Assignment of the human gene encoding the delta 1-pyrroline-5-carboxylate synthetase (P5CS) to 10q24.3 by in situ hybridization.
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1-Pyrroline-5-Carboxylate Dehydrogenase↗
Biomedical subjects
G Liu
Publications and source records attributed to G Liu.
Temperature dependence of the mitochondrial inner membrane anion channel. The relationship between temperature and inhibition by protons.
In this paper, we investigate the temperature and pH dependence of the mitochondrial inner membrane anion channel (IMAC) that is believed to be involved in mitochondrial volume homeostasis. At pH 7. 4, the flux of malonate is highly temperature-dependent with rates increasing from 1 nmol/min mg at 5 degrees C to 1900 nmol/min mg at 45 degrees C. The Arrhenius plot is nonlinear with the activation energy increasing from 21 kJ/mol (Q10 = 1.3) to 193 kJ/mol (Q10 = 13) as the temperature is decreased. This temperature dependence is unusual and not seen with solutes that are transported through the bilayer such as NH4OAc, malonamide, and KSCN (plus valinomycin) or even for cytochrome c oxidase-dependent uptake of potassium (plus valinomycin). The temperature dependence of IMAC is closely related to the inhibition of IMAC by protons. Thus, we find that the pIC50 for protons decreases from 9.3 (Hill coefficient = 1.0) at 5 degrees C to 7.1 (Hill coefficient = 2.5) at 45 degrees C. This behavior is explained on the basis of a new kinetic model for IMAC in which the net open probability is not only modulated by the binding of three protons but also by temperature via effects on the open probability of the unprotonated channel and the pK of one of the inhibitory protonation sites.
Animals↗
Improved designs for dose escalation studies using pharmacokinetic measurements.
We describe a method for incorporating pharmacokinetic (PK) data into dose escalation clinical trial designs. Doing so can improve the efficiency and accuracy of these studies. The method proposed uses a parametric dose response function that models the probability of response in each person with two effects: the dose of drug administered and an ancillary pharmacokinetic measurements. After treatment and observation of each subject (or group of subjects) for response, one calculates the dose to be administered to the next individual (or group) to yield the target probability of response from the current best estimate of the dose-response curve. This procedure is a variant of the continual reassessment method (CRM). Statistical simulations employing a logistic dose-response model (that is, we model the logit of the response probability as a linear combination of predictors), dose of drug, and the area under the time-concentration curve (AUC) demonstrate that the addition of pharmacokinetic information to the CRM is a practical and useful way to improve both dose-response modelling and the design of dose escalation studies.
Area Under Curve↗
Restoration of phosphorylation capacity to the dormant half of the alpha-subunits of Na+, K(+)-ATPase.
Purified kidney Na+, K(+)-ATPase whose alpha-subunit is cleaved by chymotrypsin at Leu266-Ala267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of alpha-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the alpha-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of alpha-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all alpha-subunits.
Adenosine Triphosphate↗
Common sequence variants of lipoprotein lipase: standardized studies of in vitro expression and catalytic function.
We have assessed the functional activity of three common sequence variants of human lipoprotein lipase (LPL). Two of these, Asn291Ser and Asp9Asn arise from missense mutations while the third, Ser447Ter, derives from a nonsense mutation, truncating LPL by two residues. As previous in vitro studies have produced conflicting results, we have re-analyzed the catalytic function of these variants using the COS cell transfection system, under optimized and standardized experimental protocols. We found the Asn291Ser variant to manifest with a decrease in catalytic activity (57% of normal) due to a reduction in secretion and stability of the active homodimeric form. The Asp9Asn variant also showed a significant decrease in catalytic activity (85% of normal), but this was found to be due to a decreased rate of secretion only, as the homodimeric form was stable. The findings for these mutants contrasted with those of the Ser447Ter truncation variant which proved to be catalytically normal; this variant also manifested normal homodimer stability. The truncated variant did however, present with a higher total secreted mass level (131%) than control LPL. This was most likely due to enhanced secretion of the monomeric form. None of these mutations exhibited defects in binding affinity to cell surface proteoglycans. Each of these variants deviated significantly from normal as regards to their secreted activity or mass levels in the COS cell transfection system.
Catalysis↗
Polymorphism of angiotensin I converting enzyme gene in the older Chinese: linked to ambulatory blood pressure levels and circadian blood pressure rhythm.
This study was undertaken to evaluate the association of polymorphism of angiotensin I converting enzyme (ACE) gene with causal and ambulatory blood pressure in the Chinese population. Genomic DNA was amplified by polymerase chain reaction (PCR) using primers flanking the polymorphic region in intron 16 of the ACE gene. Alleles were detected on agarose gels stained with ethidium bromide. Causal blood pressure was measured in the 133 normotensive and 122 essential hypertensive subjects; of the essential hypertensive subjects, 65 patients underwent non-invasive ambulatory blood pressure. There was no significant difference of ACE genotype and allele frequencies between the hypertensive and normotensive subjects. Non-significant differences were demonstrated in gender, age, causal blood pressure according to the ACE gene genotypes in the 65 patients who underwent non-invasive ambulatory blood pressure; however, effects of the ACE gene genotypes were observed on the derivatives of ambulatory blood pressure, including sleeping blood pressure levels and circadian blood pressure rhythm (p < 0.05). We suggest that insertion/deletion polymorphism of the ACE gene should be associated with blood pressure and ambulatory blood pressure might be superior to causal blood pressure at the molecular level.
