Biomedical subjects

K Leong

Publications and source records attributed to K Leong.

At least 19 recordsLinked to original sources

PubMed2000

Intergenic complementation truncation mutants of cyclin-dependent kinase.

The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.

CDC2-CDC28 Kinases↗

PubMed1999

STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors.

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell-cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Amino Acid Sequence↗

PubMed1999

Multiple organ failure: by the time you predict it, it's already there.

OBJECTIVE: Validate an at-risk population to study multiple organ failure and to determine the importance of organ dysfunction 24 hours after injury in determining the ultimate severity of multiple organ failure. METHODS: We evaluated 105 patients admitted to five academic trauma centers during a 1-year period who survived for more than 24 hours with Injury Severity Scores > or = 25 and who received 6 or more units of blood. Organ dysfunction was scored daily with a modified multiple organ failure scoring system made up of individual adult respiratory distress syndrome score, renal dysfunction, hepatic dysfunction, and cardiac dysfunction scores. Multiple organ failure (MOF) severity was quantitated using the maximum daily multiple organ failure score and the cumulative sum of daily multiple organ failure scores for the first 7 days (MOF 7) and 10 days (MOF 10). Independent variables included markers of tissue injury, shock, host factors, physiologic response, therapeutic factors, and organ dysfunction within the first 24 hours after admission. Data were subjected to a conditional stepwise multiple regression analysis, first excluding and then including 24-hour MOF as an independent variable. RESULTS: Of the 105 high-risk patients, 69 (66%) developed a maximum daily multiple organ failure score > or = 1; 50 (72%) did so on day 1 one and 60 (87%) did so by day 2. In multiple regression models, the multiple correlation coefficient increased from 0.537 to 0.720 when maximum MOF was the dependent variable, from 0.449 to 0.719 when maximum daily MOF was the dependent variable, from 0.519 to 0.812 when MOF 7 was the dependent variable, and from 0.514 to 0.759 when MOF 10 was the dependent variable. CONCLUSION: We have confirmed that the population of patients with Injury Severity Scores > or = 25 who received 6 or more units of blood represent a high-risk group for the development of multiple organ failure. Our data also indicate that multiple organ failure after trauma is established within 24 hours of injury in the majority of patients who develop it. It appears that multiple organ failure is already present at the time when most published models are trying to predict whether or not it will occur.

Adult↗

PubMed1998

Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR). As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis. Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism. The system described here may be used for further structure/function analysis of yeast DMR proteins. Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Gelatin/chondroitin 6-sulfate microspheres for the delivery of therapeutic proteins to the joint.

OBJECTIVE: To develop a biodegradable, inflammation-responsive microsphere system for the intraarticular delivery of therapeutic proteins. METHODS: Microspheres were synthesized by complex coacervation. Radiolabeled protein release and microsphere degradation were assessed by exposing the microspheres to human synovial fluids (SF) and recombinant gelatinase. Microsphere degradation was confirmed by scanning electron microscopy (SEM). Microsphere biocompatibility was evaluated in vitro by incubating the microspheres with human synoviocytes, and in vivo by injection into mouse joints. RESULTS: Optimal microsphere formulation was developed. Significant (up to 100%) release of encapsulated protein occurred in SF samples with measurable metalloprotease activity, while release was minimal in SF with negligible activity. Dissolution of microspheres exposed to gelatinase was confirmed by SEM. Microspheres were found to be noncytotoxic in vitro, and noninflammatory in vivo. CONCLUSION: Microsphere encapsulation is an inflammation-responsive and biocompatible system of protein delivery that holds promise for use in the delivery of therapeutic proteins to the joint.

Arthritis, Rheumatoid↗

PubMed1998

Risk of hepatitis C infection in neonates transfused with blood from donors infected with hepatitis C.

