Biomedical subjects
Publications and source records attributed to H Liu.
The 16S rDNAs have been amplified from four halobacteria Haloarcula vallismortis (EM201), Haloferax denitrificans (EM303). A5 and B2 through a set of specific primers using the PCR technique. The molecule of the PCR products is about 1.47 kb. The hybridization results also show that these PCR products have DNA homology with halobacteria.
Transmission of an external signal from receptors to downstream targets is often mediated by a conserved set of protein kinases that act in sequence (a kinase cascade). In haploid strains of Saccharomyces cerevisiae, a signal initiated by peptide pheromones is transmitted through this kinase cascade to a transcription factor STE12, which is required for the expression of many mating-specific genes. Here it was shown that in diploids some of the same kinases and STE12 are required for filamentous growth, but the pheromone receptors and guanosine triphosphate-binding protein are not required for filament formation. Thus, a similar kinase cascade is activated by different signals in haploids and diploids and mediates different developmental outcomes in the two cell types.
The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.
The molecular basis of the alcohol dehydrogenase (ADH)-negative deer mouse (Peromyscus maniculatus) has been investigated. Several classes of mammalian ADHs have been recognized based upon biochemical and structural properties. ADH cDNA clones identified by hybridization to a mouse class I ADH cDNA clone were obtained from a deer mouse ADH-positive liver cDNA library. This cDNA has been identified as being a class I sequence and represents the deer mouse Adh-1 gene. An additional cDNA sequence identified in both the ADH-positive and -negative deer mouse cDNA libraries was identified by weak cross-hybridization to the mouse cDNA. This cDNA encodes an amino acid sequence representing a new class of mammalian ADH, and the deer mouse gene for this ADH is named Adh-2. ADH-negative deer mice do not produce mRNA, that is detected by the Adh-1 cDNA probe. However, both stocks of deer mice produce high levels of Adh-2 mRNA in liver. Southern analysis using an essentially full-length Adh-1 cDNA probe has shown that the Adh-1 gene is deleted in the ADH-negative mice. Biochemical analysis of enzyme activity suggests at least three ADH polypeptides are expressed in different tissues and have somewhat different substrate specificities, as in the mouse.
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We have established a precise and accurate high-performance liquid chromatographic method for the simultaneous assay of carbamazepine, phenytoin, phenobarbital, primidone and their principal metabolites. This method has been used for the analysis of these drugs and the metabolites in serum, saliva and urine samples. Acetonitrile is used for the deproteinization of serum and saliva samples while solid-phase extraction is utilized for urine sample pretreatment. Samples of 2 microliters are injected onto a 3-microns ODS-Hypersil column (250 mm x 2 mm I.D.) with a column temperature of 40 degrees C. The drugs and metabolites are eluted with a mobile phase containing potassium phosphate buffer-acetonitrile-methanol (110:50:30, v/v/v) at a flow-rate of 0.2 ml/min. Signals are monitored by a photodiode-array detector at a sample wavelength of 200 nm with a bandwidth of 10 nm. These four commonly used antiepileptic drugs and their six metabolites are well separated from one another within 15 min. Within-day coefficients of variation (C.V.) are within 5% in most cases and between-day C.V. are from 2.32 to 4.75%. The recovery rates range from 95.12 to 104.42%. This method has the necessary sensitivity and linearity for routine therapeutic monitoring of both total and free drug levels and may be employed for pharmacokinetics studies of drug interactions and metabolism as well.
