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Biomedical subjects

X Lin

Publications and source records attributed to X Lin.

At least 91 records · Page 5Linked to original sources
PubMed2000

Enzymic characteristics of secreted aspartic proteases of Candida albicans.

Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosuppressive therapeutics. The secreted aspartic proteases (Saps) are known virulence factors of pernicious Candida species. The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence. Only one of these proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies. The other enzymes are present in low amounts in yeast culture and are difficult to purify. As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied. Therefore, four Saps that are known to be expressed in C. albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant zymogens and purified in large quantities. These proenzymes were autoactivated and purified as active proteases. The enzymic properties including the substrate specificities at the P(1) and P(1)' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures. All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites. At the P(1) site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu. Positively charged amino acids are also accommodated, especially by Sap2 and Sap3. The specificities at P(1)' are broader than at P(1) for all four enzymes. Sap6 prefers Ala, whereas other Saps prefer Tyr. Acidic side chains are also accommodated at this site. Analysis of substrates with a hydrophobic amino acid in P(1)' reveals that all the Saps possess a unique preference for Ala at this site. The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.

Aspartic Acid Endopeptidases↗
PubMed2000

Sleep disordered breathing in patients with cluster headache.

OBJECTIVE: To study subjects with active or inactive cluster headache (CH) for occult sleep disordered breathing (SDB). BACKGROUND: CH frequently occurs during sleep. The authors previously found that symptoms of SDB predicted reported occurrence of CH in the first half of the night, which suggested that CH could be triggered in some cases by unrecognized SDB. METHODS: The authors performed polysomnography in 25 adults (22 men) with CH. Subjects were not selected for any sleep-related complaint. In addition to standard measures, studies included monitoring of end-tidal carbon dioxide (n = 22), and esophageal pressure (n = 20). RESULTS: The rate of apneas and hypopneas per hour of sleep was >5 in 20 subjects (80%; 95% CI, 64% to 96%), minimum oxygen saturation was <90% in 10 subjects, maximum negative esophageal pressure ranged from -13 to -65 cm H2O, and maximum end-tidal carbon dioxide was > or =50 mm Hg in eight subjects. The eight subjects with active (versus inactive) CH at the time of study had higher maximum end-tidal carbon dioxide levels (50 +/- 3 versus 44 +/- 5 mm Hg; p = 0.0007). More severe oxygen desaturation was associated with reports that CH typically occurred in the first half of the nocturnal sleep period (p = 0.008). CONCLUSIONS: SDB occurred in the majority of patients with CH. Evaluation of a patient with CH should include consideration that SDB may be present.

Adult↗
PubMed2000

Isolation of respiratory bovine coronavirus, other cytocidal viruses, and Pasteurella spp from cattle involved in two natural outbreaks of shipping fever.

OBJECTIVE: To identify cytocidal viruses and Pasteurella spp that could be isolated from cattle involved in 2 natural outbreaks of shipping fever. ANIMALS: 105 and 120 castrated male 4- to 8-month-old feedlot cattle involved in 1997 and 1998 outbreaks, respectively. PROCEDURES: Nasal swab specimens and blood samples were collected, and cattle were vaccinated on arrival at an order-buyer barn from 4 local auction houses. Four days later, they were transported to a feedlot, and additional nasal swab specimens and blood samples were collected. Nasal swab specimens were submitted for virus isolation and bacterial culture; blood samples were submitted for measurement of respiratory bovine coronavirus (RBCV) hemagglutinin inhibition titers. RESULTS: 93 of 105 cattle and 106 of 120 cattle developed signs of respiratory tract disease during 1997 and 1998, respectively, and RBCV was isolated from 81 and 89 sick cattle, respectively, while at the order-buyer's barn or the day after arrival at the feedlot. During the 1997 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 4 cattle 14 days after arrival at the feedlot. During the 1998 outbreak, bovine herpesvirus 1 was isolated from 2 cattle at the order-buyer's barn and on arrival at the feedlot and from 5 cattle 7 and 14 days after arrival at the feedlot, and parainfluenza virus 3 was isolated from 1 animal the day of, and from 18 cattle 7 and 14 days after, arrival at the feedlot. Pasteurella spp was cultured from 4 and 6 cattle at the order-buyer's barn and from 92 and 72 cattle on arrival at the feedlot during the 1997 and 1998 outbreaks, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that RBCV may play a causative role in outbreaks of shipping fever in cattle. More than 80% of the sick cattle shed RBCV at the beginning of 2 outbreaks when the Pasteurella spp infection rate was low.

Animals↗
PubMed2000

Effect of HIV infection on menstrual cycle length.

