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K P Chang

Publications and source records attributed to K P Chang.

At least 19 recordsLinked to original sources
PubMed1999

Roles of free GPIs in amastigotes of Leishmania.

Glycosylated phosphatidylinositols (GPIs) are abundant cell surface molecules of the Leishmania. Amastigote-specific GPIs AmGPI-Y and AmGPI-Z, both ethanolamine (EtN)-containing glycolipids, were identified in Leishmania amazonensis. A paucity of GPI-anchored proteins in amastigotes of L. amazonensis made the kinetoplastid suitable for evaluating the importance of free (i.e. unconjugated to protein or polysaccharide) GPIs. A strain deficient in both AmGPI-Y and AmGPI-Z was produced by stable transfection of wild-type Leishmania with a GPI-phospholipase C gene. Phosphatidylinositol deficiency was not detected in the transfectants. GPI-deficient promastigotes infected murine macrophages in vitro and differentiated into amastigotes whose growth was arrested within the host cells. Cytostasis of amastigotes was also observed during axenic culture of GPI-deficient parasites. In a hamster model of leishmaniasis, GPI-deficient promastigotes produced smaller lesions with 20-fold fewer amastigotes than infections with control parasites. Together, these observations indicate that EtN-GPIs may be essential for amastigote viability, replication, and/or virulence. Implicit in these observations is the notion that drugs targeted against the GPI biosynthetic pathway might be of value in the management of human leishmaniasis.

Animals↗
PubMed1998

Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

Amino Acid Sequence↗
PubMed1998

Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene.

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

Adolescent↗
PubMed1998

Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey.

Infantile Mediterranean visceral leishmaniasis (IVL) and anthroponotic cutaneous leishmaniasis (ACL) have long been known to exist in the western and southeastern Turkey, respectively. To further study these and other related diseases, a recombinant antigen (rK39) specific to VL was used in an ELISA for serodiagnosis of selected patients and for screening dog reservoir populations in several endemic sites. Among 24 confirmed VL cases from western Turkey, the rK39 ELISA proved to be more sensitive than a combination of cultivation and microscopy of bone marrow aspirates. The specificity of rK39 for leishmaniasis was demonstrated by its lack of cross-reactivity with sera from other human diseases in the same sites. Interestingly, six of the 83 parasitologically proven ACL cases from southeast Turkey were also rK39 positive. The end point titers of the positive VL and CL cases vary from 10(-2) to 10(-5) and from 10(-2) to 10(-3), respectively. The rK39 ELISA was also used to screen 494 apparently healthy dogs from Urfa in southeast Turkey, Manisa/Alasehir near the Aegean Sea, and Karabuk near the Black Sea. Eighteen rK39-positive cases (3.6%), all from the latter two areas, were found to have varying endpoint titers (10(-2)-10(-4)). The high titers predicted increased severity and frequency of the clinical symptoms (i.e., lymphadenopathy, depilation, skin lesion, weight loss and/or death), which were manifested subsequently in 16 of these 18 cases. In addition, more positive canine cases were diagnosed by the rK39 ELISA preclinically than the procedures to detect parasites postsymptomatically in the lymph node aspirates. The use of the rK39 ELISA as a sensitive tool makes it possible to demonstrate coendemicity of canine and human VL, as expected in the case of IVL. The results also point to the possible presence of additional VL types in western Turkey and cutanovisceral type in the southeast part of this country.

Adolescent↗
PubMed1997

Stage-independent splicing of transcripts two heterogeneous neighboring genes in Leishmania amazonensis.