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The midbrain periaqueductal gray (PAG) is involved in a variety of functions including pain modulation, vocalization, autonomic control, fear and anxiety. This area contains serotonin receptors, particularly 5-HT1A that are known to play a role in the above functions. The goals of this study were to characterize the effects of 8-OH-DPAT, a selective 5-HT1A agonist, on the firing characteristics and membrane properties of PAG neurons. Both in vivo and in vitro preparations were used. The effects of 8-OH-DPAT on baseline activity of 91 neurons were tested in the in vivo preparation. In 50/91 cells, 8-OH-DPAT produced a decrease in the firing rate that ranged between 21 and 98% (mean +/- S.E.M. decrease of 49 +/- 1.9%). This inhibitory effect was dose dependent and could be blocked by spiperone. In 10/91 cells, 8-OH-DPAT produced an increase in the firing rate that ranged between 13 and 290%, with mean increase of 83 +/- 7.4%. The baseline firing rate of the remaining 31 cells was not affected by 8-OH-DPAT. In the PAG slice preparation, the effects of 8-OH-DPAT on synaptic and membrane properties of 17 PAG neurons were tested using whole-cell voltage clamp-recording procedures. In 14 cells, application of 8-OH-DPAT produced hyperpolarization that ranged between 6 and 21 mV, with mean of 8.4 +/- 2.0 mV. This hyperpolarization was associated with a decrease in membrane impedance that ranged between 8 and 45%, with mean decrease of 21.6 +/- 4.5%. The remaining three neurons did not respond to 8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent studies have demonstrated an important contribution of the A5 noradrenergic cell group of the rostral medulla in the regulation of nociceptive messages at the level of the spinal cord. These noradrenergic controls parallel those arising from the serotonin-containing neurons of the nucleus raphe magnus. In the present study, we used postembedding immunogold staining to identify GABA-immunoreactive terminals that synapse upon identified spinally projecting noradrenergic neurons of the A5 cell group in the rat. A5 projection neurons were identified by Fluoro-Gold transport from the spinal cord; sections containing retrogradely labelled cells were then immunoreacted for tyrosine hydroxylase (TH) to identify the catecholamine-containing, presumed noradrenergic, neurons. Double-labelled A5 cells were intracellularly filled with Lucifer Yellow (LY) and then the LY was photo-oxidized to an electron-dense product. Seven intracellularly filled TH-immunoreactive projection neurons were studied with postembedding immunocytochemistry. Each A5 neuron received a significant GABA-immunoreactive terminal input. Out of a pooled total of 151 terminal profiles found in apposition to intracellularly labelled somatic and dendritic profiles, 31 (20.5%) were GABA-immunoreactive. The proportion of GABA-immunoreactive terminals that contacted somatic profiles (12/72; 17%) was similar to the proportion that contacted TH-labelled dendritic profiles (19/79; 24%). There was a discernible synaptic specialization in about 50% of the labelled terminals that contacted the TH projection neuron. Both symmetric and asymmetric synaptic specializations were found. Labelled terminals contained round or pleiomorphic vesicles, but not flat vesicles; many also contained dense-core vesicles. Our results indicate that noradrenergic neurons of the A5 cell group, which contribute to both antinociceptive and cardiovascular controls through their projection to the spinal cord, are regulated by local GABAergic, presumably inhibitory, mechanisms. Whether the initiation of A5 neuron activity results from a lifting of tonic GABAergic inhibitory control, as has been proposed for the neurons of the nucleus raphe magnus, remains to be determined.
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GTPase-activating protein (GAP), a protein capable of regulating the activity of p21ras protein, is phosphorylated on tyrosine residues following the activation of tyrosine kinase(s) associated with several growth factor receptors. The present study was designed to examine potential role of phosphotyrosine phosphatase in tyrosine phosphorylation of GAP. Addition of orthovanadate, a phosphate analogue known to inhibit phosphotyrosine phosphatase, to cultured liver macrophages induced tyrosine phosphorylation of numerous cellular proteins with a range of molecular weights between 30-130 kDa; one tyrosine-phosphorylated protein was identified as the 120 kDa GAP. The effect of orthovanadate on the tyrosine phosphorylation of GAP was time- and concentration-dependent. Quantitated data indicated that approximately 4% of the total content of cellular GAP was tyrosine-phosphorylated upon orthovanadate treatment. These observations suggest a potential regulatory role of phosphotyrosine phosphatase in the tyrosine phosphorylation of GTPase-activating protein in cellular signaling mechanisms in the hepatic macrophages.
Phospholipase C (PL-C) digestion of human low density lipoprotein (LDL) results in hydrolytic cleavage of the phosphocholine head group of phosphatidylcholine, thereby generating diacylglycerol. Loss of amphiphillic surface lipids and/or accumulation of diacylglycerol causes LDL samples to develop turbidity. Examination of PL-C treated LDL by electron microscopy revealed a progressive aggregation of LDL as a function of phosphatidylcholine hydrolysis: fused particles, clusters, and multiple stacked aggregates were observed. Lipid analysis of untreated and aggregated LDL confirmed that the phosphatidylcholine content of the latter had decreased with a corresponding increase in diacylglycerol. It is likely that phospholipolysis created hydrophobic gaps within the surface monolayer of LDL, thereby inducing LDL fusion and aggregation. When amphipathic alpha-helix-containing apolipoproteins, such as human apoA-I or Manduca sexta apolipophorin III (apoLp-III) were present, PL-C treated LDL did not aggregate. Compositional analysis of apolipoprotein-containing PL-C LDL showed that phospholipolysis was not affected by the presence of apolipoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lipoproteins re-isolated following incubation with PL-C revealed an association of apoA-I or apoLp-III with PL-C digested LDL. Electron microscopy showed no major morphological differences between native LDL and apoprotein stabilized PL-C treated LDL and the average particle diameter of apoA-I stabilized PL-C LDL was 22.5 +/- 2.2 nm versus 22.8 +/- 1.6 nm for control LDL. Incubation of tritium-labeled apoLp-III with LDL and PL-C demonstrated that association of apoLp-III with PL-C LDL correlated with the extent of phospholipid hydrolysis, the apolipoproteins apparently being recruited to compensate for the increased hydrophobic surface created by conversion of phosphatidylcholine into diacylglycerol. The results suggest that transient association of amphipathic apolipoproteins with damaged or unstable LDL may provide a mechanism to obviate formation of atherogenic LDL aggregates in vivo.
