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M D Jones

Publications and source records attributed to M D Jones.

At least 55 records · Page 3Linked to original sources
PubMed1996

Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli.

Oxidative folding of recombinant human stem cell factor (rhSCF) produced in Escherichia coli was investigated in vitro. Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography. Both I-1 and I-2 contain a native-like disulfide bond, Cys4-Cys89 and Cys43-Cys138, respectively, and I-3 forms a mispaired disulfide, Cys43-Cys89. These forms appear to reach steady state equilibrium and are important folding intermediates. I-1 was found to be the prominent intermediate that directly folds into native rhSCF (N); and the thermodynamically less stable I-2 favors rearrangment into I-1. I-3 may serve as an intermediate for disulfide rearrangment between I-1 and I-2. I-4 and I-5, which are disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF folding. Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by high performance liquid chromatography, proceed with the remaining oxidative folding process after reconstitution. Iodoacetate-trapped I-1 and I-2 contain low alpha-helical content and some tertiary structure, while I-3 and reduced rhSCF have little ordered structure. Gel filtration/light-scattering experiments indicate that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating that rhSCF dimerization precedes formation of disulfide bonds. I-1, I-2, I-3, and the C43,138A analog lacking Cys43-Cys138 bond are not biologically active or exhibit significantly lower activity. The two disulfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biological activities.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of a disulfide-linked human stem cell factor dimer. Biochemical, biophysical, and biological comparison to the noncovalently held dimer.

Distinct from the noncovalently linked recombinant human stem call factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF. Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds. The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular. Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out. The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography. However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer. Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures. In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells. The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer. Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer.

Amino Acid Sequence↗
PubMed1996

Exit from G2 phase after 2 Gy gamma irradiation is faster in radiosensitive human cells with high expression of the RAF1 proto-oncogene.

We have previously noted that high endogenous expression of the protein product of the full-length RAF1 proto-oncogene is related to relative intrinsic cellular radiosensitivity in 19 human cells lines in vitro. This appeared to be unrelated to the parameters of cell kinetics. In rodent and human cell lines transfected with dominant oncogenes, including Myc and MYC, Hras and HRAS and SV40, increased radioresistance has been accompanied by increased delay in progress through the G2 phase of the cell cycle after irradiation. We have thus examined the putative relationship between RAF1 expression and postirradiation perturbation of G2 phase in six of the human cell lines for which data have been reported previously. These lines exhibit a wide range of both radiosensitivity and Raf1 protein levels as measured previously by Western blotting. We report here that the cell lines whose cells appear to exit more rapidly from G2 phase are more radiosensitive (r = 0.91, P = 0.01) and express high levels of Raf1 protein (r = -0.93, P = 0.006).

Cell Line↗
PubMed1995

Postmortem electrical excitability of skeletal muscle: preliminary investigation of an animal model.

Presented is a preliminary study of the feasibility of using an animal model to investigate the postmortem time-dependent decay in muscular response, when subjected to electrical stimulation and to develop an animal model for the assessment of the use of electrical excitability as a method for determining the postmortem period of a human corpse. Analysis of the results shows a correlation between the rate of decay of muscular response with the postmortem interval.

Animals↗
PubMed1995

Simultaneous identification of bacterial virulence genes by negative selection.

An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

Animals↗
PubMed1995

Post-translational processing of membrane-associated neu differentiation factor proisoforms expressed in mammalian cells.

Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.

Amino Acid Sequence↗
PubMed1995

Continuous fetal heart rate monitoring during bone marrow harvesting in pregnancy.

To date, our computer-assisted search failed to report any case involving a gravid patient donating her bone marrow for harvesting. It is known that bone marrow harvesting causes a significant decrease in the donor's blood volume and therefore this can be potentially detrimental to both the mother and the fetus. We report the first case of the gravid donor in which fetal heart rate (FHR) during bone marrow harvesting has been studied. Decreased beat to beat variability and disappearance of accelerations were noted. The FHR returned to normal shortly after the procedure was terminated.

Adolescent↗
PubMed1995

Cerebral lactate metabolism in near-term fetal sheep.