This look-back study was undertaken to identify newborn infants who had been infected with the hepatitis C virus (HCV) as a result of transfusions received before the introduction of routine screening in 1991 and to determine the transmission rates and persistence of transfusion-transmitted HCV infection acquired in the neonatal period. A total of 24 infants, transfused between 1980 and 1991, were identified as having received potentially infected blood from 11 blood donors. Ten of the donors had been administered batches of anti-D in 1977 known to have transmitted HCV genotype 1b infection. HCV RNA was detected in five of these donors when tested in 1994-95; the past donations of five of the donors, who had received anti-D immunoglobulin and had serological evidence of previous HCV infection but who were PCR negative when tested in 1994-95, were considered of lower risk. The source and time of acquisition of HCV infection for the one remaining donor in the study was not determined. Twenty-one (88%) of the 24 children were living at time of lookback. The median age at transfusion was 12 days. The median age at time of testing was 6.3 years. One child, who tested negative, was excluded from further analysis of HCV transmission, due to incomplete transfusion records. Overall, 12 of 20 (60%) children tested were positive for anti-HCV and seven (35%) were HCV RNA positive. Twelve (71%) of the 17 recipients of viraemic blood were ELISA positive and seven (41%) were PCR positive. Resolved HCV infection, as determined by ELISA pos, RIBA pos or indeterminate and PCR negativity, occurred in five of 12 (42%). In many instances there was more than one recipient per HCV infected donation. All of the reported children are clinically asymptomatic. However, the duration of HCV infection is relatively short and there is evidence of a degree of hepatitis in five of the seven children who are HCV RNA positive as judged by mildly elevated transaminase levels. The three who have undergone liver biopsy show mild hepatitis. The lower rates of persistence of HCV infection in this study may be due to the young age at exposure or to the source of infection which for all but one of the children was linked to one HCV genotype from female donors. Sharing of units of blood among multiple infants should be discouraged.

Adolescent↗

PubMed1996

Poly(alpha-hydroxy acids): carriers for bone morphogenetic proteins.

A broad spectrum of cells and cell products is associated with bone homeostasis and the renewal of bone following injury. The coupled interactions among cells provide the power behind sculpting of bone, sustaining form, and ensuring functionality. Local and systemic regulatory molecules (e.g. growth factors, hormones) direct cellular interactions through autocrine, paracrine, and hormonal pathways. Recently, genes for a class of osteogenic regulatory molecules have been cloned, and gene product expression has enabled investigators to assess safety and efficacy in animal studies. The molecules are known as bone morphogenetic proteins (BMPs). Therapeutic applications of BMPs depend on a carrier system. A carrier could spatially and temporally localize BMP for regional needs and be custom-tailored for acute craniofacial applications or for recalcitrant extremity non-unions. The poly(alpha-hydroxy acids) (PHAs) may be suitable for these applications. Therefore, the purposes of this paper are (i) to mention, briefly, basic concepts of the bone wound continuum and the possible therapeutic roles of BMPs; (ii) to outline several properties of selected PHAs relevant to bone regeneration dynamics; and (iii) to review selected preclinical studies with PHAs.

Animals↗

PubMed1996

Decreased growth hormone receptor expression in long bones from toothless (osteopetrotic) rats and restoration by treatment with colony-stimulating factor-1.

Growth hormone (GH) is known to regulate growth and development of skeletal tissues. This study examined the distribution of growth hormone receptor (GHR) expression in tibias from normal and osteopetrotic tl/tl rats. For normal 2 week-old rats, GHR expression was detected immunocytochemically in cells of the articular and epiphyseal cartilage, primary and secondary ossification centres, zone of resting cartilage and bone marrow. Within the marrow, GHR immunopositive cells were concentrated in the central cone and largely excluded from the zone of immature progenitors at the periphery. For the marrow haemopoietic compartment, GHR expression was almost restricted to the nucleus in large mononuclear cells, adipocytes and megakaryocytes. A population of small lymphocytelike cells in the marrow periphery expressed GHR on the plasma membrane. GHR was not detected in mature erythroid cells, macrophages, granulocytes, or osteoclasts. The expression of GHR was significantly reduced in bone marrow cells of the tl/tl rat (p < 0.001) compared with normal animals. Injection of recombinant CSF-1 into tl/tl rats every 48 hours for 2 weeks from birth restored GHR-positive cells to the central core of the marrow space. The most striking change was the appearance of substantial numbers of mononuclear cells expressing abundant GHR on the cell surface. We infer that these cells are a novel subset of CSF-1 responsive cells involved in bone resorption. The differences in relative expression of GHR by bone marrow cells in untreated and CSF-1-treated tl/tl rats suggests a CSF-1-dependent recruitment of cells bearing surface GHRs.