Aged↗
2D-NMR studies of the effects of axial substitution on two helices in horse cytochrome c.
The sequential amino acids from Gly-1 to Cys-14 in the N-terminal segment and from Lys-88 to Glu-104 in the C-terminal segment of cytochrome c-imidazole complex (Im-cyt c) have been studied by two-dimensional nuclear magnetic resonance techniques. Resonance assignments for the main-chain and side-chain protons are reported. Qualitative interpretation of nuclear Overhauser enhancement data allows the secondary structure of the two segments to be described, which indicate the patterns of NOEs found are consistent with an alpha-helix between Val-3 and Cys-14 in the N-terminus, and between Lys-88 and Asn-103 in the C-terminus. Two alpha-helices are found to be maintained. Comparison of the long-range NOEs of Im-cyt c relative to the crystal structure of native cytochrome c reveals apparent conformational changes of some side-chains especially those close to heme pocket within the N- and C-terminal helices resulting from the binding of imidazole to iron by displacing native Met-80 side-chain. The explanation for ligand-induced changes within the N- and C-helices is therefore suggested.
Amino Acid Sequence↗
Similarities and differences between the properties of native and recombinant Na+/K+-ATPases.
Progress of mutagenesis studies on the relation of the structure of Na+/K+-ATPase to its reaction mechanism has been impeded by the paucity of information on the properties of small amounts of impure recombinant enzyme obtained in the currently available expression systems, and the uncertainty of whether expression in a new environment alters the various catalytic activities of this membrane enzyme. Hence, our aim was to make a detailed comparison of the properties of the extensively studied canine kidney Na+/K+-ATPase with those of its alpha1,beta1 subunits expressed in the baculovirus-infected Sf-9 cells. The active fraction of the recombinant enzyme, containing 10-20% of the expressed a subunits, was found to have normal molar activity, all the partial reactions, and the ability to catalyze ATP-dependent Na+/K+ exchange after reconstitution into proteoliposomes. Comparison of steady-state kinetics of the hydrolytic activities of recombinant and native enzymes showed that (a) ATP and Na+ plots of Na+-ATPase were the same in the two preparations; (b) apparent K+ affinity of K+-phosphatase of recombinant enzyme was lower than that of kidney enzyme; and (c) for Na+/K+- ATPase activity, apparent K+ affinity of recombinant enzyme was lower, and its apparent Na+ and ATP affinities were higher than those of kidney enzyme. The two enzymes had similar ADP- and K+-sensitive phosphointermediates, identical affinities for ouabain, and similar ligand sensitivities of dissociation rates of ouabain-enzyme complexes. Evidently, the recombinant enzyme has reduced affinity at cytoplasmic K+ sites, but no changes at multiple Na+, ATP, and ouabain binding sites. Likely causes of this selective change include altered glycosylation state of beta and interactions among active and inactive recombinant enzymes. The present results provide the necessary database for the appropriate use of an expression system in structure-function studies on canine alpha1,beta1 isoform of Na+/K+-ATPase, and indicate the need for similar studies on recombinant Na+/K+-ATPases obtained in other expression systems.
Animals↗
Unified description of collective bands of even-even nuclei and fingerprint of the nuclear gamma shape.
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Journal Article↗
A mutation in the lipoprotein lipase gene is the molecular basis of chylomicronemia in a colony of domestic cats.
Members of a domestic cat colony with chylomicronemia share many phenotypic features with human lipoprotein lipase (LPL) deficiency. Biochemical analysis reveals that these cats do have defective LPL catalytic activity and have a clinical phenotype very similar to human LPL deficiency. To determine the molecular basis underlying this biochemical phenotype, we have cloned the normal and affected cat LPL cDNAs and shown that the affected cat has a nucleotide change resulting in a substitution of arginine for glycine at residue 412 in exon 8. In vitro mutagenesis and expression studies, in addition to segregation analysis, have shown that this DNA change is the cause of LPL deficiency in this cat colony. Reduced body mass, growth rates, and increased stillbirth rates are observed in cats homozygous for this mutation. These findings show that this LPL deficient cat can serve as an animal model of human LPL deficiency and will be useful for in vivo investigation of the relationship between triglyceride rich lipoproteins and atherogenic risk and for the assessment of new approaches for treatment of LPL deficiency, including gene therapy.
Amino Acid Sequence↗
Isolation of a tripeptide from a random phage peptide library that inhibits P1,P4-diadenosine 5'-tetraphosphate binding to its receptor.
