Biomedical subjects
Publications and source records attributed to J Chen.
The use of phosphodiesterase inhibitors such as milrinone in the treatment of severe heart failure is frequently restricted because they cause vasodilation and hypotension. In patients with decompensated heart failure with hypotension after treatment with milrinone, low doses of vasopressin restored blood pressure without inhibiting the inotropic effect of milrinone.
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In many applications, usefulness of conventional hydrogels is limited by their slow swelling. To improve the swelling property of the conventional hydrogels, we have synthesized superporous hydrogels (SPHs) which swell fast to equilibrium size in minutes due to water uptake by capillary wetting through numerous interconnected open pores. The swelling ratio was also large in the range of hundreds. The mechanical strength of the highly swollen SPHs was increased by adding a composite material during the synthesis. The composite material used in the synthesis of SPH composites was Ac-Di-Sol((R)) (croscarmellose sodium). The gastric retention property of the prepared SPH composites was tested in dogs both in fasted and fed conditions. The SPH composites were placed in a hard gelatin capsule (size 000) for oral administration. All dogs tested were fasted for 36 h before experiments. Under the fasted condition, the SPH composite remained in the stomach for 2-3 h after before breaking into two pieces and being emptied. When food was given before the experiment just once following 36 h of fasting, the SPH composite remained in the stomach for more than 24 h, even though the fed condition was maintained only for the first few hours. Our study indicated that SPH composites possessed three properties necessary for gastric retention: fast swelling; superswelling; and high mechanical strength. While more improvements need to be made, the SPH composites provide the basis for the development of effective long-term gastric retention devices.
Individuals with systemic lupus erythematosus and rheumatoid arthritis are characterized by the presence of high levels of circulating IgM and IgG autoantibodies. Although IgG autoantibodies often are pathogenic, the role of IgM autoantibodies in autoimmune disease is not clear. Using mice that are unable to secrete IgM but are able to express surface IgM and IgD and to secrete other classes of immunoglobulins, we examined the effect of the absence of secreted IgM in the development of IgG autoantibodies and autoimmune disease in lupus-prone lymphoproliferative (lpr) mice. Compared with regular lpr mice, lpr mice that lack secreted IgM developed elevated levels of IgG autoantibodies to double-stranded DNA and histones and had more abundant deposits of immune complexes in the glomeruli; they also suffered more severe glomerulonephritis and succumbed to the disease at an earlier age. Similarly, the absence of secreted IgM also resulted in an accelerated development of IgG autoantibodies in normal mice. These findings suggest that secreted IgM, including IgM autoantibodies produced naturally or as part of an autoimmune response, may lessen the severity of autoimmune pathology associated with IgG autoantibodies.
Ig class switch recombination (CSR) in activated B cells is preceded by the generation of "switch" transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile "I" exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except mu. However, all mu switch transcript mutations tested so far have left the I mu exon splice donor site intact. To test the possibility that the residual CSR activity in I mu mutants could be due to splicing of a truncated I mu exon, we generated new mutants specifically lacking the I mu splice donor site. Surprisingly, normal CSR was observed in the I mu splice donor mutants even in the absence of detectable spliced I mu transcripts. In a search for potential alternative sources of switch-like transcripts in the mu locus, we identified two novel exons which map just upstream of the Smu region and splice onto the C mu 1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Emu enhancer. These studies highlight a unique structure for the mu locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Smu. We propose that all of these transcripts directly contribute to mu class switching activity.
Interleukin 9 (IL-9) exerts its pleiotropic effects through the IL-9 receptor (IL-9R) complex, which consists of the IL-9R alpha-chain, which determines the cytokine specificity, and the IL-2 receptor gamma-chain. In the present study we used a modified yeast two-hybrid system to isolate cDNA species encoding proteins that interacted with the intracellular domain of the human IL-9R alpha-chain (hIL-9Ralpha). We have identified 14-3-3zeta as an hIL-9Ralpha-interacting protein. We also mapped residues 518-522 (Arg-Ser(519)-Trp-Thr(521)-Phe) in hIL-9Ralpha and helix I of 14-3-3zeta as being important for interaction. Moreover, peptide competition experi-ments suggested that interaction between hIL-9Ralpha and 14-3-3zeta requires the phosphorylation of Ser(519) or Thr(521). This is the first demonstration that 14-3-3 can interact with a non-tyrosine kinase receptor. The interaction between 14-3-3 and IL-9Ralpha but not IL-4Ralpha also suggests a potential role for 14-3-3 in determining cytokine specificity.
