Biomedical subjects

M Chen

Publications and source records attributed to M Chen.

At least 469 records · Page 26Linked to original sources

PubMed1997

Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apCAM in Aplysia sensory neurons.

The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM at the surface membrane of the sensory neuron. We have now used epitope tags to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked) and find that only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation.

Animals↗

PubMed1997

Antiprotozoal compounds from Asparagus africanus.

Two antiprotozoal compounds have been isolated from the roots of Asparagus africanus Lam. (Liliaceae), a new sapogenin, 2 beta, 12 alpha-dihydroxy-(25R)-spirosta-4,7-dien-3-one (1), which was named muzanzagenin, and the lignan (+)-nyasol (2), (Z)-(+)-4,4'-(3-ethenyl-1-propene-1,3-diyl)-bisphenol. The structure of the sapogenin was elucidated by MS and by 1D and 2D NMR methods and established by a single crystal X-ray analysis. (+)-Nyasol potently inhibits the growth of Leishmania major promastigotes, the IC50 being 12 microM, and moderately inhibits Plasmodium falciparum schizonts with the IC50 49 microM. These concentrations only moderately affect the proliferation of human lymphocytes. Muzanzagenin showed a moderate in vitro activity in all three tests, the IC50 against leishmania promastigotes was 70 microM, and against four different malaria schizont strains the IC50 values were 16, 163, 23, and 16 microM, respectively.

Animals↗

PubMed1997

Double coupling Edman chemistry for high-sensitivity automated protein sequencing.

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is a promising new method for the analysis of protein sequencing products. It gives 10 zmol (1 zmol = 10(-21) mol) limits of detection (3 sigma) for fluorescein thiohydantoin (FTH) amino acids. We have developed a separation for the (FTH)-amino acid products generated from 18 of the 20 coded amino acids. The extremely low volume requirement associated with CE-LIF makes it incompatible with commercial sequencers. For this reason, we have also been developing a miniaturized sequencer that can be more easily coupled to our detection system. Both the CE-LIF system and the miniaturized sequencer are described.

Amino Acid Sequence↗

PubMed1997

Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death.

Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.

Animals↗

PubMed1997

Noradrenergic activity differentially regulates the expression of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat brain.

In a previous study, it was observed that the activity of rolipram-sensitive, low-Km, cyclic AMP phosphodiesterase (PDE4) was decreased in vivo with diminished noradrenergic stimulation. The results of the present experiments indicated that the reduction in the activity may be associated with down-regulation of PDE4 protein. Immunoblot analysis using PDE4-specific, subfamily-nonspecific antibody (K116) revealed four major bands of PDE4 in rat cerebral cortex; those with apparent molecular masses of 109 and 102 kDa are variants of PDE4A. Diminished noradrenergic activity, produced by intracerebroventricular infusion of 6-hydroxydopamine (6-OHDA) or chronic subcutaneous infusion of propranolol, decreased the intensities of the protein bands for the 109- and 102-kDa PDE4A variants in rat cerebral cortex but not of the 98- or 91-kDa PDE4 forms. 6-OHDA-induced noradrenergic lesioning also decreased the content of 102-kDa PDE4A in hippocampus as labeled by PDE4A-specific antibody (C-PDE4A). Enhanced noradrenergic stimulation up-regulated PDE4 in cerebral cortex. This was indicated by the finding that repeated treatment with desipramine increased the intensity of the protein band for the 102-kDa PDE4 but not for the other variants of PDE4. These results suggest that PDE4 subtypes are differentially regulated at the level of expression, as evidenced by an apparent change in the amount of PDE4 protein, following changes in noradrenergic activity. These observations are consistent with the notion that PDE4s, especially the PDE4A variants with molecular masses of 109 and 102 kDa, play an important role in maintaining the homeostasis of the noradrenergic signal transduction system in the brain and may be involved in the mediation of antidepressant activity.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Differential effects of neuropeptide Y type 2 receptor activation on responses of rat ventral posteromedial thalamus neurons to surround vibrissae and trigeminal subnucleus interpolaris stimulation.

