Small supernumerary ring X chromosome in a four-month-old girl.
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Abnormalities, Multiple↗
Biomedical subjects
B Huang
Publications and source records attributed to B Huang.
Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.
Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.
Animals↗
Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia.
Heterozygous mutations in the CD95 (APO-1/Fas) receptor occur in most individuals with autoimmune lymphoproliferative syndrome (ALPS) and dominantly interfere with apoptosis by an unknown mechanism. We show that local or global alterations in the structure of the cytoplasmic death domain from nine independent ALPS CD95 death-domain mutations result in a failure to bind the FADD/MORT1 signaling protein. Despite heterozygosity for the abnormal allele, lymphocytes from ALPS patients showed markedly decreased FADD association and a loss of caspase recruitment and activation after CD95 crosslinking. These data suggest that intracytoplasmic CD95 mutations in ALPS impair apoptosis chiefly by disrupting death-domain interactions with the signaling protein FADD/MORT1.
Adaptor Proteins, Signal Transducing↗
A rapid, sensitive HPLC method for the determination of ganciclovir in human plasma and serum.
A method for ganciclovir determination in human serum and plasma has been developed and validated. The method has a lower limit of quantification (LLOQ) adequate for sensitive pharmacokinetic studies ( < or = 0.05 microg/ml), has run times of < or = 15 min, and uses aliquot volumes adequate for pediatric studies (0.25 ml). In the method, proteinaceous material in serum or plasma is precipitated by trichloroacetic acid. An aliquot of the supernatant is analyzed by HPLC; automated column switching removes late-eluting materials that might interfere with the analyte peak in subsequent runs. Detection and quantification of ganciclovir is by fluorescence (lambda(ex) = 278 nm; lambda(em) = 380 nm). The method has a validated range of 0.0400-4.00 microg/ml and an LLOQ of 0.0400 microg/ml. All intra- and inter-assay % C.V. values were < 8%; all recoveries (accuracy) were within 7% of nominal values. No interference was observed by mycophenolic acid or its glucuronide metabolite, by AZT, salicylic acid, acetaminophen, ibuprofen, naproxen prednisone, acyclovir, or cyclosporine. Ganciclovir is very stable in the samples and the extract during storage and sample processing. Both serum and plasma methods have been validated for use and have been successfully used to analyze samples from clinical studies.
Antiviral Agents↗
Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.
A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.
Amino Acid Sequence↗
Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding.
Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein alpha1-acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 microM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a Ki value for inhibition of thymidine uptake of 0.1 microM compared to 0.28 microM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml(-1)). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC90, by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 microM NU3076 and 1 microM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors.
Animals↗
Large genomic duplicons map to sites of instability in the Prader-Willi/Angelman syndrome chromosome region (15q11-q13).
The most common etiology for Prader-Willi syndrome and Angelman syndrome is de novo interstitial deletion of chromosome 15q11-q13. Deletions and other recurrent rearrangements of this region involve four common 'hotspots' for breakage, termed breakpoints 1-4 (BP1-BP4). Construction of an approximately 4 Mb YAC contig of this region identified multiple sequence tagged sites (STSs) present at both BP2 and BP3, suggestive of a genomic duplication event. Interphase FISH studies demonstrated three to five copies on 15q11-q13, one copy on 16p11.1-p11.2 and one copy on 15q24 in normal controls, while analysis on two Class I deletion patients showed loss of approximately three signals at 15q11-q13 on one homolog. Multiple FISH signals were also observed at regions orthologous to both human chromosomes 15 and 16 in non-human primates, including Old World monkeys, suggesting that duplication of this region may have occurred approximately 20 million years ago. A BAC/PAC contig for the duplicated genomic segment (duplicon) demonstrated a size of approximately 400 kb. Surprisingly, the duplicon was found to contain at least seven different expressed sequence tags representing multiple genes/pseudogenes. Sequence comparison of STSs amplified from YAC clones uniquely mapped to BP2 or BP3 showed two different copies of the duplicon within BP3, while BP2 comprised a single copy. The orientation of BP2 and BP3 are inverted relative to each other, whereas the two copies within BP3 are in tandem. The presence of large duplicated segments on chromosome 15q11-q13 provides a mechanism for homologous unequal recombination events that may mediate the frequent rearrangements observed for this chromosome.
