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M Lei

Publications and source records attributed to M Lei.

At least 19 recordsLinked to original sources
PubMed1999

Relation between non-uniform hemodynamics and sites of altered permeability and lesion growth at the rabbit aorto-celiac junction.

Using the rabbit's aorto-celiac junction as a representative atherosclerotic model, the hemodynamics of a bifurcating blood vessel are numerically simulated and three hemodynamic parameters are compared. The wall shear stress (WSS), the oscillatory shear index (OSI), and the spatial wall shear stress gradient (WSSG) are considered in this study. Locally enhanced wall permeabilities and intimal macrophages are generally considered to be involved in atherogenesis, and here the primary concern is with the hemodynamic influence on these early stages of the disease process. In comparing the segmental averages of the indicator functions and previously published intimal white blood cell densities, only the WSSG shows a statistically significant correlation. All three indicators have selective strengths in determining sites of early lesion growth around the aorto-celiac flow divider. At the proximal end of the flow divider on the lateral side of the orifice, there are elevated values of the OSI as well as WSSG and low WSS values. Regions of elevated wall permeabilities compare with the regions of elevated WSSG along the lateral and distal portions of the flow divider. Largely dependent upon the present input pulse with reverse flow, the OSI indicates relatively high values throughout the flow domain, however, it is important when utilized in conjunction with low WSS regions. This study presents a rationale for further quantitative correlative studies in the rabbit model based on additional histological data sets.

Animals↗
PubMed1999

Heterogeneity of 4-aminopyridine-sensitive current in rabbit sinoatrial node cells.

The electrophysiological properties of sinoatrial (SA) node pacemaker cells vary in different regions of the node. In this study, we have investigated variation of the 4-aminopyridine (4-AP)-sensitive current as a function of the size (as measured by the cell capacitance) of SA node cells to elucidate the ionic mechanisms. The 10 mM 4-AP-sensitive current recorded from rabbit SA node cells was composed of transient and sustained components (Itrans and Isus, respectively). The activation and inactivation properties [activation: membrane potential at which conductance is half-maximally activated (Vh) = 19.3 mV, slope factor (k) = 15.0 mV; inactivation: Vh = -31.5 mV, k = 7.2 mV] as well as the density of Itrans (9.0 pA/pF on average at +50 mV) were independent of cell capacitance. In contrast, the density of Isus (0.97 pA/pF on average at +50 mV) was greater in larger cells, giving rise to a significant correlation with cell capacitance. The greater density of Isus in larger cells (presumably from the periphery) can explain the shorter action potential in the periphery of the SA node compared with that in the center. Thus variation of the 4-AP-sensitive current may be involved in regional differences in repolarization within the SA node.

4-Aminopyridine↗
PubMed1998

Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice.

Experimental lesions have been used widely to induce motoneuron (MN) degeneration as a model to test the ability of different trophic molecules to prevent lesion-induced alterations. However, the morphological mechanisms of spinal MN death following different types of lesions is not clear at the present time. In this study, we have characterized the morphological characteristics of MN cell death by examining DNA fragmentation and the ultrastructural and light microscopic morphological features of MNs following different types of spinal nerve injury (i.e., axotomy and avulsion) in the developing and adult mouse. In neonatal mice, axotomy induced cell death as well as the atrophy of MNs that survived the injury. DNA fragmentation could be detected by using the terminal deoxynucleotidyl transferase (TUNEL) method during the cell death process following neonatal axotomy, whereas TUNEL labeling was not observed following either neonatal or adult avulsion. However, with the exception of TUNEL labeling, the morphological characteristics of MN death following neonatal axotomy and avulsion were similar, and both resembled most closely the form of programmed cell death termed cytoplasmic or type 3B, which exhibits similarities as well as differences with currently accepted definitions of apoptosis. By contrast, adult avulsion resulted in a type of degeneration that resembled necrosis more closely. However, even there, the morphology was mixed, showing characteristics of both apoptosis and necrosis. These results indicate that the mode of MN degeneration is complex and is related to developmental age and type of lesion.

Animals↗
PubMed1998

Adenovirus VAI RNA antagonizes the RNA-editing activity of the ADAR adenosine deaminase.

