Biomedical subjects

C H Mak

Publications and source records attributed to C H Mak.

16 recordsLinked to original sources

PubMed2006

Heat-inducible translationally controlled tumor protein of Trichinella pseudospiralis: cloning and regulation of gene expression.

To elucidate the mechanism of inducing translationally controlled tumor protein (TCTP) in stress adaptation of adenophorean nematodes, the complete coding sequence of TCTP of the infective-stage larvae of Trichinella pseudospiralis was characterized. Two cDNA clones with different 3' untranslated region were identified. Tp-TCTP contained an open reading frame of 534 bp encoding 177 residues. The gene with five introns was expressed as histidine-tagged fusion protein having a molecular mass of 17.5 kDa. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that TCTP RNA was not accumulated when the infective-stage larvae were heat-shocked for 1 h at 45 or 60 degrees C. Using enzyme-linked immunosorbent assay and antiserum against the fusion protein, the expression of TCTP was found to be up-regulated at the translational level. The data suggest that translational regulation of TCTP may play an important role in the early heat-stress adaptation of the trichinellid. Cluster analysis demonstrated that the TCTP sequence of T. pseudospiralis is closely related to that of T. spiralis, but is diverged from the secernentean species.

Amino Acid Sequence↗

PubMed2006

Large-scale simulations of the two-dimensional melting of hard disks.

Large-scale computer simulations with more than four million particles have been performed to study the melting transition in a two-dimensional hard disk fluid. The van der Waals loop previously observed in the pressure-density relationship of smaller simulations is shown to disappear systematically with increase in sample size, but even with these large system sizes, the freezing transition still exhibits what appears to be weakly first-order behavior, though the scaling of the bond orientation order is consistent with the Halperin-Nelson-Young picture. Above this freezing transition region, scaling analysis of the translational order yields a lower bound for the melting density that is much higher than previously thought and provides compelling evidence that the solid phase first melts into a hexatic phase via a continuous transition, before it goes into the isotropic phase.

Journal Article↗

PubMed2005

Cloning and characterization of the Cu/Zn superoxide dismutase of Trichinella pseudospiralis.

Copper/zinc (Cu/Zn) superoxide dismutase (SOD) activity was identified for the first time in both crude somatic extracts (CE) and excretory/secretory (E/S) products of Trichinella pseudospiralis. It was the dominant SOD in infective-stage larvae. Native polyacrylamide gel electrophoresis of CE and E/S products yielded a prominent band, which was cyanide-sensitive and was partly inhibited by hydrogen peroxide in SOD assay. Cytosolic Cu/Zn SOD was cloned. The 471-bp full-length cDNA sequence contained an open reading frame of 157 amino acids. The gene contained three introns. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of cytosolic Cu/Zn SOD was substantially higher in infective-stage larvae than in adult worms. Cluster analysis showed that the sequence of the Cu/Zn SOD of T. pseudospiralis, an adenophorean nematode, is related to those of Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Haemonchus contortus (all belonging to the sercenentean group).

Amino Acid Sequence↗

PubMed2005

Stochastic potential switching algorithm for Monte Carlo simulations of complex systems.

This paper describes a new Monte Carlo method based on a novel stochastic potential switching algorithm. This algorithm enables the equilibrium properties of a system with potential V to be computed using a Monte Carlo simulation for a system with a possibly less complex stochastically altered potential V. By proper choices of the stochastic switching and transition probabilities, it is shown that detailed balance can be strictly maintained with respect to the original potential V. The validity of the method is illustrated with a simple one-dimensional example. The method is then generalized to multidimensional systems with any additive potential, providing a framework for the design of more efficient algorithms to simulate complex systems. A near-critical Lennard-Jones fluid with more than 20,000 particles is used to illustrate the method. The new algorithm produced a much smaller dynamic scaling exponent compared to the Metropolis method and improved sampling efficiency by over an order of magnitude.

Journal Article↗

PubMed2005

Superfluidity in CH4-doped H2 nanoclusters.

We report a theoretical study of superfluidity in CH(4)-doped para-H(2) nanoclusters. Path integral simulations for clusters of 12-16 H(2) around a single CH(4) molecule were carried out at temperatures between 0.5 and 2 K to study the superfluid response of the cluster. The results indicate that a rapid increase in the superfluid response is expected to occur around 1 K. We analyzed the structures and statistics of these clusters and found that the larger permutation cycles which dominate the superfluid component tend to adopt ringlike structures on the surface of the CH(4) molecule.

Journal Article↗

PubMed2004

Cloning and characterization of the mitochondrial heat-shock protein 60 gene of Trichinella spiralis.

The full-length cDNA of mitochondrial heat-shock protein (hsp) 60 of the infective-stage larva of Trichinella spiralis was cloned by degenerative PCR and rapid amplification of cDNA end reactions. The 1,945 bp full-length cDNA sequence contained an open reading frame of 576 amino acids. A mitochondrial signal peptide was located at the N-terminal and a GGM motif at the C-terminal. The gene contained ten exons and nine introns. RT-PCR analysis indicated that thermal, cold, acidic and oxidative treatment did not elicit significant changes in the expression of mitochondrial hsp 60 in the larvae. Cluster analysis showed that the sequence of the hsp 60 gene of T. spiralis is closely related to that of Drosophila melanogaster.

