Biomedical subjects
M Ma
Publications and source records attributed to M Ma.
Validation of multiplex polymorphic STR amplification sets developed for personal identification applications.
Polymorphic short tandem repeat (STR) loci, which typically consist of variations in the number of 3-7 base pair repeats present at a site, provide an effective means of personal identification. Typing can be accomplished by amplification of genomic DNA using the polymerase chain reaction (PCR) and locus-specific primers, separation of amplified alleles using gel electrophoresis and their display using silver staining or fluorescent detection. Primers for several STR loci can be combined in a single multiplex reaction so typing of multiple loci can be accomplished rapidly and with less DNA than required if each locus were analyzed separately. Before such muliplex systems are used in forensic or paternity applications, it is desirable that they undergo testing for their reliability. This study evaluates the performance of two STR triplex systems, one containing the loci HUMCSF1PO, HUMTPOX, and HUMTH01, and the other containing HUMHPRTB, HUMFESFPS, and HUMVWFA31. Protocols for amplification of these two triplexes, and their corresponding monoplexes, were evaluated for sensitivity of detection, resistance to changes in the annealing temperature of the amplification protocol, and the ability to identify the minority contributor in amplification of mixed samples. In addition, five laboratories determined the alleles of twenty DNA samples, each extracted by one of four different extraction methods. The results illustrate that the two STR triplex systems and the monoplex systems contained within them can be used with as little as 0.25 ng of DNA template. Both triplexes amplified with 100% success using the Perkin Elmer Model 480 thermal cycler. With the GeneAmp 9600 System, the CTT triplex amplified with 100% success and the HFv triplex in 95.6% of attempts. These experiments meet many requirements for use in validation of DNA typing systems for forensic cases and paternity identification.
[Analysis of clonal origin of concomitant chronic lymphocytic leukemia and multiple myeloma in a patient with advanced age].
Concomitant chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) is a rare disease. Studies on the clonal origin of both lymphocyte and plasma cell in this disease has been few. So far it has not been reported in China. The paper reports a case of 92-year old patient with concomitant CLL and MM and the research results on the origin of lymphocyte and plasma cell by analyzing the morphologic characteristics, monoclonal antibodies, immunoglobulin Ig or TCR gene rearrangement (GR), Ig single-strand conformation polymorphsm (SSCP) fingerprint mapping and heteroduplex formation. The Ig H GR segments same in size, single strand conformation and sequence characteristics have been obtained from both peripheral blood and bone marrow DNA, even though the cells of CLL and MM had different characteristics in morphology and different secretion of IgM lambda and IgA lambda. The results suggested that the malignant cells of both CLL and MM in this patient had common origin from the B-cell.
Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.
Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.
The role of Tyr13 and Lys15 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A.
Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His13 and Thr15 with Tyr13 and Lys15 of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca2+ mobilization. As a corollary, replacement of the Tyr13 and Lys15 of the human IL-8 with His13 and Thr15 of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr13 and Lys15 sequence was found to be in the NH2-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.
Consequences and mechanisms of spike broadening of R20 cells in Aplysia californica.
We studied frequency-dependent spike broadening in the two electrically coupled R20 neurons in the abdominal ganglion of Aplysia. The peptidergic R20 cells excite the R25/L25 interneurons (which trigger respiratory pumping) and inhibit the RB cells. When fired at 1-10 Hz, the duration of the falling phase of the action potential in R20 neurons increases 2-10 fold during a spike train. Spike broadening recorded from the somata of the R20 cells affected synaptic transmission to nearby follower cells. Chemically mediated synaptic output was reduced by approximately 50% when recorded trains of nonbroadened action potentials were used as command signals for a voltage-clamped R20 cell. Electrotonic EPSPs between the R20 cells, which normally facilitated by two- to fourfold during a high frequency spike train, showed no facilitation when spike broadening was prevented under voltage-clamp control. To examine the mechanism of frequency-dependent spike broadening, we applied two-electrode voltage-clamp and pharmacological techniques to the somata of R20 cells. Several voltage-gated ionic currents were isolated, including INa, a multicomponent ICa, and three K+ currents--a high threshold, fast transient A-type K+ current (IAdepol), a delayed rectifier K+ current (IK-V), and a Ca(2+)-sensitive K+ current (IK-Ca), made up of two components. The influences of different currents on spike broadening were determined by using the recorded train of gradually broadening action potentials as the command for the voltage clamp. We found the following. (1) IAdepol is the major outward current that contributes to repolarization of nonbroadened spikes. It undergoes pronounced cumulative inactivation that is a critical determinant of spike broadening. (2) Activity-dependent changes in IK-V, IK-Ca, and ICa have complex effects on the kinetics and extent of broadening. (3) The time integral of ICa during individual action potentials increases approximately threefold during spike broadening.