Angelman Syndrome↗
[Magnetic resonance imaging in 40 cases of optic neuritis].
OBJECTIVE: To explore the clinical value of magnetic resonance imaging (MRI) in optic neuritis. METHODS: Forty patients with optic neuritis were examined with the spectral presaturation inversion recovery and fluid attenuated inversion recovery. RESULTS: The lesion was frequently seen in orbital portion (86.7%) and intracanalicular portion (29.3%), and primarily in the axial part (49.3%) and transverse section (38.7%) of the optic nerve. The longitudinal extent of lesion was from 2 to 35 mm (mean, 12.6 mm), the visual acuity of patients with optic neuritis was related to the site of the lesion, and negatively correlated with the longitudinal extent of lesion of logarithm curve. A poor visual acuity (< 0.01) was significantly associated with lesion of greater extent > 10 mm and the transverse or axial lesion, but the good visual acuity (>or= 0.3), with the lesion of smaller extent < 10 mm and the axial or peripheral lesion. In addition, paramagnetic contrast-medium, Gd-DTPA, enhancement scanning was carried out in 11 cases of optic neuritis, of them the visual acuity was increased in 10 cases after administration of corticosteroid, and in one case, visual improvement was not seen. CONCLUSION: MRI is a new and useful equipment which is helpful in the diagnosis and treatment of optic neuritis.
Adolescent↗
[The effect of simulated weightlessness on the function of spleen lymphocytes and the expression of c-fos proto-oncogene in tail-suspended mice].
Objective. To investigate the effect of simulated weightlessness on proliferation of spleen lymphocyte, production of interleukin-2 and expression of c-fos proto-oncogene in mice. Method. Mice were tail-suspended for 7 or 14 days to simulate the effect of weightlessness; proliferation of lymphocyte was measured by MTT method, production of IL-2 was determined by biological activity and expression of c-fos proto-oncogene was determined by dot blotting. Result. As compared with the control, the proliferation of spleen lymphocyte and the production of IL-2 were significantly decreased in 14 d suspended group, but the expression of c-fos proto-oncogene [correction of oncogen] was increased in both 7 d and 14 d suspended groups. Conclusion. This result suggested that simulated weightlessness could interfere with the genes regulating the lymphocyte.
Animals↗
[Preliminary experimental study on commitment differentiation of embryonic stem cells induced by corneal limbal stroma in vitro].
PURPOSE: To investigate the possibility of commitment differentiation of embryonic stem cells (ES cells) induced by corneal limbal stroma. METHODS: Superficial corneal limbal stroma and lentis epithelium from New Zealand white rabbit were used for feeder layer for ES-D3 cells culture to induce the ES cells differentiation into corneal limbal stem cells. The differentiated ES cells were determined by light microscope and electronic microscope. RESULTS: 1. The proliferation of differentiating ES cells which cultured on corneal surface stroma layer around the corneal limbus was slow. The appearance of induced ES cells were uniform cells with much larger size than undifferentiated ES cells, and the nucleoli become long and thin under electronic microscope, but the size of ES cells cultured on deep stroma maintain as small as undifferentiated ES cells. The ES cells cultivated on surface stroma layer around the corneal limbus appeared microvilli 2 weeks after grafting into subcutaneous of athymic mice. 2. Lentis epithelium can only delay but not induce the ES cells differentiation to corned limbal stem celluloid. CONCLUSION: These results suggest that the superficial corneal limbal stroma has a potential ability to induce ES cells commitment differentiation. The tertiary embryo induction may not occur in vitro.