HIV serostatus and menstrual function were examined using prospectively collected menstrual data from 802 HIV-seropositive and 273 HIV-seronegative women, ages 20 to 44, enrolled in two cohort studies of HIV infection in North American women. The associations between HIV serostatus and the probabilities of having a cycle lasting >40 days (n = 541 cycles), >90 days (n = 67 cycles), <18 days (n = 316 cycles) and mean length and variability of 18 to 40 day cycles (n = 3,634) were assessed. After adjustment for demographic characteristics, body mass index, and substance use, seropositivity increased the odds of having a very short cycle (< 18 days, odds ratio [OR], 1.45; 95% confidence interval [CI], 1.00-2.11) and a very long cycle (>90 days, OR, 1.32; 95% CI, 0.68-2.58) slightly, although the latter CIs include one. Seropositivity did not increase the odds of having a moderately long cycle (>40 days, OR, 1.14) or affect mean cycle length or variability (beta, 0.30 +/- 0.20; between-woman standard deviation [SD], 2.2 days [HIV-seronegative] and 1.9 days [HIV-seropositive]; within-woman SD, 3.5 days for both). Although seropositivity may slightly increase the probability of very short cycles, HIV serostatus has little overall effect on amenorrhea, menstrual cycle length, or variability. Among HIV-seropositive women, higher viral loads and lower CD4+ counts were associated with increased cycle variability and polymenorrhea.

Adult↗
PubMed2000

Endovascular irradiation from beta-particle-emitting gold stents results in increased neointima formation in a porcine restenosis model.

BACKGROUND: Recent studies have shown that ionizing radiation reduces neointima formation after balloon angioplasty and stent implantation in experimental models of restenosis and first clinical trials. The objective of this study was to determine the dose distribution of a new beta-particle-emitting radioactive gold stent and to evaluate the dose-dependent vascular response in the coronary overstretch pig model. METHODS AND RESULTS: Sixteen Göttinger minipigs underwent placement of 11 nonradioactive and 36 beta-particle-emitting stents with activity levels of 10.4+/-0.6, 14.9+/-2.4, 22.8+/-1.3, 35.8+/-2. 8, and 55.4+/-5.3 microCi of (198)Au. Three months after implantation, the percent area stenosis, neointimal thickness, neointimal area, and vessel injury were analyzed by quantitative histomorphometry. The lifetime radiation doses at a depth of 1 mm were 3.3+/-0.2, 4.7+/-0.5, 7.2+/-0.4, 11.4+/-0.9, and 17.6+/-1.7 Gy for the different activity groups. No dose-response relationship was observed in the radioactive stents with respect to percent area stenosis (P=0.297), mean neointimal thickness (P=0.82), or mean neointimal area (P=0.65). Significantly lower neointima formation and less luminal narrowing was seen in the control group than in the beta-particle-emitting stents (P<0.001). Multilinear regression analysis revealed that only radioactivity made a significant independent contribution to the degree of percent area stenosis (P<0. 001). CONCLUSIONS: Neointima formation in pigs is markedly increased by beta-particle-emitting stents with (198)Au as the radioisotope. This study provides evidence that dosages of 3 to 18 Gy of low-dose-rate beta-particle irradiation via endovascular stents cause pronounced luminal narrowing in the animal model at 3 months.

Animals↗
PubMed2000

Activation of metabotropic GABAB receptors inhibited glutamate responses in spiral ganglion neurons of mice.

The lateral olivocochlear efferent (LOC) fibers form axodendritic synapses with auditory nerve dendrites synapsing on inner hair cells to affect postsynaptic responses GABA is one of the primary transmitters used by the LOC efferent. However, the molecular nature and developmental expression of the GABA receptors in spiral ganglion (SG) neurons are poorly understood. We have identified the GABAB receptors in SG neurons of mice by pharmacological criteria and polymerase chain reaction (PCR) detection. Applications of GABA or baclofen increased intracellular Ca2+ concentrations and inhibited glutamate responses. The responses were blocked by saclofen, but not by bicuculline. Relative-quantitative PCR showed that GABAB receptors were expressed prenatally, and their levels did not change significantly during the maturation of SG neurons.

Age Factors↗
PubMed2000

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA.

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.

Binding Sites↗
PubMed2000

TGF-beta 2 reduces demyelination, virus antigen expression, and macrophage recruitment in a viral model of multiple sclerosis.