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OBJECTIVE: To test whether hearts from "dead," pulseless, asystolic donors could be transplanted and reanimated successfully using reperfusion manipulations. DESIGN AND INTERVENTIONS: Ten infant lambs (mean [+/- SD] weight, 4 +/- 1 kg) were anesthetized and pretreated with 50% dextrose, methylprednisolone, prostaglandin E1, and sublingual nifedipine. Five of these lambs were paralyzed; hypoxic asystolic arrest occurred 10 +/- 2 minutes later. Thirty minutes following asystole (mean, 40 +/- 2 minutes after paralysis) sternotomy was performed and the hearts were excised. Aortic blood gases were as follows: pH, 6.6 +/- 0.1; PCO2, 180 +/- 20 mm Hg; and PO2, 8 +/- 2 mm Hg. Donor hearts were given 30 mL/kg of Cardiosol (Water's Instrument Co, Danburg, Conn), a new cardioplegic agent, at 4 degrees C, and explanted into iced saline. The remaining five lambs then underwent cardiopulmonary bypass, were cooled to 20 degrees C, and hypothermic arrest was instituted. After excision of the recipient's heart, the donor heart was implanted in an orthotopic position. Total cold ischemic time was 1 hour 40 minutes +/- 10 minutes. The donor heart was retroperfused for 10 minutes with low-hematocrit, low-calcium blood via a coronary sinus catheter, then normal aortic inflow reperfusion was continued for 50 minutes. MAIN OUTCOME MEASURES: Removal of the cardiopulmonary bypass and measurement of hemodynamic parameters. RESULTS: One half hour after the bypass, mean systolic aortic pressure was 71 +/- 6 mm Hg; mean right atrial pressure was 6 +/- 2 mm Hg; mean left atrial pressure was 7 +/- 2 mm Hg; and mean pulmonary arterial pressure was 20 +/- 8 mm Hg. No inotropic drugs were given for postbypass blood pressure support. CONCLUSION: It is possible to transplant and reanimate hearts that have been dead for 30 minutes. When further developed, the use of donors who were not brain dead but allowed to die naturally could greatly increase the donor pool.
A retrospective analysis was done on 43 patients with acute promyelocytic leukemia (APL) at our hospital from June 1987 to August 1992. All-trans retinoic acid was used to induce these patients to differentiation. In the early period of induction there were risks of severe hemorrhage, which was the main cause of early death. Treatments combined with platelets and heparin or aminomethylbenzoic (PAMBA) were given to patients with abnormal coagulation. As a result only 4 out of 43 patients died of intracranial bleeding at 4-12 days when their white blood cell (WBC) counts peaked. The combination of retinoic acid (RA) and HA chemotherapy could reduce hyperleukocytosis during the RA induction course. None of 7 patients died at early stage with this treatment combination. Our studies showed that it could predict the onset of remission at early stage through observations on the successive changes of karyotypes and morphology of the bone marrow and peripheral blood cells. Our studies also showed that the growth of CFU-F could be inhibited by RA. We think that it may play a role in the RA-induced differentiation. In 4 years of follow-up the overall leukemia-free survival (LFS) was 80% with a relapse rate of 45%. Thirty-five patients out of 43 cases were still alive in remission, and one was alive in relapse. All 11 out of 43 patients relapsed within 3 years, but the relapses occurred later, after 3 years duration of remission (P < .01). Retinoic acid failed to induce 5 patients who relapsed with the continuation treatment of RA and chemotherapy alternatively. In order to overcome the resistance to RA, the continuation treatment of simple chemotherapy had been administered following CR. Two cases achieved remission in this way. The difference of resistant events to RA reached significance between these 2 groups of different continuation treatment.
