Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.

In addition to target efficacy, drug safety is a major requirement during the drug discovery process and is influenced by target specificity. Therefore, it is imperative that every new drug candidate be profiled against various liability panels that include protein kinases. Here, an effective methodology to streamline kinase inhibitor profiling is described. An accessible standardized profiling system for 112 protein kinases covering all branches of the kinome was developed. This approach consists of creating different sets of kinases and their corresponding substrates in multi-tube strips. The kinase stocks are pre-standardized for optimal kinase activity and used for inhibitor profiling using a bioluminescent ADP detection assay. We show that these strips can routinely generate inhibitor selectivity profiles for small or broad kinase family panels. Lipid kinases were also assembled in strip format and profiled together with protein kinases. We identified two specific PI3K inhibitors that have off-target effects on CK2 that were not reported before and would have been missed if compounds were not profiled against lipid and protein kinases simultaneously. To validate the accuracy of the data generated by this method, we confirmed that the inhibition potencies observed are consistent with published values produced by more complex technologies such as radioactivity assays.

Selective inhibitor of endosomal trafficking pathways exploited by multiple toxins and viruses.

Pathogenic microorganisms and toxins have evolved a variety of mechanisms to gain access to the host-cell cytosol and thereby exert virulent effects upon the host. One common mechanism of cellular entry requires trafficking to an acidified endosome, which promotes translocation across the host membrane. To identify small-molecule inhibitors that block this process, a library of 30,000 small molecules was screened for inhibitors of anthrax lethal toxin. Here we report that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone, the most active compound identified in the screen, inhibits intoxication by lethal toxin and blocks the entry of multiple other acid-dependent bacterial toxins and viruses into mammalian cells. This compound, which we named EGA, also delays lysosomal targeting and degradation of the EGF receptor, indicating that it targets host-membrane trafficking. In contrast, EGA does not block endosomal recycling of transferrin, retrograde trafficking of ricin, phagolysosomal trafficking, or phagosome permeabilization by Franciscella tularensis. Furthermore, EGA does not neutralize acidic organelles, demonstrating that its mechanism of action is distinct from pH-raising agents such as ammonium chloride and bafilomycin A1. EGA is a powerful tool for the study of membrane trafficking and represents a class of host-targeted compounds for therapeutic development to treat infectious disease.

Henipavirus receptor usage and tropism.

Nipah (NiV) and Hendra (HeV) viruses are the deadliest human pathogens within the Paramyxoviridae family, which include human and animal pathogens of global biomedical importance. NiV and HeV infections cause respiratory and encephalitic illness with high mortality rates in humans. Henipaviruses (HNV) are the only Paramyxoviruses classified as biosafety level 4 (BSL4) pathogens due to their extreme pathogenicity, potential for bioterrorism, and lack of licensed vaccines and therapeutics. HNV use ephrin-B2 and ephrin-B3, highly conserved proteins, as viral entry receptors. This likely accounts for their unusually broad species tropism, and also provides opportunities to study how receptor usage, cellular tropism, and end-organ pathology relates to the pathobiology of HNV infections. The clinical and pathologic manifestations of NiV and HeV virus infections are reviewed in the chapters by Wong et al. and Geisbert et al. in this issue. Here, we will review the biology of the HNV receptors, and how receptor usage relates to HNV cell tropism in vitro and in vivo.

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M) protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV) is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4) pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS) and the leucine-rich nuclear export signal (NES) found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors) resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50) of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Outpatient anterior cruciate ligament reconstruction: an analysis of charges and perioperative complications.

All outpatient anterior cruciate ligament (ACL) reconstructions using patellar tendon autograft performed at an accredited outpatient surgical center between 1994 and 1998 were prospectively studied. Hospital charges pertaining to the procedures were examined, and perioperative morbidities that might be attributed to an outpatient procedure were evaluated. The study group comprised 284 patients; average patient age at surgery was 28.7 years. Patients were subgrouped into group 1 (isolated ACL reconstructions; n=163), group 2 (ACL reconstructions and meniscal repair; n=48), and group 3 (ACL reconstructions and partial meniscectomy; n=73). Surgicenter facility charges, reoperation rate, complication rate, motion, pain management, hospital emergency room visits, hospital admission, and outpatient surgical facility visits were analyzed. Historical controls from our hospital and our initial outpatient pilot study (May 1994 through November 1995) were used as financial controls. The average surgical center charge for all patients was $3,443. On average, there was a $600 increase for all subgroups from May 1994 through November 1995 compared to December 1995 through August 1998. In the latter time interval, the fixed facility charges were $3,150, $4,075, and $4,275 for groups 1, 2, and 3, respectively. Overall, 19 (7%) patients required a reoperation including 7 (2.5%) patients who required arthroscopic debridement for symptomatic motion deficits. This study expands on our initial published report regarding hospital charges pertaining to an outpatient ACL reconstruction. Extended over another 4 years, we noted slight increases reflective of regional inflationary increases. Compared to our initial inpatient study (1988-1993), significant charge reductions were maintained. This study demonstrated a low complication rate and high patient subjective satisfaction level.