TGF-beta 2 is a potent immunoregulatory mediator that influences B cell, T cell, and macrophage function. To test whether this cytokine alters pathology in a model of virus-induced demyelinating disease, we treated SJL/J mice with TGF-beta 2 either before or after infection with Theiler's murine encephalomyelitis virus. Treatment continued three times weekly through day 35 postinfection. TGF-beta 2 administration resulted in significantly smaller lesions and fewer virus Ag-positive cells in the spinal cords of infected SJL/J mice. Mice treated with TGF-beta 2 had similar levels of virus-specific IgG as infected, control-treated mice. TGF-beta 2 administration significantly increased the level of non-virus-specific activated CTLs, but had no effect on virus-specific CTLs. TUNEL revealed a decrease in the number of apoptotic nuclei in the spinal cord white matter of mice treated in vivo with TGF-beta 2. Immunostaining with an Ab to F4/80 revealed that TGF-beta 2-treated mice had significantly fewer F4/80-positive cells in the white matter of the spinal cord as compared with infected control-treated mice. These data suggest that TGF-beta 2 may control virus-induced demyelination via an immunomodulatory mechanism that reduces macrophage infiltration.

Animals↗
PubMed2000

Cloning and expression of glycolipid transfer protein from bovine and porcine brain.

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.

Amino Acid Sequence↗
PubMed2000

Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein.

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the beta-secretase site of both the wild-type and Swedish mutant beta-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the beta-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of beta-secretase, which catalyzes the rate-limiting step of the in vivo production of the beta-amyloid (Abeta) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S(1)' subsite, which prefers small side chains such as Ala, Ser, and Asp.

Alzheimer Disease↗
PubMed2000

Endogenously generated spontaneous spiking activities recorded from postnatal spiral ganglion neurons in vitro.

Spontaneous spiking activities in the nervous system play an important role in the neuronal development and the coding of sensory information. Such firings could be initiated by transmitter leaked from the first-order sensory receptors or as a result of the internal operation of voltage-dependent ion channels intrinsic to the neuron. We recorded endogenously-generated spontaneous action potentials (APs) from postnatal spiral ganglion (SG) neurons of mouse in vitro. SG neurons in cultures displayed statistically stable spontaneous firings with no obvious bursting, rhythmic spiking and long silent gaps for as long as the recording configuration could be maintained. Average firing rates ranged from less than 1 to over 10 spikes/s, with most cells fired around 4 spikes/s. Interpulse interval histograms were remarkably similar to those recorded in vivo from the auditory nerve, with characteristics of a Poisson-like distribution. Resting membrane potential greatly altered the AP width and the rate of spontaneous firings. Spontaneous firing rates were also found to be controlled by the availability of the Shaw-like potassium channels. In contrast, matured SG neurons did not display any spontaneous APs, probably due to a large increase in the expression of the whole-cell potassium currents in comparison to their postnatal counterparts. This study provided the first direct evidence that postnatal SG neurons were capable of generating spontaneous APs independent of inputs from hair cells. Intracellular mechanisms for generating the spontaneous random spikes and the possible roles of such spontaneous activities in the postnatal development of SG neurons are discussed.

Action Potentials↗
PubMed2000

Human plasminogen catalytic domain undergoes an unusual conformational change upon activation.

Activation of the serine protease plasmin from its zymogen, plasminogen, is the key step in fibrinolysis leading to blood clot dissolution. It also plays critical roles in cell migration, such as in tumor metastasis. Here, we report the crystal structure of an inactive S741A mutant of human plasminogen catalytic domain at 2.0 A resolution. This structure permits a direct comparison with that of the plasmin catalytic unit. Unique conformational differences are present between these two structures that are not seen in other zymogen-enzyme pairs of the trypsin family. The functional significance of these differences and the structural basis of plasminogen activation is discussed in the light of this new structure.

Amino Acid Sequence↗
PubMed2000

Enhancement of wound healing by hyperbaric oxygen and transforming growth factor beta3 in a new chronic wound model in aged rabbits.

HYPOTHESIS: Although hyperbaric oxygen (HBO) has been used clinically for 3 decades, there have been few controlled clinical trials. Animal models have not been adequate to test the efficacy of HBO in the treatment of chronic wounds, either by itself or in combination with growth factors. We hypothesize that HBO is as efficacious as a prototype growth factor in improving wound healing in a new animal model of ischemic chronic wounds. DESIGN: Twenty-five aged rabbits and 3 young rabbits had their ears rendered chronically ischemic and ulcers were created down to the level of cartilage. These ulcers were treated in 1 of 3 ways: with HBO, 90 minutes per day, Monday through Friday, for 4 weeks; with transforming growth factor beta(3) at 1 microg/cm(2); or with both modalities combined. Controls were treated with vehicle or hyperbaric room air or both. RESULTS: This model created an aged/ischemic wound that failed to heal spontaneously up to 26 days after wounding (88% reduction compared with aged/nonischemic controls). Hyperbaric oxygen alone and transforming growth factor beta(3) alone both improved healing rate (only 38% reduction in healing compared with aged/nonischemic controls). Combined therapy produced no additional improvement over either modality by itself. CONCLUSIONS: In aged animals, HBO and transforming growth factor beta(3) were equally effective in improving wound healing. Our data suggest that HBO alone may be more effective in the chronic wound than in the acute wound. There was no additive benefit to combining modalities as has been reported in the same wound model in young rabbits.