Animals↗
[Development and evaluation of a system for pulse generation and signal processing control in a plasma atomic fluorescence spectrometer with hollow cathode lamp as excitation source].
A Z80 processor was replaced with a 386 microcomputer to control pulse generation for hollow cathode lamp and fluorescence signal processing in a Baird Plasma AFS-2000 atomic fluorescence spectrometer. An interface board was designed for control purpose. The system was evaluated with detection limits, relative standard deviations and linear regression coefficients of 15 elements, which were in good agreement with the corresponding values in the manufacturer's manual. With the higher-level microprocessor in the system, the performance of the instrument was improved.
English Abstract↗
[Atomic/ionic fluorescence in microwave plasma torch discharge excited by high current microsecond pulsed hollow cathode lamp-europium atomic/ionic fluorescence spectrometry].
Eu atomic and ionic fluorescence spectrometry in microwave plasma torch discharge excited by high current microsecond pulsed hollow cathode lamp (HCMP HCL-MPT AFS/IFS) was studied. Operating conditions were optimized. The best detection limits for AFS and IFS obtained with a desolvated ultrasonic nebulization system were 42.0 ng/mL for Eu I 462.7 nm and 21.8 ng/mL for Eu II 381.97 nm, respectively, both were better than those given by the instruction manual of a Baird ICP AFS-2000 spectrometer using pneumatic concentric nebulizer with desolvation for AFS, but were significantly higher than those obtained by using the Baird spectrometer with a mini-monochromator and a ultrasonic nebulzer system.
English Abstract↗
Three novel proteins of the syntaxin/SNAP-25 family.
Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.
Amino Acid Sequence↗
Identification of an unusual marker chromosome by spectral karyotyping.
We ascertained a newborn girl with multiple congenital anomalies including severe hypotonia, cardiovascular defects, hearing loss, central nervous system anomalies, and facial anomalies. The infant died at 12 days. Cytogenetic analysis showed a de novo supernumerary marker chromosome. Fluorescence in situ hybridization (FISH) with a combination of chromosome specific alpha-satellite probes and an all-human centromere probe failed to show hybridization to the marker, indicating that the marker chromosome lacked detectable alpha satellite sequences. Spectral karyotyping (SKY) was performed and showed that the marker was chromosome 15 in origin. This was confirmed by FISH with a 15q specific subtelomerie probe, which showed hybridization to both ends of the marker chromosome. Based on FISH information and G-banding pattern, the marker was determined to be an inverted duplication of 15q25-qter, leading to partial tetrasomy for chromosome 15. Although the marker chromosome lacked detectable centromeric alpha-satellite sequences, it seemed to have a functional centromere as it is mitotically stable. This observation is consistent with previous studies on acentric marker chromosomes, which suggested that the DNA sequence at the breakpoint could function similarly to alpha-satellite sequences once activated through marker formation.
Abnormalities, Multiple↗
[Expression of human SRY gene and the DNA-binding property of its product].
OBJECTIVE: To investigate the role which human SRY gene plays in the regulation of the downstream gene. METHODS: The fragment of SRY (sex determinating region on the Y chromosome) HMG domain was cloned into the expressing vector pET-15b. The hSRY gene recombinant plasmid-pETSY was transformed and expressed in E.coliBL21. The target protein was purified by pET His. Tag system. The DNA-binding retardation test and its competitive reaction were conducted between SRY protein and the fragment of Mullerian inhibiting substance (MIS) promoter. RESULTS: The molecular weight of the expressed hSRY protein was shown to be approximately 21kD. The specific DNA binding property of SRY protein to the fragment of MIS was confirmed in the retardation test and its competitive reaction. CONCLUSION: The results suggest that the product of SRY gene can bind the MIS promoter region and may initiate the transcription of MIS.
DNA-Binding Proteins↗
Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation.