The present study was designed to see if lactate can cross the blood-brain barrier of the near-term fetal sheep and replace glucose as an oxidative substrate during normoglycemia and acute insulin-induced hypoglycemia. Cerebral uptake of glucose, oxygen, lactate, and [14C]lactate as well as cerebral production of 14CO2 were measured under three conditions: 1) normoglycemia-normolactemia, 2) acute hypoglycemia-normolactemia, and 3) hypoglycemia-steady-state hyperlactemia. Although uptake of tracer [14C]lactate was consistent, there was no net uptake of unlabeled lactate during either normoglycemia or hypoglycemia. When arterial lactate concentration was raised from 2.2 +/- 0.5 to 3.3 +/- 0.4 (SE) mM by sodium lactate infusion, however, lactate was taken up. Comparison of cerebral [14C]lactate uptake with 14CO2 production indicated that the principal metabolic fate of lactate is oxidation. At increased concentrations, exogenous lactate accounted for approximately 7% of cerebral oxygen consumption. This study demonstrates that lactate crosses the blood-brain barrier of the near-term fetal sheep, is oxidized, and at elevated concentrations can partially replace glucose as an oxidative substrate during acute hypoglycemia.

Animals↗
PubMed1995

Hypoxia, hypercapnia, and hypertension: their effects on pulsatile cerebral blood flow.

Pulsatile cerebral blood flow reflects characteristics of arterial blood pressure as well as the structure and mechanical properties of the cerebrovascular network. Although the effects of changes in systemic blood gases and blood pressure on mean cerebral flow are established, their effects on pulsatile cerebral blood flow are unknown. These studies assessed the effects of hypoxia-hypercapnia (combined; both arterial PO2 and PCO2 approximately 55 Torr) and acute hypertension (+30-35 mmHg by aortic occlusion) on pulsatile cerebral blood flow in ketamine-anesthetized rabbits. We characterized the relationship between pulsatile systemic blood pressure (Millar catheter) and cerebral cortical capillary blood-flow (laser-Doppler) by calculating the transfer function, a frequency-domain expression that relates amplitudes and phase angles of flow output to those of the pressure input. During hypoxia-hypercapnia, mean flow increased 17% (P < 0.001), but the amplitude and contour of pulsatile cortical blood flow were unchanged (P > 0.10). Although aortic occlusion, during hypoxia-hypercapnia as well as during normoxia-normocapnia, increased systemic pulse pressure by 40%, the amplitude of cortical flow pulsations was unaffected. Changes in dynamic properties of the cerebral vasculature (P < 0.0001 by analysis of the transfer function) minimized alterations in pulsatile cortical blood flow and thus intrabeat vessel wall stress during acute hypertension; on the basis of analysis of an electrical analogue, we propose that these changes reflect alterations in both resistance and compliance.

Animals↗
PubMed1994

Transformation by polyoma virus middle T-antigen involves the binding and tyrosine phosphorylation of Shc.

Polyoma virus middle T-antigen converts normal fibroblasts to a fully transformed, tumorigenic phenotype. It achieves this, at least in part, by binding and activating one of the non-receptor tyrosine kinases, pp60c-src, pp62c-yes or pp59c-fyn (reviewed in refs 2 and 3). As a result, middle T-antigen itself is phosphorylated on tyrosine residues, one of which (Tyr 315) acts as a binding site for the SH2 domains of phosphatidylinositol-3'OH kinase 85K subunit. Here we show that another tyrosine phosphorylation site in middle T-antigen (Tyr 250; refs 4, 5) acts as a binding region for the SH2 domain of the transforming protein Shc. This results in Shc also becoming tyrosine-phosphorylated and binding to the SH2 domain of Grb2 (ref. 10). This probably stimulates p21ras activity through the mammalian homologue of the Drosophila guanine-nucleotide-exchange factor Sos (reviewed in ref. 11). We suggest that middle T-antigen transforms cells by acting as a functional homologue of an activated tyrosine kinase-associated growth-factor receptor.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Peptide map analysis of recombinant human granulocyte colony stimulating factor: elimination of methionine modification and nonspecific cleavages.