Single cell recording, receptive field analysis, and microejection techniques were used to evaluate the effects of neuropeptide Y (NPY) type 2 (Y2) receptor activation on the responses of vibrissae-sensitive ventral posteromedial thalamic (VPM) neurons. Activation of this receptor with the C-terminal fragment, NPY[13-36] had no significant effect on responses evoked by deflection of principal or surround vibrissae or electrical stimulation of the trigeminal principal sensory nucleus. However, it did significantly decrease (p < 0.01) responses evoked by electrical stimulation of subnucleus interpolaris (SpI) by a mean (+/- standard error) of -31.0 +/- 8.7%). The finding that responses to surround vibrissa stimulation were not significantly decreased by a manipulation that did significantly attenuate SpI- evoked responses suggests that this nucleus is not the sole source of surround vibrissae input to VPM neurons.

Animals↗

PubMed1997

A multiple comparison procedure to control the strong stagewise family error rate in comparing test treatments and a control.

Multiple comparisons are commonly seen in clinical trials and many other fields. An example, which is the focus of this paper, is the comparison of several test treatments (possibly different doses of a compound) with placebo (control). It is well known that steps must be taken to control the type I error rate when multiple testing is performed. We introduce the concept of the strong stagewise family error rate and propose a multiple comparison procedure to control this error rate. The proposed procedure is compared to Dunnett's step-down procedure when there are two test treatment groups and a placebo group.

Clinical Trials as Topic↗

PubMed1997

Lyme borreliosis in transgenic mice tolerant to OspA from Borrelia burgdorferi 25015.

Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface protein A (OspA) from the weakly virulent B. burgdorferi 25015 was assessed because the response to OspA has been implicated in protective immunity and is suspected in disease pathogenesis. B. burgdorferi 25015 is infectious but does not cause significant arthritis in C3H/HeN mice. Tolerance of mice to B. burgdorferi N40 OspA had no effect on disease. Since B. burgdorferi N40 is more virulent than and genetically distinct from B. burgdorferi 25015, the generality of these findings was investigated. OspA-25015-transgenic mice were tolerant to OspA-25015 and did not develop OspA antibodies or cellular responses following immunization with OspA-25015. The development and resolution of Lyme arthritis was similar in transgenic and nontransgenic littermates, suggesting that ablation of the OspA-25015 response does not cause B. burgdorferi 25015 to become more arthritogenic.

Animals↗

PubMed1997

The novel oxygenated chalcone, 2,4-dimethoxy-4'-butoxychalcone, exhibits potent activity against human malaria parasite Plasmodium falciparum in vitro and rodent parasites Plasmodium berghei and Plasmodium yoelii in vivo.

Previous studies have shown that licochalcone A, an oxygenated chalcone, exhibits antileishmanial and antimalarial activities. The present study was designed to examine the antimalarial activity of an analog of licochalcone A, 2,4-dimethoxy-4'-butoxychalcone (2,4mbc). 2,4mbc inhibited the in vitro growth of both a chloroquine-susceptible (3D7) and a chloroquine-resistant (Dd2) strain of Plasmodium falciparum in a [3H]hypoxanthine uptake assay. The in vivo activity of 2,4mbc was tested in mice infected with Plasmodium berghei or Plasmodium yoelii and in rats infected with P. berghei. 2,4mbc administered either orally, intraperitoneally, or subcutaneously for 5 days protected the mice from otherwise lethal infections of these parasites. 2,4mbc administered orally for 5 days reduced parasitemia in the rats infected with P. berghei. These results demonstrate that 2,4mbc exhibits potent antimalarial activity and might be developed into a new antimalarial drug.

Animals↗

PubMed1997

Genetic evidence for mother-to-infant transmission of hepatitis G virus.