BACKGROUND: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. METHODS: A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype. RESULTS: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases. CONCLUSIONS: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.
By the use of partial least squares (PLS) method and 11 quantum chemical descriptors computed by PM3 Hamiltonian, Quantitative Structure-Property Relationships (QSPRs) for direct photolysis quantum yields of selected polycyclic aromatic hydrocarbons (PAHs) were obtained. Direct photolysis quantum yields were predicted for PAHs for which experimental quantum yield values were lacking. Based on the QSPR models, significant PAH molecular characters governing their direct photolysis quantum yields were identified. It can generally be concluded that PAHs with large average molecular polarizability, molecular weight, and heat of formation values tend to have small photolysis quantum yields. PAHs with large values of the energy of the lowest unoccupied molecular orbital (Elumo), small values of the energy of the highest occupied molecular orbital (Ehomo), and large Elumo-Ehomo values, tend to have great photolysis quantum yields.
The process of polyglutamylation mediated by folylpolyglutamate synthetase (FPGS) in mammalian cells has nutritional and pharmacological importance. In murine cells, FPGS expression is controlled by two promoters that, as we show here, vary substantially in their efficiency, at least in the context of a reporter gene assay. Characteristics of the most efficient promoter (promoter B) were examined in the present studies. Insertion in pGL3 of a 1635 bp segment of upstream sequence including the most upstream exon (B1c), intron B1c and only 26 bp of the more downstream exon Bla resulted in a 15-20-fold increase in transcription in NIH3T3 and Hep1-6 cells compared with the promoterless vector. Deletion analysis of DNA sequence upstream of exon B1c showed that transcription was regulated by putative cis active elements only within two distally located upstream segments which when deleted cumulatively increased transcription three- to four-fold. However, deletion of the 56 bp intron B1c immediately downstream of the most upstream exon (Blc) resulted in 1/10 the rate of transcription. Primer extension analysis with NIH3T3 cells revealed start sites for transcription appreciably upstream of and within exon B1c as well as downstream in exon B1a. This result is consistent with the frequent occurrence in murine cells of an FPGS variant (variant III) incorporating exon B1c [Roy et al., J. Biol. Chem. 271 (1996) 23820; 272 (1997) 5587]. Site-directed mutagenesis and DNAse I footprinting revealed that three canonical GC boxes, either overlapping or tightly clustered within intron B1c, bound Sp1 and markedly enhanced transcription, accounting for the maximal promoter B activity. Moreover, in a cellular background devoid of Sp1 activity, we demonstrate that Spl can induce high levels of promoter B activity in pGL3 transfectants, but only when intron B1c is included within the reporter gene construct used. These results suggest that the unusually tight cluster of active Sp1 sites within intron B1c are essential and sufficient for maximal activity of this promoter. These tightly clustered sites appear to act as an enhancer element in promoting transcription and efficiently stabilize transcription initiation complexes at both distal and proximal start sites.