The virus-associated VAI RNA of adenovirus is a small highly structured RNA that is required for the efficient translation of cellular and viral mRNAs at late times after infection. VAI RNA antagonizes the activation of the interferon-inducible RNA-dependent protein kinase, PKR, an important regulator of translation. The RNA-specific adenosine deaminase, ADAR, is an interferon-inducible RNA-editing enzyme that catalyzes the site-selective C-6 deamination of adenosine to inosine. ADAR possesses three copies of the highly conserved RNA-binding motif (dsRBM) that are similar to the two copies found in PKR, the enzyme in which the prototype dsRBM motif was discovered. We have examined the effect of VAI RNA on ADAR function. VAI RNA impairs the activity of ADAR deaminase. This inhibition can be observed in extracts prepared from interferon-treated human cells and from monkey COS cells in which wild-type recombinant ADAR was expressed. Analysis of wild-type and mutant forms of VA RNA suggests that the central domain is important in the antagonism of ADAR activity. These results suggest that VAI RNA may modulate viral and cellular gene expression by modulating RNA editing as well as mRNA translation.

Adenosine Deaminase↗
PubMed1998

Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases.

The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

Amino Acid Sequence↗
PubMed1998

Inhibition by Compound II, a sotalol analogue, of delayed rectifier current (iK) in rabbit isolated sino-atrial node cells.

The effects of Compound II, a sotalol analogue, on spontaneous electrical activity and on three membrane currents (the delayed rectifier current, iK, the long-lasting inward calcium current, i(Ca,L) and hyperpolarization activated inward current, i(f)) were investigated in rabbit isolated sino-atrial node cells by whole cell clamp with amphotericin-permeabilised patches. A submaximal concentration of Compound II (50 nM) had a significant effect on the time and voltage dependent activation of iK and caused a positive shift of the iK activation curve. As well as blocking i(Kr), it caused some degree of block of i(Ks). Block of iK by Compound II was found to be concentration dependent with an IC50 of approximately 40 nM. 1 microM Compound II nearly completely blocked iK without significantly affecting the peak current or I/V relationships of i(Ca,L) or i(f). 50 nM Compound II caused a significant prolongation of APD100 and of cycle length. It also decreased diastolic depolarization rate without significantly affecting MDP and action potential amplitude. It is concluded that Compound II, a sotalol analogue, slows spontaneous activity of isolated rabbit SA node cells through a selective inhibition of iK.

Action Potentials↗
PubMed1998

Swelling-induced decrease in spontaneous pacemaker activity of rabbit isolated sino-atrial node cells.

The heart responds to an increase in sino-atrial node wall stress with an augmentation in rate of contraction. It has been suggested that swelling-activated ion channels may play a key role in that response. This paper investigates directly the effects of cell swelling on spontaneous activity of rabbit isolated sino-atrial node pacemaker cells. The main finding is that sino-atrial node cells, studied in current clamp mode using amphotericin-permeabilized patches, decrease their spontaneous pacemaker rate by 24.2 +/- 7.8% (P < 0.01, n = 9) during 75% hyposmotic swelling. This response is opposite to the predicted impact of volume-activation of sarcolemmal ion conductances. Computer modelling (OXSOFT Heart v4.8) suggests that swelling-induced dilution of the cytosol, reduction in intracellular potassium concentration, and decrease in the delayed rectifier potassium current, IK, are leading mechanisms in the response. This is supported by voltage-clamp data that show a swelling-induced positive shift in the reversal potential of IK by between 5 and 10 mV (n = 7) and a reduction in amplitude of its rapidly activating component, IKr, (n = 6). Thus, spontaneously active sino-atrial node cells reduce pacemaking rate during swelling. This response cannot be explained by the known volume-activated sarcolemmal ion conductances, but appears to be dictated by other mechanisms including dilution of the cytosol and reduction in IK. The results re-enforce the view that cardiac responses to cell volume changes may be quite different from those to longitudinal stretch.

Animals↗
PubMed1997

Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis.