Procedures for HPLC peptide map analysis of recombinant human granulocyte colony stimulating factor include reduction and S-carboxymethylation of the denatured protein, as well as protease digestion with Staphylococcus aureus endoproteinase Glu-C followed by reverse-phase liquid chromatographic separations. Under nonoptimized experimental conditions analytical problems including methionine modification during carboxymethylation, as well as generation of large, insoluble fragments and nonspecific cleavages during proteolytic digestion, occurred. These problems have complicated the analysis of peptide digests and affected the performance of HPLC columns. This report describes the elimination of these problems by optimizing peptide mapping procedures. We found that mild reduction and alkylation conditions can prevent methionine modification, while protease digestion in the presence of urea at room temperature alleviates generation of peptides derived from incomplete digestion and nonspecific cleavage by endoproteinase Glu-C. Peptide maps generated using the optimized procedures contain fewer peptide peaks with higher recovery. Elimination of incomplete digestion, which generates fewer larger, insoluble peptides, substantially extends the life of reverse-phase columns. The optimized method reproducibly produced peptide maps suitable for routine analysis.

Amino Acid Sequence↗
PubMed1994

Characterization and tissue-specific expression of the human 'very low density lipoprotein (VLDL) receptor' mRNA.

A cDNA has been isolated from human heart that is homologous to a member of the low density lipoprotein (LDL) receptor gene family recently identified in rabbit. It was named the very low density lipoprotein (VLDL) receptor, although its physiological function is not yet known. The predicted human protein shows 97.4% sequence homology to the rabbit protein, much more than the approximately 75% observed between their LDL receptor proteins. The sequence is also highly conserved in the hamster and the African green monkey. The mRNA was identified as a 3.9 kb transcript by Northern blotting in Hep G2 cells, cultured arterial smooth muscle cells and human skin fibroblasts, where its level was unaffected by sterols. The mRNA was not detected in EBV-lymphoblasts or in monocyte-macrophages by Northern blotting or by RT-PCR. In human tissues in vivo, the mRNA was expressed predominantly in heart and skeletal muscle, and also in ovary and kidney, but not in the liver. Although the 3.9 kb mRNA was the major transcript, a larger variant of 5.2 kb was also detectable and was predominant in skeletal muscle. Amplification of the mRNA from cultured human cells also revealed a potential splice variant that lacked 84 bp coding for a region equivalent to the O-linked sugars domain of the LDL receptor. It was a minor component in most cell types, but was predominant in Hep G2 cells.

Amino Acid Sequence↗
PubMed1994

Cerebrovascular response to carbon dioxide in lambs receiving extracorporeal membrane oxygenation.

OBJECTIVE: To determine if the institution of extracorporeal membrane oxygenation (ECMO) alters the cerebrovascular response to changes in PaCO2. DESIGN: Prospective, randomized, controlled animal trial. SUBJECTS: Anesthetized 1- to 7-day-old lambs of mixed breed (n = 16). SETTING: University research laboratory. INTERVENTIONS: The experimental group was placed on ECMO. Both experimental and control groups (n = 8) were exposed to three concentrations of PaCO2 (hypocarbia, normocarbia, and hypercarbia) by varying mechanical ventilation and by adding carbon dioxide to the ventilator gases. MEASUREMENTS AND MAIN RESULTS: Cerebral blood flow was measured by the radiolabeled microsphere method. Arterial blood gases and sagittal sinus blood gases were drawn at the time of cerebral blood flow measurement so that cerebral metabolism, cerebral oxygen transport, and extraction could be calculated. In the control group, as PaCO2 increased from 34 +/- 2 (SD) to 53 +/- 4 torr (4.5 +/- 0.3 to 7.1 +/- 0.5 kPa), cerebral blood flow increased from 53 +/- 12 to 147 +/- 50 mL/min/100 g. This increase in cerebral blood flow was not different from that of the ECMO group, where PaCO2 increased from 33 +/- 2 to 56 +/- 3 torr (4.4 +/- 0.3 to 7.5 +/- 0.4 kPa) and cerebral blood flow increased from 48 +/- 17 to 106 +/- 38 mL/min/100 g. As PaCO2 decreased from 34 +/- 2 to 19 +/- 2 torr (4.5 +/- 0.27 to 2.5 +/- 0.27 kPa), cerebral blood flow decreased from 53 +/- 12 to 43 +/- 8 mL/min/100 g in the control group. This decrease was not different from that of the ECMO group, where cerebral blood flow decreased from 48 +/- 17 to 39 +/- 10 mL/min/100 g as PaCO2 decreased from 33 +/- 2 to 22 +/- 3 torr (4.4 +/- 0.3 to 2.9 +/- 0.4 kPa). When regional cerebral blood flow was analyzed, no regional differences in the cerebrovascular responses to PaCO2 between ECMO and control groups were found. The cerebral metabolic rate was not different between ECMO and control groups at any level of PaCO2, nor was the cerebral metabolic rate affected by changes in PaCO2. Oxygen extraction increased with hypocarbia and decreased with hypercarbia in a similar fashion in both ECMO and control groups. CONCLUSION: The cerebrovascular response to changes in PaCO2 was unaffected by ECMO.