Vaccination of chinchillas with nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) conjugates protected against otitis media. Correlations between the levels of conjugate-induced LOS antibodies (Abs) in sera and middle ear fluids (MEFs) and Ab-mediated biological functions and protection were examined. Following parenteral vaccination and middle ear challenge, all vaccinated animals, but none of the controls, had high titers of anti-LOS in their sera and MEFs. There was a correlation between the levels of anti-LOS IgG + M, IgG or IgA in the sera and in the MEFs (P < 0.001). An inverse correlation was found between the level of serum IgG + M and bacterial counts and between the levels of MEF Abs and bacterial counts at the early postchallenge stage (P < 0.05). Of the 39 vaccinated animals, 44% showed complete protection against otitis media, 46% (18/39) of their sera inhibited adherence of NTHi to human epithelial cells, 49% (19/39) demonstrated bactericidal activity and 49% (19/39) showed opsonophagocytic activity. In contrast, none of the controls (19) were protected, none of their sera inhibited bacterial adherence or had bactericidal activity and only 21% showed opsonophagocytosis. Our interpretation is that vaccine-induced LOS Abs transuded into the middle ear and conferred immunity to NTHi by binding to LOS of NTHi, inhibition of NTHi adherence to epithelial cells and complement-mediated bacteriolysis (or opsonophagocytosis).
IL-17 is a T cell-derived cytokine that may play an important role in the initiation or maintenance of the proinflammatory response. Whereas expression of IL-17 is restricted to activated T cells, the IL-17 receptor is found to be widely expressed, a finding consistent with the pleiotropic activities of IL-17. We have cloned and expressed two novel human cytokines, IL-17B and IL-17C, that are related to IL-17 ( approximately 27% amino acid identity). IL-17B mRNA is expressed in adult pancreas, small intestine, and stomach, whereas IL-17C mRNA is not detected by RNA blot hybridization of several adult tissues. No expression of IL-17B or IL-17C mRNA is found in activated T cells. In a survey of cytokine induction, IL-17B and IL-17C stimulate the release of tumor necrosis factor alpha and IL-1beta from the monocytic cell line, THP-1, whereas IL-17 has only a weak effect in this system. No induction of IL-1alpha, IL-6, IFN-gamma, or granulocyte colony-stimulating factor is found in THP-1 cells. Fluorescence-activated cell sorter analysis shows that IL-17B and IL-17C bind to THP-1 cells. Conversely, IL-17B and IL-17C are not active in an IL-17 assay or the stimulation of IL-6 release from human fibroblasts and do not bind to the human IL-17 receptor extracellular domain. These data show that there is a family of IL-17-related cytokines differing in patterns of expression and proinflammatory responses that may be transduced through a cognate set of cell surface receptors.
BACKGROUND: Atrial fibrillation (AF) has traditionally been described as aperiodic or random. Yet, ongoing sources of high-frequency periodic activity have recently been suggested to underlie AF in the sheep heart. Our objective was to use a combination of optical and bipolar electrode recordings to identify sites of periodic activity during AF and elucidate their mechanism. METHODS AND RESULTS: AF was induced by rapid pacing in the presence of 0.1 to 0.5 micromol/L acetylcholine in 7 Langendorff-perfused sheep hearts. We used simultaneous optical mapping of the right and left atria (RA and LA) and frequency sampling of optical and bipolar electrode recordings (including a roving electrode) to identify sites having the highest dominant frequency (DF). Rotors were identified from optical recordings, and their rotation period, core area, and perimeter were measured. In all, 35 AF episodes were analyzed. Mean LA and RA DFs were 14.7+/-3.8 and 10.3+/-2.1 Hz, respectively. Spatiotemporal periodicity was seen in the LA during all episodes. In 5 of 7 experiments, a single site having periodic activity at the highest DF was localized. The highest DF was most often (80%) localized to the posterior LA, near or at the pulmonary vein ostium. Rotors (n=14) were localized on the LA. The mean core perimeter and area were 10.4+/-2.8 mm and 3.8+/-2.8 mm(2), respectively. CONCLUSIONS: Frequency sampling allows rapid identification of discrete sites of high-frequency periodic activity during AF. Stable microreentrant sources are the most likely underlying mechanism of AF in this model.