Animals↗

PubMed1995

Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences.

Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation.

Alternative Splicing↗

PubMed1993

Controlled release, biodegradable cytokine depots: a new approach in cancer vaccine design.

Experimental studies using murine tumor models have demonstrated that potent systemic immunity can be generated using tumor vaccines engineered by gene transfer to secrete certain cytokines. The underlying physiological principle behind these strategies involves the sustained release of high doses of cytokine at the site of the tumor. In some cases, this paracrine approach appears to enhance tumor antigen presentation and avoids systemic cytokine toxicity. The widespread clinical use of autologous cytokine gene transduced tumor vaccines may be limited by the technical difficulty and labor intensity of individualized gene transfer. We have therefore explored an alternate approach to generating sustained release of cytokines local to the tumor cells. High doses of granulocyte-macrophage colony-stimulating factor encapsulated in cell-sized gelatin-chondroitin sulfate microspheres were mixed with irradiated tumor cells prior to s.c. injection. This vaccination scheme resulted in systemic anti-tumor immune responses comparable to granulocyte-macrophage colony-stimulating factor gene transduced tumor vaccines.

Animals↗

PubMed1993

Fetal neuroblastoma: prenatal diagnosis and natural history.

Obstetrical sonography has helped diagnose and define the features of some congenital malformations and tumors. We present five fetal neuroblastomas detected by routine prenatal sonography. All were adrenal tumors diagnosed between 26 and 39 weeks gestation. All 5 tumors were completely resected postnatally and the patients have remained disease free from 2 months to 10 years after resection without adjuvant therapy. A literature review collated 16 other cases of fetal neuroblastoma detected by sonography between 29 and 38 weeks gestation. These cases included 1 cervical, 1 thoracic, and 14 adrenal tumors. Thirteen neonates had Evans stage I or II tumors, and three had more advanced disease. Eleven mothers did not have hypertension or preeclampsia during the pregnancy, and the neonates all had stage I or II disease. Four mothers had hypertension or preeclampsia. Three of these neonates had stage IV or IVS disease with liver metastases, and all three had fetal hydrops. Review of the congenital neuroblastoma literature documented 71 cases diagnosed soon after birth, and several of these cases had unusual features that could have been detected by prenatal ultrasound. Four of the tumors were so large that dystocia resulted and fetal dismemberment was required for delivery. Eight of the tumors metastasized to the placenta, and 1 metastasized to the umbilical cord with subsequent fetal death. We conclude that fetal neuroblastoma can be diagnosed by prenatal sonography. Accurate staging is difficult by sonography, but in mothers with no preeclampsia symptoms the chance of widely disseminated disease is small.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1993

The use of magnetic resonance imaging to track controlled drug release and transport in the brain.

A method has been developed to track controlled drug release and transport in the brain. This method entails the use of a polymeric implant to release, over time, a paramagnetically labelled compound into the brain. Magnetic resonance imaging is used to determine the evolving concentration distribution. This method is well suited to other types of intracranial drug delivery systems as well as to track transport in other organs of the body.

Agar↗

PubMed1991

In vitro release of hydrophobic drugs from polyanhydride disks.

We have evaluated the feasibility of using polyanhydride disks containing hydrophobic antiproliferative agents for controlled drug release after glaucoma filtration surgery. Taxol and VP-16 were the most potent inhibitors of fibroblast proliferation tested, with ID50s of 3 ng/mL and 200 ng/mL, respectively. In vitro release of taxol occurred at concentrations exceeding its ID50 for at least 100 days; VP-16 was released for 31 days. The media into which the drugs were released were able to inhibit fibroblast proliferation in vitro, indicating that the bioactivity of the drugs withstood incorporation into, and release from, the polyanhydride. Polyanhydride disks containing taxol and VP-16 merit testing in animal models of glaucoma filtration surgery.

