Biomedical subjects

L Lai

Publications and source records attributed to L Lai.

At least 37 records · Page 2Linked to original sources

PubMed2001

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig oocytes.

Animals↗

PubMed2001

Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes.

The objective of this study was to evaluate in vitro and in vivo development of porcine in vitro matured (IVM) porcine oocytes fertilised by intracytoplasmic sperm injection (ICSI) and the possibility of producing transgenic embryos and offspring with this procedure. Activated ICSI oocytes had a higher pronuclear formation than non-activated ICSI oocytes (mean 64.8+/-17.3% vs 28.5+/-3.4%, p<0.05). When the zygotes with two pronuclei were cultured to day 2, there was no difference (p<0.05) in the cleavage rate (mean 60.0+/-7.0% vs 63.3+/-12.7%) between the two groups. The blastocyst rate in the activation group was significantly higher than that in the non-activation group (mean 30.0+/-11.6% vs 4.6+/-4.2%, p<0.05). After injection of the sperm transfected with DNA/liposome complex, destabilised enhanced green fluorescent protein (d2EGFP) expression was not observed on day 2 in either cleaved or uncleaved embryos. But from day 3, some of the embryos at the 2-cell to 4-cell stage started to express d2EGFP. On day 7, about 30% of cleaved embryos, which were in the range of 2-cell to blastocyst stage, expressed d2EGFP. However, for the IVF oocytes inseminated with sperm transfected with DNA/liposome complex, and for oocytes injected with sperm transfected with DNA/liposome complex, and for oocytes injected with DNA/liposome complex following insemination with sperm not treated with DNA/liposome complex, none of the embryos expressed d2EGFP. Sixteen day 4 ICSI embryos derived from sperm not treated with DNA/liposome complex were transferred into a day 3 recipient. One recipient delivered a female piglet with normal birthweight. After transfer of the ICSI embryos derived from sperm transfected with DNA/liposome complex, none of the four recipients maintained pregnancy.

Animals↗

PubMed2001

Protein ligand docking based on empirical method for binding affinity estimation.

An empirical protein-ligand binding affinity estimation method, SCORE, was incorporated into a popular docking program, DOCK4. The combined program, ScoreDock, was used to reconstruct the 200 protein-ligand complex structures and found to give good results for the complexes with high binding affinities. A quality assessment method for docking results from ScoreDock was developed based on the whole test set and tested by additionally selected complexes. The method significantly improves the docking accuracy and was shown to be reliable in docking quality assessment. As a docking tool in structural based drug design, ScoreDock can screen out final hits directly based on the predicted negative logarithms of dissociation equilibrium constants of protein-ligand complexes, and can explicitly deal with structure water molecules, as well as metal atoms.

Computer Simulation↗

PubMed2001

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Agriculture↗

PubMed2001

Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector.

Here we report the production of transgenic pigs that express enhanced green fluorescent protein (eGFP). Porcine oocytes were matured in vitro in a serum-free, chemically defined maturation medium, subsequently infected with a replication deficient pseudotyped retrovirus, fertilized and cultured in vitro before being transferred to a recipient female. Two litters were born from these embryo transfers; one pig from each litter was identified as transgenic and both expressed eGFP. From a tool in basic research to direct applications in production agriculture, domestic livestock capable of expressing foreign genes have many scientific applications.

Agriculture↗

PubMed2001

Dynamic events are differently mediated by microfilaments, microtubules, and mitogen-activated protein kinase during porcine oocyte maturation and fertilization in vitro.

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

Actin Cytoskeleton↗

PubMed2001

Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs.

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.

Animals↗

PubMed2001

Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

Animals↗

PubMed2001

Developmental potential of porcine nuclear transfer embryos derived from transgenic fetal fibroblasts infected with the gene for the green fluorescent protein: comparison of different fusion/activation conditions.

The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42-44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3-5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 microsec (group A), or one pulse of 1.6 kV/cm for 30 microsec followed in 30 min by one pulse of 1.2 kV/cm for 30 microsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean +/- SEM, 68.4% +/- 3.9%) was higher (P < 0.05) than in group B (59.4% +/- 2.3%). The rates of cleavage (50.1% +/- 4.6% to 62.8% +/- 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% +/- 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% +/- 1.7% and 4.9% +/- 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% +/- 4.2%) was higher (P < 0.05) than in group C (58.9% +/- 2.7%). The cleavage rates were not different among control and treatment groups (58.1% +/- 8.1% to 73.6% +/- 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% +/- 1.7%) was lower (P < 0.05) than in controls and group C (11.4% +/- 2.0% to 16.4% +/- 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% +/- 1.4%) was higher (P < 0.05) than in the group without CB (70.9% +/- 0.2%). The cleavage rate of the control group (85.5% +/- 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% +/- 2.7% and 63.9% +/- 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% +/- 2.5% to 19.1% +/- 6.0%) and the mean cell number of blastocysts (29.4 +/- 5.2 to 45.7 +/- 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.

Animals↗

PubMed2001

Assessment of two hand hygiene regimens for intensive care unit personnel.