Cationic antimicrobial peptides, such as polymyxin and cecropin, activated transcription of osmY and micF in growing Escherichia coli independently of each other. The micF response required the presence of a functional rob gene. It is intriguing that in this and other assays an identical response profile was also seen with hyperosmotic salt or sucrose gradient, two of the most commonly used traditional food preservatives. The osmY and micF transcription was not induced by hypoosmotic gradient, ionophoric peptides, uncouplers, or with other classes of membrane perturbing agents. The antibacterial peptides did not promote transcription of genes that respond to macromolecular or oxidative damage, fatty acid biosynthesis, heat shock, or depletion of proton or ion gradients. These and other results show that the antibacterial cationic peptides induce stasis in the early growth phase, and the transcriptional efficacy of antibacterial peptides correlates with their minimum inhibitory concentration, and also with their ability to mediate direct exchange of phospholipids between vesicles. The significance of these results is developed as the hypothesis that the cationic peptide antimicrobials stress growth of Gram-negative organisms by making contacts between the two phospholipid interfaces in the periplasmic space and prevent the hyperosmotic wrinkling of the cytoplasmic membrane. Broader significance of these results, and of the hypothesis that the peptide mediated contacts between the periplasmic phospholipid interfaces are the primary triggers, is discussed in relation to antibacterial resistance.
We show that blockage of hyperosmotic shock induced plasmolysis by polymyxin B (PxB) is related to its selective antimicrobial action against Gram-negative organisms. The rapid wrinkling of the cytoplasmic membrane induced by the hyperosmotic shrinkage of cytoplasmic volume due to the water efflux is monitored as an increase in the 90 degrees light scattering. The rapid scattering response is complete within 1 min after the addition of hyperosmolar NaCl. PxB decreases the amplitude of the rapid increase in the light scattering due to the shrinkage of the cytoplasmic volume by hyperosmotic shock. The amplitude is highest with cells in the early log phase of growth. The effect of PxB is induced rapidly and the maximum effect is seen within 1 min preincubation of cells. The effect of PxB is concentration dependent, and about 50% decrease in the amplitude is seen in the range of the growth inhibitory concentrations of PxB. The effect of PxB is not seen if added after the onset of the up-shock. As a heuristic model we suggest that PxB forms contacts between the two phospholipid interfaces that enclose the periplasmic space. The plasmolytic response results with osmY(-) mutant suggest that, like PxB, the osmY gene product in the periplasmic space prevents the shrinkage of the cytoplasmic compartment. Since PxB induces osmY transcription, we propose that, as a possible locus for the origin of the PxB induced stress, a contact between the phospholipid interfaces surrounding the periplasmic space triggers the metabolic changes leading to bacterial stasis.
Wild type p53 accumulates in the cytoplasm in a subset of tumors such as neuroblastomas and breast carcinomas through an unknown mechanism. Exclusion of p53 from the nucleus may lead to inactivation of p53 during tumor development. We present evidence that MDM2 plays a significant role in promoting the degradation of nuclear p53 in tumor cells with a cytoplasmic p53 phenotype. Inhibition of MDM2 expression using antisense oligonucleotide, inhibition of MDM2 function by the tumor suppressor ARF or a MDM2 deletion mutant result in the accumulation of nuclear p53. p53 point mutants deficient in MDM2 binding have increased nuclear localization. Inhibition of nuclear export by leptomycin B also results in retention of nascent p53 in the nucleus, suggesting that cytoplasmic distribution of p53 results from efficient export of nuclear p53 in combination with MDM2-mediated degradation. These results suggest that MDM2 is an important determinant of p53 subcellular distribution and may contribute to p53 inactivation without overexpression.
To study the role of adenosine triphosphate (ATP) P2x-purinoceptor in the persistent nociceptive response induced by subcutaneous (s.c.) bee venom injection, we used a selective P2x receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), to evaluate whether spinal P2x receptor play a role in development of spontaneous persistent pain. Injection s.c. of bee venom into the plantar surface of one hindpaw in the conscious rat produces a monophasic, prolonged persistent nociception characterized by continuously flinching reflex of the injected paw for 1-2 h. Intrathecal (i.t.) pretreatment with PPADS at two lower doses of 5 and 10 microg resulted in suppression of the flinching reflex in a dose dependent manner with the inhibitory rate 37 and 44%, respectively, when compared with the control group; whereas i.t. PPADS at a higher dose of 30 microg failed to produce any inhibitory effect. This result suggests that activation of P2x-purinoceptor in the spinal cord contributes to the induction of bee venom-induced prolonged persistent pain. However, the antinociceptive effect of ATP P2x-purinoceptor antagonist such as PPADS on clinical pathological pain seems to be limited due to its lack of effectiveness at higher dose.