Gene expression in trypanosomatid protozoa is largely regulated posttranscriptionally, e.g., 5' splice leader addition and 3' polyadenylation of mRNAs. We examined these events in Leishmania by mapping the splice sites of the transcripts from two different, but closely linked single-copy genes 2.3 kb apart. The coding regions of the approx. 1 kb upstream gene (P36) and the approx. 1.4 kb downstream gene (NAGT) produce approx. 2 and 3 kb mRNAs, respectively. Both genes were overexpressed in cells that were transfected with this bicistronic unit (> or = 7.5 kb), taking advantage of the NAGT as a selectable marker for tunicamycin-resistance. The transcripts from both genes were spliced constitutively at both ends, irrespective of their episomal or chromosomal expression in both leishmanial stages. Primer extension of the 5' UTRs and S1 nuclease protection of the 3' UTRs initially identified the major splice sites, corresponding to the genomic sequence at -205 bp and + approx. 900 bp of P36, and -1012 bp and + approx. 600 bp of NAGT. These splice sites, consistent with the size of the major transcripts, are among those mapped precisely by sequencing RT-PCR amplified 5' and 3' UTRs. The additional sites mapped by the latter are minor alternatives, especially abundant for transcripts of the downstream NAGT. All these minor splice sites are closer than the major splice sites to the coding region, indicating that the most distant splice sites are preferentially used. This preference creates a 387 bp 'gap' with polypyrimidine tracts in the intergenic region consistent with the model coupling splice leader addition with polyadenylation in pre-mRNA processing. The stage-independence of these events suggests that the 7.5 kb dicistronic unit is suitable for constructing Leishmania-specific constitutive expression vectors.

Animals↗
PubMed1997

Intracellular Ca2+ pool content and signaling and expression of a calcium pump are linked to virulence in Leishmania mexicana amazonesis amastigotes.

Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.

Amino Acid Sequence↗
PubMed1997

Primary cerebellar extramedullary myeloid cell tumor mimicking oligodendroglioma.

Extramedullary myeloid cell tumors (EMCTs) are tumors consisting of immature cells of the myeloid series that occur outside the bone marrow. Most of them are associated with acute myelogenous leukemia or other myeloproliferative disorders, and a small number occur as primary lesions, i.e., are not associated with hematological disorders. Occurrence inside the cranium is rare, and there has been only one case of primary EMCT involving the cerebellum reported in the literature. The case we report here is a blastic EMCT occurring in the cerebellum of a 3-year-old boy who had no signs of leukemia or any hematological disorder throughout the entire course. The cerebellar tumor was at first misdiagnosed as an "oligodendroglioma" because of the uniformity and "fried egg" artifact of the tumor cells. The tumor disappeared during chemotherapy consisting of 12 treatments. However, it recurred and metastasized to the cerebrospinal fluid (CSF) shortly after the therapy was completed. A diagnosis of EMCT was suspected because of the presence of immature myeloid cells in the CSF, and was confirmed by anti-myeloperoxidase and anti-lysozyme immunoreactivity of the cerebellar tumor. The patient succumbed 1 year and 3 months after the first presentation of the disease.

Cerebellar Neoplasms↗
PubMed1996

Posttranslational regulation of a Leishmania HEXXH metalloprotease (gp63). The effects of site-specific mutagenesis of catalytic, zinc binding, N-glycosylation, and glycosyl phosphatidylinositol addition sites on N-terminal end cleavage, intracellular stability, and extracellular exit.

Leishmanolysin (EC 3.4.24.36) (gp63) is a HEXXH metalloprotease, encoded by multicopied genes in Leishmania and implicated in the infectivity of these parasitic protozoa. We examined posttranslational regulation of gp63 expression by site-specific mutagenesis of the predicted catalytic/zinc-binding sites in the H264EXXH motif, the potential sites of N-glycosylation and glycosyl phosphatidylinositol addition. Mutant and wild-type genes were cloned into a Leishmania-specific vector for transfecting a deficient variant, which produced gp63 approximately 20-fold less than wild-type cells. The selective conditions chosen fully restored this deficiency in transfectants with the wild-type gene. Under these conditions, all transfectants were found comparable in both the plasmid copy number per cell and elevation of gp63 transcripts. Mutant and wild-type products in the transfectants were then compared quantitatively and qualitatively by specific immunologic and protease assays. The results indicate the following. 1) Glu-265 in the HEXXH motif is indispensable for the catalytic activity of gp63. The propeptide of the inactive mutant products was cleaved, suggestive of a non-intramolecular event. 2) Substitution of either His residue in HEXXH leads to apparent intracellular degradation of the mutant products, pointing to a role for zinc binding in in vivo stability of gp63. 3) The three potential sites of N-glycosylation at Asn-300, Asn-407, and Asn-534 are all utilized and contribute to intracellular stability of gp63. 4) Substitution of Asn-577 causes release of all mutant products, indicative of its specificity as a glycosyl phosphatidylinositol addition site for membrane anchoring of gp63. It is suggested that expression of gp63 as a functional protease is regulated by these posttranslational modification pathways.