The initiation of DNA synthesis is an important cell cycle event that defines the beginning of S phase. This critical event involves the participation of proteins whose functions are regulated by cyclin dependent protein kinases (Cdks). The Mcm2-7 proteins are a family of six conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes. In Saccharomyces cerevisiae, members of the Mcm2-7 family undergo cell cycle-specific phosphorylation. Phosphorylation of Mcm proteins at the beginning of S phase coincides with the removal of these proteins from chromatin and the onset of DNA synthesis. In this study, we identified DBF4, which encodes the regulatory subunit of a Cdk-like protein kinase Cdc7-Dbf4, in a screen for second site suppressors of mcm2-1. The dbf4 suppressor mutation restores competence to initiate DNA synthesis to the mcm2-1 mutant. Cdc7-Dbf4 interacts physically with Mcm2 and phosphorylates Mcm2 and three other members of the Mcm2-7 family in vitro. Blocking the kinase activity of Cdc7-Dbf4 at the G1-to-S phase transition also blocks the phosphorylation of Mcm2 at this defined point of the cell cycle. Taken together, our data suggest that phosphorylation of Mcm2 and probably other members of the Mcm2-7 proteins by Cdc7-Dbf4 at the G1-to-S phase transition is a critical step in the initiation of DNA synthesis at replication origins.

Cell Cycle Proteins↗
PubMed1997

Computational design of a bypass graft that minimizes wall shear stress gradients in the region of the distal anastomosis.

PURPOSE: Recent experimental and theoretic studies show that large wall shear stress gradients characterize disturbed flow patterns associated with the location of myointimal hyperplasia, atheroma, or both. Graft-to-artery anastomoses that minimize wall shear stress gradients may reduce the degree of myointimal development and the propensity for thrombosis. This study analyzes the distribution of distal anastomotic wall shear stress gradients for conventional geometries and for the optimized geometry assuming idealized merging of the graft with the artery. METHODS: A validated computational fluid dynamics program was used to solve the transient three-dimensional partial differential equations and auxiliary equations that describe laminar incompressible blood flow. Time-averaged wall shear stresses and wall shear stress gradients were calculated for three distal graft-artery anastomoses: a standard end-to-side, a Taylor patch, and an optimized geometry. The latter was obtained iteratively by minimizing the local wall shear stress gradients and was analyzed under resting and exercise inflow waveforms. RESULTS: Both the standard and Taylor patch anastomoses have relatively high wall shear stress gradients in the regions of the toe and heel. For all flow inputs studied nonuniform hemodynamics in the optimized graft design are largely eliminated, and the time-averaged wall shear stress gradients are greatly reduced throughout the anastomotic zone. At resting flow the Taylor patch produces slightly lower wall shear stress gradients in the anastomotic region than the standard end-to-side anastomosis. The optimized design reduces wall shear stress gradients to almost one half of that of the standard and Taylor patch geometries. At exercise flow wall shear stress gradients almost triple in the standard anastomosis and increase approximately 30% in the Taylor patch. In contrast, the geometrically optimized design is basically independent of the type of flow input waveform in terms of time-averaged wall shear stress gradients and disturbed flow patterns. CONCLUSION: This study demonstrates that it is possible to design a terminal graft geometry for an end-to-side anastomosis that significantly reduces wall shear stress gradients. If the wall shear stress gradient is confirmed to be a major hemodynamic determinant of intimal hyperplasia and restenosis, these results may point to the design of optimal bypass graft geometries.

Algorithms↗
PubMed1997

Hemodynamic simulations and computer-aided designs of graft-artery junctions.

Severe occlusion of graft-artery junctions due to restenosis, e.g., excessive tissue overgrowth and renewed plaque formation, may occur within a few months or years after bypass surgery. Our hypothesis is that nonuniform hemodynamics, represented by large sustained wall shear stress gradients, trigger abnormal biological processes leading to rapid restenosis and hence early graft failure. In turn, this problem may be significantly mitigated by designing graft-artery bypass configurations for which the wall shear stress gradient (WSSG) is approximately zero and hence nearly uniform hemodynamics are achieved. Focusing on the distal end of several femoral artery bypass junctions, a validated finite volume code has been used to compute the transient three-dimensional velocity vector fields and its first and second surface derivatives in order to test the idea. Specifically, it is shown that the Taylor patch, which generates higher patency rates than standard end-to-side anastomoses, exhibits lower WSSG levels than standard configurations, and that further geometric design improvements reduce the WSSG in magnitude and local extent even more.