Amino Acid Sequence↗

PubMed2003

Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis.

Inhibitor sensitivity assays using azocaesin and FTC-caesin as substrates showed that the excretory/secretory (E/S) products of the infective-stage larvae of Trichinella spiralis contained serine, metallo-, cysteine and aspartic proteinases. The activity of the metallo-proteinase was zinc ion dependent (within a range of ZnSO(4) concentrations). Gelatin-substrate gel electrophoresis revealed two bands of molecular mass 48 and 58 kDa which were sensitive to the metallo-proteinase inhibitor EDTA. The former peptide was probably a cleavage product of the latter. The authenticity of the 58 kDa metallo-proteinase as an E/S product was confirmed by immunoprecipitation. Using PCR and RACE reactions, a complete nucleotide sequence of the metallo-proteinase gene was obtained. It comprised 2,223 bp with an open reading frame encoding 604 amino acid residues. The 3' untranslated region consisted of 352 bp, including a polyadenylation signal AATAA. A consensus catalytic zinc-binding motif was present. The conserved domains suggest that the cloned metallo-proteinase belongs to the astacin family and occurs as a single copy gene with 11 introns and 10 exons. Cluster analysis showed that the sequence of the metallo-proteinase gene of T. spiralis resembles those of Caenorhabdites elegans and Strongyloides stercoralis.

Amino Acid Sequence↗

PubMed2001

Identification of some heat-induced genes of Trichinella spiralis.

Three heat-induced genes of the infective-stage larvae of Trichinella spiralis were successfully identified by the suppression subtractive hybridization (SSH) technique. As indicated by reverse Northern blotting, 19 of 25 clones were scored as differentially transcribed in the heat-shocked infective-stage larvae. The sequencing data showed the presence of 12 different genes. Three were homologous to histone H3, histone H2B and translationally controlled tumour protein (TCTP). A 0.6 kb cDNA of histone H3 was generated by the RACE method and sequenced. It contained an open reading frame of 136 amino acids that demonstrated 94% identity with genes from Drosophila hydei. Semi-quantitative RT-PCR indicated that after heat-shock treatment, the expression levels of histone H3, histone H2B and TCTP increased 4.8, 27 and 5.7-fold, respectively. Northern analysis confirmed the upregulation of histone H3, histone H2B and TCTP transcripts. The upregulation of these genes during stress conditions has not been reported in parasitic organisms. The stress proteins may play an active role to sustain the parasite after exposure to hostile host factors.

Amino Acid Sequence↗

PubMed2001

DNA-binding activity in the excretory-secretory products of Trichinella pseudospiralis (Nematoda: Trichinelloidea).

A novel DNA-binding peptide of Mr approximately 30 kDa was documented for the first time in the excretory-secretory (E-S) products of the infective-stage larvae of Trichinella pseudospiralis. Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E-S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of the Mr of proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E-S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E-S product.

Animals↗

PubMed2000

Single-stranded endonuclease activity in the excretory--secretory products of Trichinella spiralis and Trichinella pseudospiralis.

A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory-secretory (E-S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E-S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to high concentrations of NaCl. Zymographic analysis indicated that it was encoded by at least 3 peptides of Mr approximately 50-60 kDa. The rate of hydrolysis of single-stranded targets by the E-S products was substantially higher than that of the double-stranded molecule. Due to the differences in peptide profile, divalent cation dependence, and species-specific expression, the single and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.

Animals↗

PubMed1999

Regulation by phosphorylation of the zinc finger protein KRC that binds the kappaB motif and V(D)J recombination signal sequences.

The DNA binding protein KRC (forkappaB binding andrecognitioncomponent of the V(D)J recombination signal sequence) belongs to a family of large zinc finger proteins that bind to the kappaB motif and contains two widely separated DNA binding structures. In addition to the kappaB motif, KRC fusion proteins bind to the signal sequences of V(D)J recombination to form highly ordered complexes. Here, we report that KRC may be regulated by post-translational modifications. Specific protein kinases present in the nucleus of pre-B cells phosphorylated a KRC fusion protein at tyrosine and serine residues. Such protein modifications increased DNA binding, thereby providing a mechanism by which KRC responds to signal transduction pathways. KRC is a substrate of epidermal growth factor receptor kinase and P34cdc2 kinase in vitro. Our results suggest that activation of the KRC family of transcription factors may provide a mechanism by which oncogenic tyrosine kinases regulate genes with kappaB-controlled gene regulatory elements.

Amino Acid Sequence↗

PubMed1999

Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis.

Double-stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double-stranded breaks to the native DNA. The ES double-stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective-stage larvae of T. spiralis collected at 37 degrees C and was present in much smaller amounts in samples collected at 43 degrees C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated approximately 25, 30 and 58 kDa, respectively.

Animals↗

PubMed1998

KRC transcripts: identification of an unusual alternative splicing event.