Animals↗
PubMed1996

Quality of life and life satisfaction in families after a child has undergone corpus callostomy.

Epilepsy is a chronic neurological condition which can have a detrimen tal effect on patients' quality of life (QOL). We evaluated the overall satisfaction with QOL and changes in QOL in 25 families with children who had undergone corpus callostomy for severe epilepsy. The mean age of seizure onset was 3.4 years, and the mean period from seizure onset to operation was 5.2 years. Mean follow-up was 19.3 months. Twenty of 22 patients had various degrees of mental retardation. Mean reduction in severity of seizure activity was 64%. Nineteen of 25 (76%) parents were satisfied with the surgical result. Eighteen of 25 parents (72%) described a good level of satisfaction with their families QOL after callostomy. Reduction in severity of seizure activity was significantly correlated with how successful families considered the surgery to be and how satisfied they were with their QOL (r = 0.72 and 0.77, respectively). Life domains showing one greatest improvement after callostomy included level of self-care, family life, and school performance. Improvements in level of self-care, family life, and school performance were associated with higher levels of life satisfaction (r = 0.69, 0.60, and 0.59, respectively). Hyperactivity, attention span, and social skills improved significantly in 11 patients after surgery. Callostomy did not improve mental performance. We conclude that improved seizure control after callostomy is also associated with improved QOL.

Achievement↗
PubMed1996

Kinetics of entry of virulent and avirulent strains of Leishmania donovani into macrophages: a possible role of virulence molecules (gp63 and LPG).

Specific receptors may be involved in the process of attachment of Leishmania donovani promastigotes to macrophage surfaces and their subsequent internalization. Two virulent strains of Indian L. donovani (AG83 and GE-I) were found to enter into macrophages much faster than the avirulent ones (UR6). These virulent promastigotes express surface glycoprotein (gp63) and lipophosphoglycan (LPG) to a greater extent than avirulent strains. We examined their interaction with macrophages as a function of time by preblocking the macrophage receptors with the exogenous addition of gp33 or LPG. In experiments where gp63 was used as the blocking agent, the entry of one virulent strain (GE-I) was affected. In other experiments where LPG was used, the entry of another virulent strain (AG83) was affected. Entry of the avirulent strain (UR6) was unaffected by either of these treatments. Exposed LPG or gp63 on the surface of promastigotes thus appear to expedite their recognition and entry into the host cell. To assess the role of gp63 further in the entry of Leishmania into the macrophages, an avirulent UR6 strain was transfected with the gp63 gene cloned from L. amazonensis. The transfected UR6 as expected expressed more GP63 at a faster rate and entered into the macrophages like the virulent strain when compared to the nontransfected UR6 or UR6 transfected with vector alone. Thus, the expression of the gp63 gene is involved in the recognition and intracellular entry of visceral Leishmania into the macrophages in addition to the cutaneous species demonstrated previously.

Animals↗
PubMed1995

Role of the Leishmania surface protease gp63 in complement fixation, cell adhesion, and resistance to complement-mediated lysis.

The Leishmania surface protease gp63 has been identified as a parasite virulence factor. To better define the role of gp63 in Leishmania infectivity, the interaction of recombinant gp63 with complement and complement receptors was examined. On Leishmania, gp63 was not necessary for complement fixation. Complement activation occurred on transfected organisms expressing varying amounts of gp63 and on organisms expressing a mutant form of gp63 devoid of proteolytic activity. However, organisms expressing wild-type gp63 on their surface fixed only small amounts of the terminal complement components and were dramatically more resistant to lysis by complement than were those lacking functional gp63. Organisms expressing wild-type gp63 more rapidly converted C3b on their surface to a form that exhibited the neoantigen of iC3b and interacted avidly with cells expressing Mac-1, the receptor for iC3b. Complement fixation by transfected mammalian cells expressing recombinant Leishmania gp63 on their surface was also examined. The presence of Leishmania gp63 on the surface of these cells converted them into efficient activators of complement. Cells expressing gp63 on their surface fixed complement and bound avidly to the human complement receptors. The proteolytic activity of this molecule was not necessary for complement activation or adhesion to complement receptors. Thus, gp63 may contribute to parasite virulence by exerting a novel type of control over complement fixation. Organisms expressing gp63 can exploit the opsonic properties of complement while avoiding its lytic effects.