Pyruvate dehydrogenase phosphatase (PDP) is one of the few mammalian phosphatases residing within the mitochondrial matrix space. It is responsible for dephosphorylation and reactivation of the pyruvate dehydrogenase complex (PDC) and, by this means, is intimately involved in the regulation of utilization of carbohydrate fuels in mammals. PDP is a dimeric enzyme consisting of catalytic and regulatory subunits. The catalytic subunit of PDP is a Mg2+-dependent enzyme homologous to the cytosolic phosphatases of the 2C family. In the present study, we isolated two cDNAs encoding for mitochondrial phosphatases. The first cDNA is highly homologous to the previously identified cDNA encoding for the catalytic subunit of PDP (PDP1). The second cDNA encodes a previously unknown catalytic subunit of PDP (PDP2). The new phosphatase, expressed as the recombinant protein in Escherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain alpha-ketoacid dehydrogenase as substrate. Like PDP1, PDP2 is a Mg2+-dependent enzyme, but its sensitivity to Mg2+ ions is almost 10-fold lower than that of PDP1. In contrast to PDP1, PDP2 is not regulated by Ca2+ ions. Instead, it is sensitive to the biological polyamine spermine, which, in turn, has no effect on the enzymatic activity of PDP1. Western blot analysis of PDP extracted from mitochondria isolated from liver and skeletal muscle revealed that PDP1 is predominantly expressed in mitochondria from skeletal muscle, whereas PDP2 is much more abundant in the liver rather than muscle mitochondria. Both isoenzymes are expressed in mitochondria from 3T3-L1 adipocytes, but the level of expression of PDP2 is considerably higher. These observations are consistent with previous findings on the enzymatic parameters of PDP in adipose tissue. Thus, our results provide the first evidence that there are at least two isoenzymes of PDP in mammals that are different with respect to tissue distribution and kinetic parameters and, therefore, are likely to be different functionally.
3T3 Cells↗
NMR structure and mutagenesis of the FADD (Mort1) death-effector domain.
When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.
Adaptor Proteins, Signal Transducing↗
Alterations in cardiac gene expression during ventricular remodeling following experimental myocardial infarction.
Following myocardial Infarction (MI) the heart undergoes a process of remodeling characterized by considerable hypertrophy of the non-infarcted myocardium. We have recently characterized the molecular basis of key electrophysiologic alterations that may provide insight into the arrhythmogenecity of post-MI remodeled hypertrophied myocardium. To further characterize other key alterations in the pattern of cardiac gene expression in a time-dependent manner, we have measured mRNA and immunoreactive protein levels of selective cardiac genes in the remodeled hypertrophied left-ventricular (LV) myocardium of rats, 3 and 21 days after left-coronary ligation and compared the results with sham-operated rats. RNase protection assay was performed to assess the expression of c-fos, atrial natriuretic factor (ANF), brain natriuretic factor (BNF), alpha2/3 isoform of Na-K ATPase, cardiac alpha/beta isoform of myosin heavy chain (MHC). Compared to the sham group, the expression of c-fos was increased 10-fold (P<0.02) in the MI group on day 3, but unlike other overload hypertrophy models, the expression remained elevated by three-fold on day 21. Similar to other overload models, the ANF and BNF expression increased significantly. No alterations were observed in the expression of cardiac alpha-actin. There was reexpression of the fetal isogene form of MHC and Na-K ATPase after MI. The beta-MHC mRNA levels, the fetal isoform of MHC, returned to basal levels after 21 days. After an initial five-fold decrease the adult isoform of alphaNa-K ATPase, alpha2 Na-K ATPase mRNA, returned to control levels and similar changes were seen in the corresponding protein levels. These findings indicate that during LV remodeling and hypertrophy following MI, there is an upregulation of early response genes and fetal isogene expression. The pattern of activation, however, is distinct from that observed in other overload models, indicating the possible involvement of alternate signal transduction pathways.
Animals↗