Animals↗
PubMed1994

Cerebrovascular autoregulation during fetal development in sheep.

There are scant data regarding the development of cerebrovascular autoregulation in fetuses. We tested the hypothesis that a decrease in cerebrovascular resistance (CVR) at reduced cerebral perfusion pressure (CPP) is absent in midgestation and near-term fetal sheep. Catheters were chronically implanted for microsphere determination of cerebral blood flow (CBF) in 9 fetuses at 92 days and in 10 fetuses at 132 days gestation (full term = 145 days). CPP was reduced by ventricular infusion of artificial cerebrospinal fluid. In 92-day fetuses, CPP was reduced stepwise from 35 to 25 and 18 mmHg and CBF decreased from 52 +/- 5 to 43 +/- 4 and 27 +/- 5 (SE) ml.min-1 x 100 g-1, respectively. Half of the immature fetuses showed some reduction in CVR at moderate reduction in CPP; however, there was no significant change in CVR in the group as a whole (from 0.72 +/- 0.06 to 0.61 +/- 0.04 and 0.89 +/- 0.20 mmHg.ml-1.min.100 g). In 132-day fetuses, CPP was reduced from 45 to 33 and 28 mmHg and CBF was unchanged (from 105 +/- 7 to 97 +/- 11 and 89 +/- 8 ml.min-1 x 100 g-1). CVR decreased from 0.45 +/- 0.05 to 0.41 +/- 0.08 and 0.33 +/- 0.03 mmHg.ml-1.min.100 g. There were no significant changes in arterial blood gases at reduced CPP in either age group. We conclude that cerebrovascular autoregulation at reduced CPP is not well developed at 92 days (0.63 gestation) in fetal sheep but that autoregulatory capacity is evident near term. We speculate that poor autoregulation may place the premature fetal brain at risk for injury.

Animals↗
PubMed1994

Cerebral responses to single and multiple cocaine injections in newborn sheep.

Newborn infants exposed to cocaine near birth display a wide range of neurologic abnormalities, but the mechanism or mechanisms for these injuries remain unknown. We studied the cerebral effects of a single acute dose (4 mg/kg; n = 7) and multiple binge doses (4 mg/kg hourly for 5 h; n = 7) of i.v. cocaine in unanesthetized newborn (5 +/- 1 d old) sheep. We measured cerebral blood flow, mean arterial blood pressure, arterial blood gases, and cerebral O2 metabolism. Measurements were made at baseline; 30 s; and 5, 15, and 60 min after a single injection of cocaine in the acute group and at the same time intervals after the 5th dose of cocaine in the binge group. CBF increased by 98 +/- 68% (mean +/- SD) at 30 s after a single acute dose and by 97 +/- 94% at 30 s after the 5th of five hourly binge doses. Although it returned to baseline by 5 min in the acute group, cerebral blood flow remained elevated 5, 15, and 60 min after the 5th cocaine dose in the binge group. At 30 s, mean arterial blood pressure increased by 57 +/- 21% in the acute group and 46 +/- 15% in the binge group. In both groups, mean arterial blood pressure remained elevated at 5 min. Although no change occurred in cerebral O2 metabolism in the acute group, an increase in cerebral O2 consumption (7.4 +/- 1.3 mL/100 g/min versus 5.5 +/- 1.1 at baseline) was observed at 5 min in the binge group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Identification and analysis of the transport/capsid assembly protein (tp/cap) gene of human herpesvirus-6 (HHV6).

The transport/capsid assembly protein (tp/cap) gene of human herpesvirus 6 (HHV6) strain U1102 has been identified and localized on the restriction enzyme map of the viral genome, to the EcoRI-Q fragment. The complete DNA sequence of the tp/cap gene was determined. The tp/cap gene encodes a protein product of 726 amino acids and has the strongest similarity with the homologous gene (HCMV UL56) from HCMV. Upstream of the tp/cap open reading frame is the gene for the major DNA binding protein (mdbp) and downstream is the glycoprotein B (gB) gene. This gene block arrangement is common to all herpesviruses.

Amino Acid Sequence↗