[G gamma/A gamma, A gamma I/A gamma T ratios of fetal hemoglobin of Han, Hui, Uygur and Kazak newborns in Xinjiang and gene mapping analysis of two cases with abnormal ratio].
The G gamma/A gamma, A gamma I/A gamma T ratios of fetal hemoglobin of 372 cord blood samples of newborns (Han 102, Hui 102, Uygur 99, and Kazak 69) in Urumchi were determined by HPLC. The means of G gamma ratios of 4 ethnic groups (Han, Hui, Uygur, and Kazak) were 66.83%, 68.33%, 70.44% and 69.70%, respectively. 4 cases (Hui 3 and Kazak 1) with low G gamma (< 50%), and 25 cases (Han 5, Hui 4, Uygur 11 and Kazak 5) with high G gamma (> 80%) were found. The occurrences of low G gamma in Hui and Kazak were 2.94% and 1.45% while in Han and Uygur no single case with low G gamma was found. Those of high G gamma in 4 ethnic groups were 4.9% (Han), 3.9% (Hui), 11.1% (Uygur) and 7.2% (Kazak), respectively. A gamma T heterozygotes in Han 21, Hui 23, Uygur 26 and Kazak 29 cases, and one A gamma T homozygote in each ethnic group were found. The average values of A gamma I were 56.83%, 55.58%, 50.94% and 54.68% respectively. The frequencies of A gamma T gene were 0.113, 0.123, 0.141 and 0.225, respectively. The gene arrangement of a low G gamma case was identified as-GA gamma I-/-G gamma-A gamma I-, and that of a high G gamma case as -G gamma-AG gamma-A gamma I(A gamma T)-/-G gamma-A gamma T(A gamma I)-.
Characterization of a novel binding site for the human immunodeficiency virus type 1 envelope protein gp120 on human fetal astrocytes.
125I-labeled recombinant gp120 bound to primary cultures of human fetal astrocytes with a single class of 260-kDa binding sites, with a Kd of 26 nM and maximal number of binding sites of 29.9 fmol/4 x 10(4) cells. Neither CD4 nor galactocerebroside was detectable on astrocytes, and 125I-labeled recombinant gp120 binding to astrocytes was not blocked by antibodies against galactocerebroside or the gp120 binding domain of CD4.
[Effect of the changes of amino acids on both signal peptide C-terminal and mature protein N-terminal region to the secretion of alpha-amylase in B. subtilis].
By site-directed mutagenesis, G and C have taken the place of T and G at nucleotide sequence 287 and 291 of B. licheniformis alpha-amylase gene to generate pAm-y413B and the N-terminal sequence of mature protein have been changed from 7Leu 8Met to 7Arg8Ile. By the insertion of polylinker into the C-terminal of the signal sequence of alpha-amylase gene of pAmy413, the signal peptide of alpha-amylase produced by pAmy413L is 13 amino acids more than the pAmy413 (which is 29 amino acids long) and also, a new recognition cleavage sequence for signal peptidase I (Ala-Gln-Ala decreases Ser) is created; The secondary structure of the signal peptide has been analyzed by computer programs. The alpha-amylase relative activity of the two mutant strains is 3% and 36% of pAmy413, respectively. The molecular weight of extracellular alpha-amylase is the same as pAmy413. Terminal analysis shows that the N-terminal amino acid of mature protein is Ala, not Ser, and suggests that SPase I prefers to cleavage at the wild type recognition site (Ala-Ala-Ala decreases Ala). Therefore, all of the above results show that the secretion of alpha-amylase in B. subtilis is in accordance with the co-translational transportation model.