Mother-to-infant transmission of hepatitis G virus (HGV [or GBV-C]) was studied in sera from 42 mothers at high risk for bloodborne infections and from their 45 infants (3 twin pairs). Seven (17%) of the mothers had HGV RNA in serum by a polymerase chain reaction assay. One of the 8 (12.5%) infants born to HGV-infected mothers became positive for HGV at 3 months of age. He remained HGV-infected throughout the study (42 months), with no signs of liver disease. His twin sister remained HGV-negative despite the presence of serum and salivary HGV in both the mother and the brother. Analysis of HGV sequences from the infected mother and the infected child confirmed a genetic link between the virus of the mother and the infected child. Thus, mother-to-infant transmission of HGV, presumably occurring at partus, may cause persistent HGV viremia.

Amino Acid Sequence↗

PubMed1997

Protection of hematopoietic progenitors from ultraviolet C by interleukin-1 and tumor necrosis factor-alpha.

Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) can protect hematopoietic progenitors from the toxicity of 4-hydroperoxycyclophosphamide (4-HC) and gamma radiation. We hypothesize that IL-1 and TNF-alpha may be inducing a universal stress reaction in hematopoietic progenitors. In this study, we examined their protective effects against ultraviolet C (UVC) compared with that seen against 4-HC using colony formation assays and flow cytometric analysis. We demonstrated that 20 h preincubation with IL-1 or TNF-alpha or both protected normal hematopoietic colony-forming cells (CFCs) from UVC. Colony formation assays and flow cytometric analysis of the cells protected from either 4-HC or UVC revealed that similar proportions of hematopoietic progenitors are protected in the IL-1 and TNF-alpha group in comparison to control. Furthermore, at least 20 h of preincubation with the two cytokines was needed for optimal protection. The addition of 2 micrograms/ml cycloheximide, a protein synthesis inhibitor, during the 20 h preincubation completely abolished the protection observed for CFCs. In conclusion, IL-1 and TNF-alpha can protect normal hematopoietic progenitors from UVC as well as from 4-HC and gamma radiation, and, therefore, a global response to DNA damaging treatments induced by IL-1 and TNF-alpha needs to be further investigated.

DNA Damage↗

PubMed1997

Early neuropathologic effects of mild or moderate hypoxemia after controlled cortical impact injury in rats.

Hypoxemia has detrimental effects after traumatic brain injury (TBI) in both experimental models and humans. The purpose of this study was to determine the effect of mild or moderate hypoxemia on early histologic and motor functional outcome after controlled cortical impact (CCI) in rats. Anesthetized rats underwent CCI and were randomized to receive mild (FiO2 = 13%, n = 6), moderate (FiO2 = 11%, n = 9), or no (FiO2 = 33%, n = 6) hypoxemia for 30 min after trauma. Sham-operated rats without hypoxemia (n = 7) were used as controls. Motor function (beam balance latency) was assessed on days 0-5. Rats were killed 7 days after injury and their brains removed for assessment of survival of hippocampal neurons and contusion volume. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) was performed on brain sections from rats killed at 6, 24, and 72 h after CCI and moderate hypoxemia to assess DNA fragmentation in situ. Mild and moderate hypoxemia augmented motor function deficits after CCI in a dose-dependent manner. Moderate hypoxemia after CCI reduced 7-day survival of CA3 neurons but not CA1 neurons vs. sham (55 [46-86] vs. 99 [95-130], p < 0.05, and 79 [63-86] vs. 101 [81-123], NS, respectively; % uninjured hemisphere, median [range]). The addition of mild or moderate hypoxemia did not increase contusion volume. TUNEL-positive neurons were seen in ipsilateral cortex and dentate gyrus at 6, 24, and 72 h after trauma, and in ipsilateral CA3 hippocampal neurons and thalamus at 24 and 72 h. Moderate hypoxemia augments CA3 neuronal death and early motor functional deficits after CCI. The pattern of DNA fragmentation in selectively vulnerable neurons suggests that apoptosis may play a role in the delayed neuronal death seen after TBI.

Analysis of Variance↗

PubMed1997

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Induction of mitochondrial manganese superoxide dismutase in macrophages by oxidized LDL: its relevance in atherosclerosis of humans and heritable hyperlipidemic rabbits.