Extracellular P1,P4-diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have previously demonstrated specific receptors for Ap4A at the surface of cardiac myocytes (Walker et al., 1993a). In addition, we have isolated a monoclonal antibody (mAb TL4) that recognized the Ap4A receptor and inhibited binding of Ap4A to its receptor (Walker & Hilderman, 1993). As part of our effort to characterize the Ap4A receptor building domain, we screened a random phage peptide library with mAb TL4. After affinity purification of specifically bound phage, we isolated 38 individual phage clones. Twenty-eight of these clones bound mAb TL4 in ELISA and dot blot analyses. Twenty-two of the twenty-eight individual clones contained inserts with an RGS tripeptide sequence. Synthetic RGS peptide specifically inhibits the binding of mAb TL4 to its membrane receptor. Furthermore, the RGS peptide also inhibits [3H]Ap4A binding to its receptor. These data are consistent with the RGS peptide mimicking part of the mAb TL4 recognition site on the Ap4A receptor. The The RGS peptide may be used to help characterize the Ap4A receptor binding domain and to help determine the physiological significance of the interaction between Ap4A and its receptor.
Amino Acid Sequence↗
Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture.
Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a 60Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germline transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally.
Animals↗
Single-shot diffusion MRI of human brain on a conventional clinical instrument.
A single-shot diffusion MRI technique on a standard clinical 1.5T scanner is presented. The method incorporates the following elements: (a) an inversion RF pulse followed by a delay of 1.3 s to null cerebral spinal fluid (CSF) signal, (b) a stimulated echo sequence (TE = 56 ms, TM = 100 ms) to obtain strong diffusion weighting, (c) a single-shot gradient- and spin-echo (GRASE) sequence for imaging with a modified k-space trajectory and Carr-Purcell Meiboom-Gill (CPMG)-phase cycle. The trace of the diffusion coefficient obtained with this approach is in good agreement with values reported for animal brain, and for recent human studies. It is demonstrated that single-shot diffusion imaging of human brain is feasible on an unmodified standard instrument without high-gradient slew rate or extreme field homogeneity.
Brain↗
pH, EF-1alpha and the cytoskeleton.
One of the unexpected cellular components found interacting with the cytoskeleton is elongation factor 1 alpha (EF-1alpha). How this interaction is regulated is not clear, but pH may be a potent regulator. Interestingly, pH also regulates the amount of protein translation occurring in many cell systems. In this paper, the authors suggest that sequestration of EF-1alpha in the cytoskeleton may play a key role in regulating the spatial distribution of macromolecular assembly in a way that is dependent on cytoplasmic pH.
Journal Article↗
PCR differential display of immune gene expression in Trichoplusia ni.
The immune state of insects is defined by a set of proteins that is absent in the naive state. To explore the immune system of Trichoplusia ni in more detail we have employed a PCR differential display technique to compare the mRNA population of untreated last instar larvae to that of immunized animals. In the primary display, more than one hundred bands seemed induced upon bacterial challenge. When they were used as probes in Northern blots, 35% of these probes detected inducible mRNA species. Such probes were used to screen a cDNA library from immunized larvae. We isolated clones for T. ni homologs of cecropin A, lysozyme and attacin. One differentially expressed band hybridized to clones for BJHSP1, a hemacy-anin-related protein which is hormonally up-regulated in last instar larvae; this induction is probably not related to the bacterial infection. Still other probes recognized inducible mRNAs of 1.6 and 1.0 kb. The corresponding cDNA clones did not show strong sequence homology to any known proteins. We have demonstrated the potential of this PCR technique to display both known and unknown genes specific for the immune state of whole insects against a background of genes involved in larval development.
Amino Acid Sequence↗
F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction.
The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin-binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F-actin, we have studied the effect of F-actin on the ability of EF-1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa-tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF-1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus.
Actins↗
Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends.
Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non-membrane-associated protein translation may be occurring in vivo.
Actin Cytoskeleton↗
Orthotopic liver transplantation in two patients with hypertrophic obstructive cardiomyopathy.
Orthotopic liver transplantation (OLT) in patients with end-stage liver disease is a procedure associated with high cardiac output, low systemic vascular resistance (SVR), coagulopathy and the potential for significant blood loss. A feature of hypertrophic obstructive cardiomyopathy (HOCM) is left ventricular outflow tract obstruction which may be exacerbated by reduced SVR, reduced filling pressures, tachycardia and positive inotropy. We report two cases of OLT in patients with HOCM. Our anaesthetic technique involved the use of halothane and vecuronium and avoidance of drugs causing tachycardia and positive inotropy. Management was aided by intraoperative transoesophageal echocardiography which showed that filling pressures poorly reflected end-diastolic volumes. Volume administration, vasoconstrictors and avoidance of inotropes and chronotropes reduced the outflow tract obstruction which was particularly severe in the reperfusion period.
Anesthesia, General↗