Alkaloids↗

PubMed1990

High-level transcription from the adenovirus major late promoter requires downstream binding sites for late-phase-specific factors.

The adenovirus major late promoter (MLP) is active during both the early and late phases of infection. During the early phase the activity of the MLP is similar to those of the other early viral promoters, but during the late phase the rate of transcription from the MLP becomes much greater by comparison. We report here that sequence-specific binding proteins are induced during the late phase which interact with three regions in the first intron of the MLP transcription unit from positions +37 to +68, +80 to +105, and +105 to +125 relative to the transcription initiation site. To measure the significance of these binding sites for transcription during the late phase, we constructed MLP-beta-globin fusions and substituted them for early region 3 in adenovirus recombinants. Deletion of the binding sites caused significant reductions in the rate of transcription, specifically during the late phase of infection. Deletion of all three sites reduced the rate of transcription 25- to 50-fold and the accumulation of cytoplasmic MLP-beta-globin RNA 200-fold. These results indicate that the high rate of transcription from the MLP during the late phase of infection results from the interaction of virus-induced transcription factors with three binding sites in the first intron of the major late transcription unit.

Adenoviruses, Human↗

PubMed1989

Ultrasound-enhanced polymer degradation and release of incorporated substances.

The effect of ultrasound on the degradation of polymers and the release rate of incorporated molecules within those polymers was examined. Up to 5-fold reversible increases in degradation rate and up to 20-fold reversible increases in release rate of incorporated molecules were observed with biodegradable polyanhydrides, polyglycolides, and polylactides. Up to 10-fold reversible increases in release rate of incorporated molecules within nonerodible ethylene/vinyl acetate copolymer were also observed. The release rate increased in proportion to the intensity of ultrasound. Temperature and mixing were relatively unimportant in effecting enhanced polymer degradation, whereas cavitation appeared to play a significant role. Increased release rates were also observed when ultrasound was applied to biodegradable polymers implanted in rats. Histological examination revealed no differences between normal rat skin and rat skin that had been exposed to ultrasonic radiation for 1 hr. With further study, ultrasound may prove useful as a way of externally regulating release rates from polymers in a variety of situations where on-demand release is required.

Animals↗

PubMed1988

Polyanhydride microsphere formulation by solvent extraction.

A novel process based on solvent extraction was developed to produce drug-loaded polyanhydride microspheres for controlled-release applications. The technique consists of adding a chloroform solution of polyanhydride and drug into a stirred silicone oil phase containing suspended droplets of surfactant. No chemical reaction, heating, nor contact with water was required in this process. The microspheres produced were capable of releasing various dyes for prolonged periods of time.

Anhydrides↗

PubMed1988

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗

PubMed1986

Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts.

Protein encoded by adenovirus early region 1A (E1A) stimulates transcription from adenovirus promoters in vivo. Here we show that this effect can be observed in vitro. In a run-off transcription assay from the adenovirus serotype 2 (Ad2) major late promoter, extracts prepared 20 hr postinfection were 5-15 times more active than mock-infected-cell extracts prepared in parallel. Similar results were observed for in vitro transcription from the protein IX and E3 adenovirus promoters, whereas a 2-fold increase was observed for the human beta-globin promoter. The increased activities of infected-cell extracts did not depend on the expression of viral late proteins or the small E1A-encoded proteins but did require expression of the large E1A protein. These results are consistent with the large E1A protein stimulating transcription in vitro as it does in vivo. By limiting in vitro transcription to one initiation per template, we found that the higher activity of an infected-cell extract was due to an increase in the number of templates transcribed. These results suggest that the large E1A protein either increases the number of active transcription factors in infected cells or facilitates the interaction of cellular transcription factors with promoter DNA.

Adenovirus Early Proteins↗