal mesenchymal chondrosarcoma is a rare tumor. It has been noted to occur most commonly in muscle and the central nervous system. It has never been reported to occur in the soft tissues of the anogenital region. We report the first such case, occurring in a 40-year-old woman who presented with a 1-year history of a mass originating in the left labium majus. After extensive local resection and lymph node dissection she remains disease free after 40 months follow-up.

Adult↗
PubMed1996

Breath-by-breath determinations of airway occlusion pressure in the developing lamb.

The ventilatory effects of breath-by-breath measurements of airway occlusion pressure, i.e., airway pressure determined 100 ms after initiation of inspiration (P0.1) were evaluated in seven lambs studied sequentially between 7 and 28 days after birth. P0.1 was determined by computer-aided, on-line regression analysis of the inspiratory pressure versus time (dP/dt) by means of a pneumatic occlusion valve that allowed occlusion times to vary in proportion to respiratory rate. No significant changes were found in minute ventilation, tidal volume, respiratory rate or end-tidal CO2 concentration when the valve was operating as a oneway valve (opening pressure 0.02 kPa or 0.2 cmH2O) compared to when in occlusion mode [opening pressure 0.18-0.2 kPa or 1.8-2.0 cmH2O, mean occlusion time 44 (25) ms]. The calculated P0.1 values correlated well with those obtained from manual occlusions (r = 0.87, P < 0.0001). This new technique, which detects and discards irregular or non-linear (r < 0.95) inspiratory pressure profiles, enables breath-by-breath determinations of inspiratory drive in rapidly breathing lambs with minimal impact on respiratory pattern and ventilation.

Aging↗
PubMed1996

The effect of xenobiotic acetylation on interorgan metabolism of glucose in fasted rats.

The effect of cytosolic acetylation on interorgan glucose metabolism was studied by arteriovenous (AV) difference and tracer kinetic techniques in fasted, ketamine-anesthetized rats. The administration of sulfamethazine (SMZ, 2 mmol/kg, i.p.) resulted in 39% and 313% increases in hepatic contents of glucose and lactate, respectively. Plasma concentrations of glucose and lactate in the aorta, portal vein and hepatic vein also increased (33-43% for glucose, and 86-200% for lactate). Net hepatic release of glucose was not significantly different from the control. Net hepatic uptake of lactate increased 72-151% in the SMZ-treated rats. The concentration and hepatic gradient of alanine were little changed in the SMZ-treated rats. The rates of turnover for plasma glucose, estimated from [3-3H]-glucose, were not significantly different between the control and SMZ-treated rats. The rate of glucose recycling, estimated from the difference in the rates of turnover between [3-3H]-and[U-14C]-glucose, decreased by 42% in the SMZ-treated rats. Muscle glycogen in the SMZ-treated rats also decreased (33%). In conclusion, our data indicate that cytosolic acetylation can affect not only ketogenesis, as we have previously reported, but also interorgan metabolism of glucose. Although direct evidence is not available, increases in the levels of plasma and liver glucose suggest that gluconeogenesis is increased in the SMZ-treated rats. A net loss of lactate from muscle glycogen store to the liver is indicated by the increase in hepatic uptake of lactate and the decrease in the rate of glucose recycling in the rats treated with SMZ.

Acetylation↗
PubMed1996

Shifts in children's ear asymmetry during verbal and nonverbal auditory-visual association tasks: a "virtual stimulus" effect.

Dichotic consonant-vowel-consonant (CVC) syllables were presented to 96 right-handed children between the ages of 8 and 12 years. Children were assigned either to a "code" condition that entailed translating the CVCs into English words or to a "bird" condition in which the CVCs had to be matched to cartoons of birds. A differential ear asymmetry for the code and bird tasks developed linearly across four blocks of trials. By Block 4, the code task yielded a significant right-ear advantage and the bird task yielded no ear advantage. The results are inconsistent with any model that attributes ear asymmetries entirely to fixed structural characteristics of the nervous system. Instead, ear asymmetries are influenced by the subject's categorization of the stimuli, i.e., by "virtual stimuli". These appear to be constructed over time (blocks of trials).

Association Learning↗
PubMed1996

Cystic tuberculosis of the bone mimicking osteogenic sarcoma.

Hong Kong has a relatively high incidence of tuberculosis in comparison with other developed cities, possibly due to the influx of mainland Chinese immigrants and Vietnamese boat people. However, primary, solitary cystic tuberculous infection of the bone is rare and few cases have been reported. Radiological appearances of this disease can mimic several bone conditions, including bone cyst, osteoblastoma and even osteosarcoma. We report on a case of a healthy fifteen-year-old who presented with swelling to the left elbow; biopsy confirmed this as cystic tuberculosis of the bone.

Adolescent↗
PubMed1996

Chondromyxoid fibroma-like osteosarcoma: a distinct variant of low-grade osteosarcoma.

Chondromyxoid fibroma-like osteosarcoma is a recently described, extremely rare subtype of low-grade osteosarcoma. Two such cases were encountered among 102 cases of osteosarcoma seen in the Prince of Wales Hospital, Hong Kong, between 1984 and 1994. The first patient, a 39-year-old woman, presented with a mass in her right maxilla which was resected and mistaken as a myxoma. The tumour recurred locally four years later and she now has extensive local recurrent disease six years after initial presentation and is amenable to support treatment only. The second patient, a 28-year-old man, had a pelvic tumour which recurred in the form of a polypoid left atrial tumour and pulmonary nodules six years after operation. The left atrial tumour recurred one year after operation, and led to sudden death of the patient seven years after initial presentation. Radiologically, the tumours in both cases appeared as expansile osteolytic lesions with erosion of adjacent bone and infiltration into soft tissue. Histologically, they consisted of lobules of spindle, stellate or polygonal tumour cells showing mitotic activity and with moderate nuclear pleomorphism and hyperchromatism, set in a highly myxoid stroma superficially mimicking chondromyxoid fibroma. The histological hallmark was the direct production of osteoid by tumour cells. Chondromyxoid fibroma-like osteosarcoma merits recognition as a distinct variant of low-grade osteosarcoma for which early appropriate surgery is indicated.

Adult↗
PubMed1996

cDNA sequence analysis of a novel cardiotoxin-like protein from Taiwan banded krait.

The cDNAs encoding a novel protein was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus by reverse transcription-polymerase chain reaction. The deduced amino acid sequence of this novel protein contained 65 amino acid residues with 8 cysteine residues. Comparative sequence analysis showed that it was structurally related to cardiotoxin rather than neurotoxins. These results suggest that the venom of Bungarus multicinctus may contain cardiotoxin(s) which was not noticed before.

Amino Acid Sequence↗