Gaussian theory of superfluid-Bose-glass phase transition.
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Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.
To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.
Molecular cloning of cDNA encoding the phenol/aryl form of sulfotransferase (mSTp1) from mouse liver.
The cDNA sequence of the mouse liver phenol/aryl form of sulfotransferase (mSTp1) has been determined. The cloned cDNA consists of 1269 base pairs (bp) and contains an 897 nucleotide open reading frame (ORF) beginning at nucleotide 65, which encodes a 298 amino acid polypeptide of 34.7 kDa. Alignment of mSTp1 to other sulfotransferases shows overall identities of 87% to r-STp, 37% to r-STa, 48% to r-STe, 51% to b-STe, and 37% to h-STa, at the deduced amino acid level.
Molecular cloning of the alcohol/hydroxysteroid form (mSTa1) of sulfotransferase from mouse liver.
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Molecular cloning of two cDNAs encoding the mouse bilirubin/phenol family of UDP-glucuronosyltransferases (mUGTBr/p).
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Karyotypic Variation within Clonal Lineages of the Rice Blast Fungus, Magnaporthe grisea.
We have analyzed the karyotype of the rice blast fungus, Magnaporthe grisea, by using pulsed-filed gel electrophoresis. We tested whether the electrophoretic karyotype of an isolate was related to its pathotype, as determined by infection assays, or its genetic lineage, as determined by DNA fingerprinting. Highly reproducible electrophoretic karyotypes were obtained for a collection of U.S. and Chinese isolates representing a diverse collection of pathotypes and genetic lineages. Chromosomes ranged in size from 3 to 10 Mb. Although chromosome number was largely invariant, chromosome length polymorphisms were frequent. Minichromosomes were also found, although their presence was not ubiquitous. They ranged in number from 1 to 3 and in size from 470 kb to 2.2 Mb. Karyotypes were sufficiently variable as to obscure the obvious relatedness of isolates on the basis of pathogenicity assays or genetic lineage analysis by DNA fingerprinting. We documented that the electrophoretic karyotype of an isolate can change after prolonged serial transfer in culture and that this change did not alter the isolate's pathotype. The mechanisms bringing about karyotype variability involve deletions, translocations, and more complex rearrangements. We conclude that karyotypic variability in the rice blast fungus is a reflection of the lack of sexuality in wild populations which leads to the maintenance of neutral genomic rearrangements in clones of the fungus.
Multicellular oxidant defense in unicellular organisms.
Although catalase is thought to be a major defense against hydrogen peroxide (H2O2), the catalase activity within individual Escherichia coli fails to protect against exogenous H2O2. Contrary to earlier reports, we find that dilute suspensions of wild-type and catalase-deficient E. coli are identical in their sensitivity to H2O2, perhaps because even wild-type, catalase-positive E. coli cannot maintain an internal/external concentration gradient of this highly diffusible oxidant. However, concentrated suspensions or colonies of catalase-positive E. coli do preferentially survive H2O2 challenge and can even cross-protect adjacent catalase-deficient organisms. Furthermore, high-density catalase-positive--but not catalase-negative--E. coli can survive and multiply in the presence of competitive, peroxide-generating streptococci. These observations support the concept that bacterial catalase may defend colonial, but not individual, E. coli against environmental H2O2. Group protection by the activity of enzymes that mitigate oxidative stress may have been a driving force in the evolution of multicellular organisms.
Finite-size scaling analysis of the random-Tc model with anisotropy.
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Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.
A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.