The incidence of residual neoplastic cells on prostatic biopsy following conventional external beam radiotherapy is reported to range from 40-90%. As a result, it has been stated that current modalities of radiotherapy may carry an unacceptable local failure rate even in patients irradiated for low stage disease. In order to assess the potential benefits of three-dimensional (3-D) treatment planning, an unselected, consecutive group of patients with localized adenocarcinoma of the prostate was evaluated. This study was designed to determine the frequency of residual cancer in the prostate two years following definitive external beam radiotherapy designed, using a 3-D planning system. Between February 1988 and February 1989, 30 consecutive patients with localized (Stage T1-T3NxMo) adenocarcinoma of the prostate received definitive external beam radiotherapy. All treatment fields were designed with a computed tomography (CT)-based 3-D treatment planning system, resulting in a static conformal radiotherapy plan. The minimum dose delivered to the target volume, which included the prostate, periprostatic tissues, and a 1 cm margin, was between 65 and 69 cGy. Twenty-six patients had Stage T1, T2NxMo primary tumors and four were T3NxMo. Two years following the completion of treatment, all patients underwent digital rectal examination, transrectal ultrasound examination of the prostate with multiple biopsies, bone scan, and serum prostate specific antigen (PSA) determinations. Residual prostate cancer was proven by biopsy in six of 30 patients (20%). Four of 26 (15%) with Stage T1 and T2 tumors had a positive biopsy. However, two of the four Stage T3 tumors had postradiation biopsies positive for cancer (50%). Only one patient with a positive biopsy had an abnormal rectal examination. Five of the eight patients with elevated serum PSA levels after two years had residual neoplasia identified on biopsy. One of six patients with an abnormal postradiation ultrasound had residual tumor. Only one of the 22 patients (5%) with a normal serum PSA at two years had a positive postradiation biopsy. In patients with localized prostate cancer, the use of 3-D static conformal radiotherapy followed by multiple ultrasound guided biopsies confirmed the efficacy of external beam radiotherapy in low stage disease. We believe that the low incidence of positive biopsies in this study resulted from the benefits of 3-D treatment planning as well as the fact that all patients were evaluated, whereas past studies have been in selected patient groups when suspicion of residual disease existed prior to biopsy.(ABSTRACT TRUNCATED AT 400 WORDS)
A new synthetic route was utilized to prepare 6-substituted 3,4-benzocoumarins where the substituents were iodo, fluoro, trifluoromethyl, bromo, chloro, isopropyl, ethyl, t-butyl, methyl, hydrogen, amino, phenyl, or nitro; 3,4-naphthocoumarin was also synthesized. The relative affinities of these congeners for the aryl hydrocarbon (Ah) receptor were determined using rat hepatic cytosol and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) as the radioligand. In addition, the Ah receptor agonist activity of the 6-substituted 3,4-benzocoumarins was determined from their concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast with many other structural classes of halogenated aromatics, there was not a correlation between the structure-binding versus structure induction relationships for the 6-substituted 3,4-benzocoumarins. These results suggested that some of these congeners may exhibit partial Ah receptor antagonist activities and this was investigated by determining the inhibitory effects of 6-substituted 3,4-benzocoumarins on TCDD-induced EROD activity in rat hepatoma H4II E cells in culture. Only four compounds (6-isopropyl, 6-phenyl, 6-fluoro, and 6-t-butyl) inhibited the TCDD-induced response (21.7 to 64.4% inhibition) and the mechanism of action of the most active inhibitor, 6-t-butyl-3,4-benzocoumarin, was further investigated. In contrast, with other partial Ah receptor antagonists such as alpha-naphthoflavone, cotreatment of rat hepatoma H4II E cells with 1 nM TCDD plus 1 and 10 microM 6-t-butyl-3,4-benzocoumarin did not result in decreased levels of the Ah receptor complex (liganded with TCDD). In addition, there was not significant inhibition of TCDD-induced CYP1A1 mRNA levels or protein as determined by Northern and Western blot analyses. The results suggest that 6-t-butyl-3,4-benzocoumarin or one of its metabolites is a post-translational inhibitor of CYP1A1-dependent enzyme (EROD) activity in this cell line and thus represents a novel Ah receptor-independent inhibition of CYP1A1.
With time-resolved spectroscopy, we develop an experimental approach by sample substitution to measure the absorption (mu a) and reduced scattering (mu's) coefficients of small-volume biological samples. To investigate the method, small-volume control samples are substituted into a large-size host medium during increases in the absorber (or scatterer) concentration of the host. By characterizing the deviation of the spectra taken with and without the sample, we determine the matching points where the sample and surrounding medium are optically identical. We show that this method can result in correct values of the mu a and mu's for the sample within 6% error if the matching conditions for both the mu a and mu's are fully realized. The results also indicate that this method can give approximate values of the mu a and mu's in a reasonable range if either the mu a or the mu's matching between the two media is realized. This method has been applied to the studies of absorption properties of a human finger and of scattering properties of yeast.