The objective of the study was to analyze the intracellular antioxidative response of macrophages (Mphi) exposed to increased levels of low density lipoprotein (LDL). We studied manganese superoxide dismutase (MnSOD) and, in part, GSH in cultured human and rabbit Mphi, and in atheromatous arterial tissue of humans and heritable hyperlipidemic (HHL) rabbits. Incubation of human Mphi with oxidized-LDL (ox-LDL) resulted in an induction of MnSOD mRNA production as shown by RT-PCR. MnSOD immunoreactivity (IR) was found to be located in the mitochondria of Mphi. In HHL rabbits, MnSOD activity and GSH concentration were significantly increased in atherosclerotic intima compared to the media of the aorta, but significantly decreased (P<0.01) in larger plaques compared with smaller ones, resulting in a significant inverse correlation of MnSOD activity (r=-0.67, P<0.001) and GSH concentration (r=-0.57, P<0.01) with plaque size. Immunohistology of the atherosclerotic intima revealed MnSOD-IR in Mac-1 (CD 11b/CD 18)-immunoreactive (ir) Mphi of human arteries and, similarly, in RAM-11-ir Mphi of rabbit ones. The relation of MnSOD-ir Mphi decreased with plaque advancement, which is consistent with biochemical findings. Most MnSOD-ir Mphi in atherosclerotic plaques revealed TUNEL-positive nuclei, indicating DNA strand breaks, and p53-IR. We conclude that mitochondrial antioxidants such as MnSOD are induced in Mphi in vitro and in atherosclerotic arteries as a reply to increased mitochondrial oxidation. As normal consequences of an increased oxidative stress due to the exposure to ox-LDL nuclear DNA strand breaks occur, which are suggested to be a signal to increase p53 protein levels. Reactive oxygen species-mediated mitochondrial-dependent pathways are suggested as major contributing pathomechanisms to nuclear damage, which eventually may result in apoptosis. A common response to increased oxidative stress due to modified LDL is presumed in rabbit and human atherosclerotic plaques.

Animals↗

PubMed1997

Blood pressure trends, plasma insulin levels and risk factors in rural and urban elderly populations of north India.

OBJECTIVE: To compare the prevalence of hypertension and its risk factors with age-specific blood pressures in rural and urban subjects. DESIGN AND SETTING: A cross-sectional survey of two randomly selected villages and 20 randomly selected streets in Moradabad, north India. SUBJECTS AND METHODS: The subjects were 255 rural people (140 men, 115 women) and 311 urban people (172 men, 139 women) aged 60-84 years. The survey methods were questionnaires, blood pressure and anthropometric measurements and electrocardiography. RESULTS: The mean +/- SD blood pressures, both systolic (137.7 +/- 13 versus 131.2 +/- 12 mmHg) and diastolic (89.8 +/- 41 versus 85.8 +/- 9 mmHg) were significantly higher in urban men than they were in rural men. Similar differences between systolic (135.6 +/- 11 versus 129.2 +/- 10 mmHg) and diastolic (90 +/- 10 versus 87.6 +/- 9 mmHg) blood pressures were found among urban and rural women, respectively. A significant correlation between systolic blood pressures and increasing age was observed both for rural and for urban subjects of both sexes. The overall prevalences of hypertension based on World Health Organization criteria (17.6 versus 5.0%) and Joint National Committee fifth report criteria (34.0 versus 10.1%) were significantly higher among urban than they were among rural subjects. Multivariate logistic regression analysis revealed that age, body mass index, central obesity, glucose intolerance, 2 h plasma insulin and triglyceride level were associated independently with hypertension. CONCLUSIONS: The findings indicate that urban subjects had higher blood pressures than did rural subjects and that age, body mass index, central obesity and 2 h plasma insulin levels were significant risk factors for hypertension in an elderly population.

Age Factors↗

PubMed1997

Immune responses to the hepatitis C virus NS4A protein are profoundly influenced by the combination of the viral genotype and the host major histocompatibility complex.