OBJECTIVE: To compare skin condition and skin microbiology among intensive care unit personnel using one of two randomly assigned hand hygiene regimens: a 2% chlorhexidine gluconate (CHG)-containing traditional antiseptic wash and a waterless handrub containing 61% ethanol with emollients (ALC). DESIGN: Prospective, randomized clinical trial. SETTING: Two critical care units (medical and surgical) in a large, metropolitan academic health center in Manhattan. SUBJECTS: Fifty staff members (physicians, nurses, housekeepers, respiratory therapists) working full time in the intensive care unit. INTERVENTIONS: One of two hand hygiene regimens randomly assigned for four consecutive weeks. MEASUREMENTS AND MAIN RESULTS: The two outcomes were skin condition (measured by two tools: Hand Skin Assessment form and Visual Skin Scaling form) and skin microbiology. Samples were obtained at baseline, on day 1, and at the end of wks 2 and 4. Participants in the ALC group had significant improvements in the Hand Skin Assessment scores at wk 4 (p = 0.04) and in Visual Skin Scaling scores at wks 3 (p = 0.01) and 4 (p = 0.0005). There were no significant differences in numbers of colony-forming units between participants in the CHG or ALC group at any time period. The ALC regimen required significantly less time than the CHG regimen (mean: 12.7 secs and 21.1 secs, respectively; p = 0.000) and resulted in a 50% reduction in material costs. CONCLUSIONS: Changes in hand hygiene practices in acute care settings from the traditional antiseptic wash to use of plain, mild soap and an alcohol-based product should be considered. Further research is needed to examine the association between use of antiseptic products for hand hygiene of staff and reductions in nosocomial infection rates among patients.

Adult↗

PubMed2001

Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.

Alcaligenes↗

PubMed2001

Moloney murine leukemia virus integrase protein augments viral DNA synthesis in infected cells.

Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (DeltaIN) or 34 C-terminal amino acid residues (Delta34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Delta34 and DeltaIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Delta34 and DeltaIN mutants in trans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of DeltaIN mutant virions could not be complemented with the Delta34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Base Sequence↗

PubMed2001

Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro.

The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

Actin Cytoskeleton↗

PubMed2000

Analysis of human immunodeficiency virus type 1 containing HERV-K protease.

The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.

Cell Line↗

PubMed2000

Organic cosolvents and hen egg white lysozyme folding.

Studies on the influence of organic cosolvents on lysozyme folding have been reported. As most of the researches are confined to a few specific molecules and focus on equilibrium states, less is known about the effect on folding dynamics. We have studied the influence of six soluble organic cosolvents on hen egg white lysozyme heat induced denaturation and refolding dynamics. It was found that trifluoroethanol (TFE) can change the folding pathway significantly. With the presence of TFE, the overshot phenomenon generally observed in lysozyme folding at 222 nm disappears. The common mechanism of how organic cosolvents influence folding is analyzed. The heat induced denaturation temperature was found to have a quantitative relationship with the slow phase rate constant during folding. We discuss this finding and hypothesize that it is due to the similar influence of organic cosolvent on the transition state of heat denaturation and refolding.

Animals↗

PubMed2000

An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer.

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.

Alpha Particles↗

PubMed2000

Crystal structure of archaeal RNase HII: a homologue of human major RNase H.

BACKGROUND: RNases H are present in all organisms and cleave RNAs in RNA/DNA hybrids. There are two major types of RNases H that have little similarity in sequence, size and specificity. The structure of RNase HI, the smaller enzyme and most abundant in bacteria, has been extensively studied. However, no structural information is available for the larger RNase H, which is most abundant in eukaryotes and archaea. Mammalian RNase H participates in DNA replication, removal of the Okazaki fragments and possibly DNA repair. RESULTS: The crystal structure of RNase HII from the hypothermophile Methanococcus jannaschii, which is homologous to mammalian RNase H, was solved using a multiwavelength anomalous dispersion (MAD) phasing method at 2 A resolution. The structure contains two compact domains. Despite the absence of sequence similarity, the large N-terminal domain shares a similar fold with the RNase HI of bacteria. The active site of RNase HII contains three aspartates: Asp7, Asp112 and Asp149. The nucleotide-binding site is located in the cleft between the N-terminal and C-terminal domains. CONCLUSIONS: Despite a lack of any detectable similarity in primary structure, RNase HII shares a similar structural domain with RNase HI, suggesting that the two classes of RNases H have a common catalytic mechanism and possibly a common evolutionary origin. The involvement of the unique C-terminal domain in substrate recognition explains the different reaction specificity observed between the two classes of RNase H.

Amino Acid Sequence↗

PubMed2000

Vaccination of patients with chronic myelogenous leukemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses.

Chronic myelogenous leukemia (CML) presents a unique opportunity to develop therapeutic strategies using vaccination against a truly tumor-specific antigen that is also the oncogenic protein required for neoplasia. CML is characterized by the t(9;22) that results in the bcr-abl fusion oncogene and in the expression of a chimeric protein product p210. Previously we have shown that peptides derived from amino acid sequences crossing the b3a2 fusion breakpoint in p210 elicit class I restricted cytotoxic T lymphocytes and class II responses, respectively, in vitro. Such sequences may thus comprise absolutely tumor-specific antigens in a peptide-based vaccine. We evaluated the safety and immunogenicity of a multidose, bcr-abl breakpoint peptide vaccine in 12 adults with chronic-phase CML. Cohorts of 3 patients each received either 50 microg, 150 microg, 500 microg, or 1500 microg total peptide mixed with 100 microg QS-21 as an immunological adjuvant. Delayed-type hypersensitivity (DTH), humoral responses, and unprimed ex vivo autologous proliferation ((3)H-thymidine incorporation) and cytotoxicity (chromium-51 release) responses were measured. All 68 vaccinations were well tolerated without significant adverse effects. In 3 of the 6 patients treated at the 2 highest dose levels of vaccine, peptide-specific, T-cell proliferative responses (n = 3) and/or DTH responses (n = 2) were generated that lasted up to 5 months after vaccination. Cytotoxic T lymphocytes have not been identified. In conclusion, a tumor-specific, bcr-abl derived peptide vaccine can be safely administered to patients with chronic-phase CML and can elicit a bcr-abl peptide-specific immune response despite the presence of active disease in these patients and approximately 10(12) leukemia cells. (Blood. 2000;95:1781-1787)

Adjuvants, Immunologic↗