OBJECTIVES: Burma produces approximately 60% of the world's heroin, Laos is the third leading producer. Recent outbreaks of injecting drug use and HIV-1 in Burma, India, China, and Vietnam have been associated with Burmese and Laotian overland heroin trafficking routes. We analyzed findings from narcotics investigations, molecular epidemiology studies of HIV-1, and epidemiologic and behavioral studies of injecting drug use, to evaluate the roles that the heroin export routes play in the spread of drug use and HIV-1 in south and south-east Asia. METHODS: We reviewed the medical and narcotics literature, the molecular epidemiology of HIV, and did key informant interviews in India, China, and Burma with injecting drug users, drug traffickers, public health staff, and narcotics control personnel. RESULTS: Four recent outbreaks of HIV-1 among injecting drug users appear linked to trafficking routes. Route 1: From Burma's eastern border to China's Yunnan Province, with initial spread of HIV-1 subtype B, and later C. Route 2: Eastern Burma to Yunnan, going north and west, to Xinjiang Province, with B, C, and a B/C recombinant subtype. Route 3: Burma and Laos, through northern Vietnam, to China's Guangxi Province, subtype E. Route 4: Western Burma, across the Burma-India border to Manipur, predominant subtype C, and B and E. CONCLUSIONS: Overland heroin export routes have been associated with dual epidemics of injecting drug use and HIV infection in three Asian countries and along four routes. Molecular epidemiology is useful for mapping heroin routes. Single country narcotics and HIV programs are unlikely to succeed unless the regional narcotic-based economy is addressed.
BACKGROUND AND OBJECTIVES: The value of secondary cytoreductive surgery is still controversial, especially in patients with recurrent epithelial ovarian cancer. In this retrospective study, we investigated the effect on survival of secondary cytoreduction for recurrent disease and variables influencing redebulking surgical outcome. METHODS: Between 1986 and 1997, 60 patients who received primary cytoreductive surgery and platinum-based chemotherapy for stage III and IV epithelial ovarian cancer experienced disease recurrence at least 6 months after completion of primary therapy, and secondary surgical cytoreduction was performed. The optimal residual disease cutoff was 1.0 cm. The Cox proportional regression model and Logistic stepwise regression were used in statistical processing of the data. RESULTS: The median progression-free interval between the two operations was 13 months (range, 6-56 months). Optimal secondary cytoreduction was achieved in 23 patients (38.33%). There was a significant difference in survival between patients who were optimally cytoreduced compared to those suboptimally cytoreduced, with an estimated median survival in the optimal group of 19 months vs. 8 months in the suboptimal group (chi(2) = 22.04, P = 0.0000). Prognosis of survival for individuals with progression-free interval >12 months was better than that of those with the interval </=12 months (chi(2) = 5.22, P = 0.0224). Patients with ascites at disease recurrence suffered a pessimistic outcome, with an estimated median survival of 6 vs. 13 months in those without ascites (chi(2) = 13.99, P = 0.0002). Multivariate analysis strongly suggested that residual disease after second operation, ascites at disease recurrence, and progression-free interval were independent prognostic factors of survival. Logistic stepwise regression revealed that recurrent ascites (P = 0.0072, relative risk = 20.36) and residual disease after the second operation (P = 0.0096, relative risk = 5.16) were important determinants of secondary surgical outcome. CONCLUSIONS: Secondary cytoreductive surgery significantly lengthened survival for patients with recurrent epithelial ovarian cancer. Patients with ascites at disease recurrence, however, were not suitable for aggressive secondary surgery, and redebulking surgery for those with residual disease of >1.0 cm after primary operation should be considered prudently.