Algorithms↗
PubMed1997

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

Alleles↗
PubMed1997

Airway smooth muscle orientation in intraparenchymal airways.

Airway smooth muscle (ASM) shortening is the central event leading to bronchoconstriction. The degree to which airway narrowing occurs as a consequence of shortening is a function of both the mechanical properties of the airway wall as well as the orientation of the muscle fibers. Although the latter is theoretically important, it has not been systematically measured to date. The purpose of this study was to determine the angle of orientation of ASM (theta) in normal lungs by using a morphometric approach. We analyzed the airway tree of the left lower lobes of four cats and one human. All material was fixed with 10% buffered Formalin at a pressure of 25 cmH2O for 48 h. The fixed material was dissected along the airway tree to permit isolation of generations 4-18 in the cats and generations 5-22 in the human specimen. Each airway generation was individually embedded in paraffin. Five-micrometer-thick serial sections were cut parallel to the airway long axis and stained with hematoxylin-phloxine-saffron. Each block yielded three to five sections containing ASM. To determine theta, we measured the orientation of ASM nuclei relative to the transverse axis of the airway by using a digitizing tablet and a light microscope (x250) equipped with a drawing tube attachment. Inspection of the sections revealed extensive ASM crisscrossing without a homogeneous orientation. The theta was clustered between -20 degrees and 20 degrees in all airway generations and did not vary much between generations in any of the cats or in the human specimen. When theta was expressed without regard to sign, the mean values were 13.2 degrees in the cats and 13.1 degrees in the human. This magnitude of obliquity is not likely to result in physiologically important changes in airway length during bronchoconstriction.

Animals↗
PubMed1997

Nitric oxide can increase heart rate by stimulating the hyperpolarization-activated inward current, I(f).

We investigated the chronotropic effect of increasing concentrations of sodium nitroprusside (SNP, n = 8) or 3-morpholinosydnonimine (SIN-1, n = 6) in isolated guinea pig spontaneously beating sinoatrial node/atrial preparations. Low concentrations of NO donors (nanomolar to micromolar) gradually increased the beating rate, whereas high (millimolar) concentrations decreased it. The increase in rate was (1) enhanced by superoxide dismutase (50 to 100 U/mL, n = 6), (2) prevented by the guanylyl cyclase inhibitors 6-anilino-5,8-quinolinedione (5 mumol/L, n = 6) or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (10 mumol/L, n = 6), and (3) mimicked by 8-bromo-cGMP (n = 6) with no additional positive chronotropic effect of SIN-1 (n = 5). The response to 10 mumol/L SNP (n = 28) or 50 mumol/L SIN-1 (n = 16) was unaffected by IcaL antagonism with nifedipine (0.2 mumol/L) but was abolished after blockade of the hyperpolarization-activated inward current (I(f)) by Cs+ (2 mmol/L) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride (1 mumol/L). The effect on I(f) was further evaluated in rabbit isolated patch-clamped sinoatrial node cells (n = 21), where we found that 5 mumol/L SNP or SIN-1 caused a reversible Cs(+)-sensitive increase in this current (+130% at -70 mV and +250% at -100 mV). In conclusion, NO donors can affect pacemaker activity in a concentration-dependent biphasic fashion. Our results indicate that the increase in beating rate is due to stimulation of I(f) via the NO-cGMP pathway. This may contribute to the sinus tachycardia in pathological conditions associated with an increase in myocardial production of NO.

Animals↗
PubMed1996

Altered development of spinal cord in the mouse mutant (Patch) lacking the PDGF receptor alpha-subunit gene.