Aging↗
PubMed2000

Characterization and distribution of somatostatin binding sites in goldfish brain.

Somatostatin (SRIF) binding sites were characterized in goldfish brain. Binding of (125)I-[Tyr(11)]-SRIF-14 to a brain membrane preparation was found to be saturable, reversible, and time-, temperature-, and pH-dependent. Binding was also displaceable by different forms of SRIF. Under optimal conditions (22 degrees C, pH 7.2), the equilibrium binding of (125)I-[Tyr(11)]-SRIF-14 to goldfish brain membranes was achieved after 60 min incubation. Analysis of saturable equilibrium binding revealed a one-site model fit with K(a) of 1.3 nM. SRIF-14, mammalian SRIF-28, and salmon SRIF-25 displaced (125)I-[Tyr(11)]-SRIF-14 binding with similar affinity, whereas other neuropeptides, e.g., substance P, were unable to displace (125)I-[Tyr(11)]-SRIF-14. Autoradiography studies demonstrated that (125)I-[Tyr(11)]-SRIF-14 binding sites are found throughout the goldfish brain. A high density of (125)I-[Tyr(11)]-SRIF-14 binding sites was found in the forebrain, including the nucleus preopticus, nucleus preopticus periventricularis, nucleus anterioris periventricularis, nucleus lateralis tuberis, nucleus dorsomedialis thalami, nucleus dorsolateralis thalami, nucleus ventromedialis thalami, and nucleus diffusus lobi inferioris. In midbrain, (125)I-[Tyr(11)]-SRIF-14 binding sites were found in the optic tectum. The facial and vagal lobes and the mesencephalic-cerebellar tract were found to have a high density of binding sites. This study provides the first characterization and distribution of specific binding sites for SRIF in a fish brain.

Animals↗
PubMed2000

An immune algorithm for resolution of multicomponent overlapping chromatograms.

A novel immune algorithm for resolution and quantitative determination of the components in overlapping chromatograms was proposed by imitating biological immune systems. The algorithm takes an overlapping chromatogram as its input and subtracts the chromatograms of standard samples from the input by iteration of a network. When the residual does not change, the network will converge and chromatographic information of the components in overlapping chromatogram will be obtained. Both simulated and experimental data sets were investigated by the method. Results showed that both resolved results and recoveries of quantitative determination are satisfactory. Comparing with conventional least-square method, the immune algorithm is fast in calculation.

Algorithms↗
PubMed2000

Molecular cloning and expression of cDNA encoding a brain bombesin/gastrin-releasing peptide-like peptide in goldfish.

A complementary DNA (cDNA) of 928 bp encoding a bombesin (BBS)/gastrin-releasing peptide (GRP) precursor was identified from goldfish brain. Goldfish BBS/GRP messenger RNA (mRNA) encodes a 157 amino acid precursor, which contains a signal peptide sequence, the 22 amino acid putative BBS/GRP-like peptide, and a carboxy-terminal extension peptide. Reverse transcription-polymerase chain reaction (PCR) (RT-PCR) demonstrated that the mRNA for this precursor has a widespread distribution in goldfish brain, and is also present in skin, gastrointestinal tract, gonad, and gill. Phylogenetic analysis of BBS/GRP-like peptide precursors in vertebrates shows that goldfish BBS/GRP is more closely related to the known GRP precursors than to BBS precursors.

Amino Acid Sequence↗
PubMed2000

Preprotachykinin gene expression in goldfish brain: sexual, seasonal, and postprandial variations.

Recently, we described the complete nucleotide sequence of gamma-preprotachykinin (gamma-PPT) mRNA and the deduced amino acid sequence of the precursor on the basis of molecular cloning and sequence analysis of cDNA from goldfish brain. In the present study, gamma-PPT gene expression in the brain of goldfish was examined using quantitative Northern blot analysis. The results showed that the gamma-PPT gene is highly but differentially expressed in the olfactory bulbs, hypothalamus, and posterior brain regions. There are sexual dimorphism and seasonal variations in gamma-PPT gene expression. In addition, the postprandial changes in gamma-PPT gene expression in the olfactory bulbs and hypothalamus suggest that tachykinin peptides are involved in regulation of feeding behavior in goldfish.

Animals↗