The interaction between the host major histocompatibility complex (MHC) and the genotype of the hepatitis C virus (HCV) was analysed using synthetic full-length non-structural (NS) 4A proteins, residues 1658-1712, of genotypes 1b, 2b, 3a, 4a and 5a. Human and murine antibodies specific for the five NS4A genotypes analysed focused on residues 1688-1707. In immunized B10 H-2 congenic mice, the H-2d, H-2f and H-2s haplotypes were good responders to NS4A, irrespective of the viral genotype. In contrast, the H-2k haplotype was a low or non-responder to all NS4A genotypes, except for genotype 2b. Also, H-2f- and H-2s-restricted NS4A genotype 1b-specific T-cells focused on residues 1670-1679 and 1683-1692, respectively, whereas H-2k-restricted NS4A genotype 2b-specific T-cells focused on the carboxy terminus. Interestingly, H-2f-restricted genotype 1b-specific T-cells did not cross-react with T-cell site analogues of seven other genotypes, whereas the H-2s-restricted, genotype 1b-specific T-cells cross-reacted with genotypes 1a, 4a and 5a. Thus the combination of viral genotype and host MHC profoundly influences the ability to mount an HCV NS4A-specific immune response.

Amino Acid Sequence↗

PubMed1997

Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an acquired blistering skin disease characterized by the presence of IgG autoantibodies to type VII collagen. EBA autoantibodies recognize four major immunodominant epitopes localized within the amino-terminal, noncollagenous (NC1) domain. In this study, we developed a rapid, quantitative enzyme-linked immunosorbent assay (ELISA) to detect autoantibody activity against the complete NC1 domain of type VII collagen with the use of an eukaryotic-expressed, recombinant human NC1 antigen. With the ELISA, we tested serum from patients with EBA (n = 24), bullous systemic lupus erythematosus (BSLE) (n = 3), bullous pemphigoid (n = 16), pemphigus (n = 11), and normal controls (n = 12). All EBA and BSLE serum, including four sera that were negative by indirect immunofluorescence, demonstrated reactivity with immobilized NC1 in the ELISA. In contrast, none of the sera from healthy control subjects or patients with unrelated blistering skin diseases reacted with NC1. The EBA sera also reacted with recombinant NC1 by immunoblot analysis but with less sensitivity. Thus, the newly developed ELISA using recombinant NC1 is a sensitive, specific assay and a useful tool for rapidly screening EBA and BSLE serum.

Autoantibodies↗

PubMed1997

Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid.

We characterized basement membrane zone (BMZ) autoantigens targeted by autoantibodies (AAb) from patients with cicatricial pemphigoid. Serum from a patient with severe oral cicatricial pemphigoid contained IgG anti-BMZ AAb. The AAb labeled a lower BMZ component on salt-split skin and localized to the lower lamina lucida/lamina densa by direct and indirect immunoelectron microscopy (IEM) but did not label blood vessels. The AAb did not react with EHS laminin-1 and type IV collagen, pepsinized human type IV collagen, recombinant entactin, or NC1 domain of type VII collagen by dot blotting and western blotting. We focused our studies on the laminin family, as laminin-5 was identified as an autoantigen in cicatricial pemphigoid. Culture-conditioned media from normal keratinocytes (containing laminin-6 and laminin-5) and JEB keratinocytes (containing laminin-6 but not laminin-5) were studied by western blotting. Under nonreducing conditions, the patient's AAb recognized a 600-kDa protein (laminin-6) intensely and a 400-kDa protein (laminin-5) weakly in normal keratinocyte medium even though abundant laminin-5 was present. InJEB keratinocyte medium, however, the 600-kDa protein (laminin-6) alone was recognized by the patient's AAb. The AAb also immunolabeled BMZ of JEB skin that lacked laminin-5. The AAb from this patient and two other patients with anti-laminin-5 cicatricial pemphigoid immunoprecipitated both laminin-6 and laminin-5. Taken together, the results of IEM, non-reducing western blotting, immunoprecipitation, and JEB skin BMZ immunolabeling indicate that laminin-6, as well as laminin-5, is identified by the AAb from a subset of cicatricial pemphigoid patients. We propose the name "anti-laminin cicatricial pemphigoid" for this subset.

Adult↗