Animals↗
PubMed1995

Cloning and sequencing a putative pyrophosphate-dependent phosphofructokinase gene from Entamoeba histolytica.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) gene from Entamoeba histolytica was cloned from its genomic library and sequenced. The open reading frame has 1149 bp and codes for a protein of 41.5 kDa. The deduced amino acid sequence of E. histolytica PPi-PFK has 25 to 28% identity to the PPi-PFKs from Propionibacterium freudenreichii, Naegleria fowleri and potato. The amino acid residues known to contribute to the active site of PPi-PFK from P. freudenreichii are conserved.

Amino Acid Sequence↗
PubMed1995

Hypothalamic hamartoma and gelastic epilepsy: a case report.

We studied a 6-year-old girl who presented with inappropriate and uncontrollable laughing episodes since age 3. Physical examination revealed a precocious puberty. The luteinizing hormone-releasing hormone (LH-RH) stimulation test showed an increased level of follicle-stimulating hormone (FSH). The interictal electroencephalogram (EEG) was normal. Several laughing fits were documented during video/EEG monitoring. During laughing, the ictal EEG showed a diffuse suppression of background rhythm, prominent over the left mesial temporal region. A mass lesion about 2 x 2 cm in size was found over the suprasellar cistern with a broad base attached to the hypothalamus, which was isodense on a computed tomography (CT) scan, isointense to gray matter on T1-weighted magnetic resonance (MR) imaging and hyperintense on T2-weighted MR imaging. The findings were suggestive of a hypothalamic hamartoma. A variety of anticonvulsants had been used with little or no response to the frequency or duration of the laughing seizures.

Child↗
PubMed1995

Diagnostic and prognostic value of K39 recombinant antigen in Indian leishmaniasis.

The recombinant product (rK39) of the 39 amino acid repeats encoded by a kinesin-like gene of visceral Leishmania spp. was further evaluated by enzyme-linked immunosorbent assay (ELISA) for its diagnostic potential in Indian kala-azar (VL) and post kala-azar dermal leishmaniasis (PKDL). Anti-rK39 antibodies were highly positive in 20 symptomatic cases, including 6 resistant to single or double chemotherapy, but became negligible or absent in 9 recently cured patients. Endpoint titration of samples from the 20 active cases showed that the anti-rK39 IgG titers fell within a wide range of 10(-2) to > 10(-6), and that their mean was > 1 order of magnitude higher than in VL reported previously. The anti-rK39 IgG titers were correlated with parasite burden found in the patients and remained undiminished in those refractory to chemotherapy. These results indicate that: (1) the K39 epitope is conserved in Indian strains of Leishmania donovani, (2) the extremely high levels of K39 antibodies in both VL and PKDL suggest the application of rK39 for sensitive and specific serodiagnosis, and (3) rK39 ELISA is also valuable for prognostic evaluation of both diseases.

Adolescent↗
PubMed1995

Startle epilepsy presenting as drop attacks: a case report.

A case of startle epilepsy, induced by an unexpected touch on the left shoulder of a 3.5 year old boy, was investigated. The startle epilepsy manifested as an atonic drop attack. Neurological examination revealed a mild hemiparesis on the left side. Computed tomography (CT) scanning demonstrated an enhancement over the right parieto-frontal region, suggesting a hemangioma. Interictal electroencephalography (EEG) showed diffuse slowing and asymmetry of background activities with lower amplitude over the right centro-parieto-temporal region. The ictal EEG showed a paroxysmal bilaterally synchronized high amplitude single spike followed by a few slow waves lasting about one second over the bilateral centro-parieto-temporal regions. The seizure resisted anticonvulsant therapy and no significant responses were observed despite the use of combined therapy with valproic acid, phenytoin, primidone and clonazepam.

Child, Preschool↗