Mouse KRC is a large zinc finger protein that binds to the kappaB motif of gene transcription and to the recognition signal sequences for the somatic recombination of the immunoglobulin and T-cell receptor gene segments. The mouse KRC gene is more than 70 kilobases (kb) in size, and contains at least seven exons, with the largest transcript being approximately 9.5 kb. Multiple differentially spliced transcripts of KRC were identified in thymus and brain, which would result in the production of multiple KRC protein isoforms with different N-termini and number of DNA binding domains. Alternative splicing events leading to the production of these multiple transcripts have been elucidated. Of particular interest are the exclusions in some transcripts of sequences from a gigantic exon of 5487 base pairs (bp), or from an exon of 176 bp. Both potentially deleted exons code for zinc finger motifs that are essential components of the N-terminal and C-terminal DNA binding domains, respectively. Another intriguing phenomenon found in some KRC transcripts is the skipping of a 459 bp fragment within the gigantic exon that would code for the N-terminal DNA binding domain. Bacterial fusion proteins derived from this fragment bind specifically to KRC target DNAs. Apparently, distinct alternative splicing events could eliminate the N-terminal DNA binding domain of KRC.

Alternative Splicing↗

PubMed1996

The mouse DNA binding protein Rc for the kappa B motif of transcription and for the V(D)J recombination signal sequences contains composite DNA-protein interaction domains and belongs to a new family of large transcriptional proteins.

Rc is a DNA binding protein with dual specificities for the V(D)J recombination signal sequences and for the B motif of the immunoglobulin kappa chain gene enhancer. The largest Rc transcript present in lymphoid cells/tissues is approximately 9 kb. Molecular cloning and sequence determination for 8822 bp of mouse Rc cDNA revealed an open reading frame of 2282 amino acids and long 5'- and 3'-untranslated regions. The derived amino acid sequence contains multiple DNA and protein interaction domains. Composite ZAS structures with tandem zinc fingers, an acidic motif, and a Ser/Thr-rich segment are located near the N-terminal and the C-terminal regions. The middle region of Rc contains a lone zinc finger, an acidic motif, a Ser-rich region, a nucleus localization signal, and GTPase motifs. Cloning and characterization of a mouse Rc gene show that the Rc cDNA corresponds to seven exons located in a genomic region spanning 70 kb. Exon 2 is exceptionally large, with 5487 bp. cDNA cloning and Northern blot analyses revealed multiple Rc transcripts, probably generated by alternative splicings. Sequence comparisons show that Rc belongs to a ZAS protein family that is involved in gene transcription and/or DNA recombination. The major histocompatibility complex class I gene enhancer binding proteins MBP1 and MBP2 are other representatives of this ZAS protein family.

Amino Acid Sequence↗

PubMed1994

The V(D)J recombination signal sequence and kappa B binding protein Rc binds DNA as dimers and forms multimeric structures with its DNA ligands.

The murine DNA binding protein Rc binds to the heptamer motif of the V(D)J recombination signal sequences and to the kappa B motif of the immunoglobulin enhancer. Bacterial fusion proteins for Rc and DNA ligands of Rc form multiple protein-DNA complexes in electrophoretic mobility shift assays (EMSA). Large complexes formation is favored by an increased Rc concentration. In order to determine the architecture of these complexes, the apparent molecular weights of the protein-DNA complexes were first determined by their gel mobilities. The data suggest that Rc binds to its DNA ligands as dimers, tetramers, and multiples of tetramers. The inference that Rc binds DNA as dimers was substantiated by the formation of chimeric complexes when two electrophoretically distinguishable Rc proteins were employed in EMSA. Methylation interference experiments show that there are no contiguous protein binding sites evident in the DNA of the larger complexes. Apparently, multimerization occurs via protein-protein interactions. Such interaction was demonstrated by the formation of Rc dimers and tetramers in a chemical crosslinking experiment. Significantly, the multimerization of DNA-bound Rc could be involved in bringing the variable region gene segments together for the somatic V(D)J recombination.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of a zinc finger protein which binds to the heptamer of the signal sequence for V(D)J recombination.

The somatic V(D)J recombination for the assembly of the Ig and TCR genes is mediated by the recombination signal sequences (Rss) and the V(D)J recombinase. A cDNA clone was isolated from a lambda gt11 expression library made from mouse thymocyte poly(A)+ RNA, using the Rss as a ligand. The deduced amino acid sequence of the putative protein, designated Recognition component (Rc), reveals a pair of Cys2-His2 zinc fingers followed by a Glu- and Asp-rich acidic domain. In addition, there are five copies of the Ser/Thr-Pro-X-Arg/Lys sequence, which are putative DNA binding units. The zinc finger-acidic domain structures present in Rc are also found in several enhancer binding proteins, such as those for the kappa B motif of the Ig kappa light chain enhancer or related sequences. Bacterial fusion proteins for Rc bind preferentially to the Rss heptamer and to the kappa B motif. The dual affinities of Rc for the Rss heptamer and the kappa B motif suggest a possible link between Ig transcription and somatic recombination. The formation of multiple 'gel-shifted' DNA-protein complexes for Rc and its DNA ligand suggests that these complexes tend to multimerize.

Amino Acid Sequence↗