The platelet-derived growth factor receptor alpha subunit (PDGFR alpha) is expressed by glial precursors, glial cells, and some peripheral neurons during normal rodent development. Its ligands are expressed ubiquitously in neurons, including sensory and motor neurons. Thus, neuronally secreted PDGF-A may play a paracrine role in the development of both glial cells and peripheral neurons. The Patch (Ph) mutation, which is a deletion of the PDGFR alpha, is a homozygous embryonic lethal mutation in the mouse. Previously, several developmental abnormalities, including deficiencies in connective tissues in many organs, aberrant neural crest cell migration, and defects in non-neuronal derivatives of crest cells, have been shown to be associated with the Patch mutation. However, whether and the extent to which motor and sensory neurons are affected by the mutation are not known. Here, we have examined the survival and/or morphological differentiation of spinal motor and sensory (dorsal root ganglion) neurons during the period of naturally occurring cell death, i.e., between E14 and E18, in control and Ph/Ph mice. The results show a 65-70% decrease in motor and sensory neuron numbers in Ph/Ph mice, compared to controls, at all stages examined. Furthermore, motoneurons in Ph/Ph mice were significantly smaller than those in controls. Because of the bidirectional nature of neuron-glial cell interactions, these results suggest that PDGFR alpha plays an important role in glial cell development and, thus, indirectly in neuronal cell development or, alternatively, that PDGF and the PDGFR alpha are directly involved in peripheral neuron survival and development by an autocrine/paracrine mechanism.

Animals↗
PubMed1996

Accumulation of S100 beta mRNA and protein in cerebellum during infancy in Down syndrome and control subjects.

S100 protein is a 20 kDA calcium-binding protein that accumulates during CNS maturation in mammals. The human gene coding for the beta subunit of S100 protein (S100 beta) is located on chromosome 21, in a subtelomeric position in 21q22.3. In order to investigate the effect of trisomy 21 on S100 beta gene expression, we performed Southern, Northern and Western blot analysis on DNA, RNA and protein, respectively, extracted from the cerebellum of control and Down syndrome (DS) subjects aged 1-18 months. Southern blot analysis revealed a novel EcoRI polymorphism in the S100 beta gene in two of 15 DNA samples examined, and a 1.5 gene dosage for S100 beta in DS. Northern and Western blot analysis showed an approximately 10-fold increase in S100 beta mRNA and protein levels between 1 and 18 months. No differences in the rates of accumulation of S100 beta mRNA and protein were observed between DS and normal subjects. These results demonstrate an increase in S100 beta mRNA and protein levels during infancy indicative of postnatal astrocytic maturation and show that there is no gross deregulation in the expression of the S100 beta gene in DS as a consequence of trisomy 21.

Age Distribution↗
PubMed1996

A focal stress gradient-dependent mass transfer mechanism for atherogenesis in branching arteries.

A new arterial wall permeability function, based on the local wall shear stress gradient, has been developed and employed to simulate enhanced low density lipoprotein transfer across the endothelium. the atherosclerotic model used is that of the aorto-celiac junction of rabbits. The experimentally validated computer simulation model for convection mass transfer provides further evidence that the wall shear stress gradient is a reliable predictor of critical atherogenic sites in branching arteries. Some of the underlying biological aspects of atherogenesis due to locally significant and sustained wall shear stress gradient values are briefly discussed.

Animals↗
PubMed1996

Two components of the delayed rectifier potassium current, IK, in rabbit sino-atrial node cells.

The delayed rectifier current was studied in rabbit isolated sino-atrial (SA) node cells using the whole-cell voltage clamp technique with amphotericin-permeabilized patches. The envelope of tails test indicated that in SA node cells the decay of IK (IK.tall) comprises two distinct current components similar to the specific fast and slow components of IK (IKr and JKs) that have been found in atrial and ventricular myocytes. Dofetilide, a Class III antiarrhythmic agent and a selective blocker of IKr. separated the delayed rectifier current into drug-sensitive current (IKr) and drug-insensitive current (IKs). The dofetilide-sensitive current activated rapidly and it showed two components of deactivation, the larger of which was very slow, while the dofetilide-insensitive current activated more slowly and deactivated quickly. The effects of propofol, an IKs blocker, on the delayed rectifier current were also investigated. The results show that IKs contributes to IK.tall and that the more positive the membrane potential, the more the contribution of IKs. The ratio of IKs to IKr in tail currents at -40 mV after a 1 s clamp pulse to +40 mV was 0.3-0.4:1. Dofetilide slowed spontaneous activity, suggesting that IKr contributes to the pacemaker activity of the SA node cell.

Animals↗