The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure.

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.

[The clinical significance of lung resistance protein (LRP) gene expression in patients with acute leukemia].

OBJECTIVE: To investigate the relationship between the expression of lung resistance protein (LRP) gene and drug resistance in patients with acute leukemias (AL). METHODS: Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)was used to examine the expression of LRP gene in AL patients and 15 normal subjects. Beta(2) microglobulin (beta(2)MG) was used as internal reference. LRP/beta(2)MG ratio >or= 0.3 was defined as LRP positive. RESULTS: The positivity percentage of LRP gene expression in newly diagnosed group was 32.4%. The first complete remission rate was 84.0% and 33.0% in LRP negative and LRP positive patients, respectively. The difference was significant (P < 0.005). The expression level of LRP mRNA and the positivity percentage of LRP in relapsed/refractory group were significantly higher than that in newly diagnosed group (P < 0.01). The expression level of LRP gene in normal subjects and long-term survival groups was very low and correlated with FAB subtypes. The mdr-1 gene was examined simultaneously in 61 AL patients. No significant correlation was found between the expression of LRP and mdr-1 gene (P > 0.5). Coexpression of LRP and mdr-1 genes in the same AL patient might result in the worst prognosis. CONCLUSION: High expression of LRP gene leads to clinical drug resistance and is an unfavorable factor to AL patients of prognosis.

[The construction of a general adeno-associated virus vector].

The general adeno-associated virus(AAV) vector pACR-Neo was constructed by substituting AAV's construct gene with cassette that was composed of CMV-IE promoter, multiple cloning sites and SV40-polyA. After transfecting pACR-Neo into recombinant AAV's packaging cell line AE1201, which was infected by Adenovirus 5 before transfection, we got rAAV/ACR-Neo at the titer of 4.2 x 10(5) CFU/ml. Using rAAV/ACR-Neo infected host cell A549, the recombinant AAV could transfer reporter gene Neo into host cell and mediate the integration of viral genome into host cell's chromosome DNA. By extracting chromosome DNA of host cell A549 that had been infected by rAAV/ACR-Neo, and digestion respectively with restriction endonuclease PvuII and HindIII, each of which has only one cutting site within rAAV/ACR-Neo's genome. We did Southern blot to check the hybridization of endonuclease digested chromosome to digoxin labelled Neo gene probe. After analyzing the experimental results, we found that rAAV/ACR-Neo's genome had integrate into host cell's chromosome compared to wild type AAV's site-specific integration. Through this work, we constructed a general AAV vector successfully. It lays foundation for research on AAV vector and application on gene therapy.

[Adeno-associated virus vector mediated gene transfer of HSVI-TK and its effect on killing cancer cell].

The plasmid pACTK-19 was constructed by inserting HSVI-TK gene into the multiple cloning sites of the general adeno-associated virus(AAV) vector pACR-Neo. When plasmid pACTK-19 was transfected to recombinant AAV's packaging cell line AE1201, which was exposed to Adenovirus 5 for two hours before transfection, we got rAAV/ACTK at the titer of 3.4 x 10(5) CFU/ml. After infecting human lung cancer cell A549 with rAAV/ACTK, we extracted host cell's chromosome cDNA and amplified part of the HSVI-TK sequence by a pair of HSVI-TK's primers and then hybridized to digoxin labelled HSVI-TK gene probe. We got corresponding positive band and it proved the integration of HSVI-TK into host cell's chromosome cDNA. By reverse transcripting rAAV/ACR-Neo infected A549 total cell RNA, we amplified part of HSVI-TK sequence and hybridized to Neo gene probe. The corresponding positive band demonstrated the expression of HSVI-TK. So through rAAV/ACTK, HSVI-TK was transferred into host cells and was expressed in them, the rAAV's genome was integrated into host cell's chromosome DNA. Connecting with Ganciclovir (GCV), the HSVI-TK gene transfer mediated by rAAV/ACTK, could inhibit the synthesis of chromosome DNA and lead to the killing of the lung cancer cell. This work proved that recombinant AAV could be used as vector for gene transduction to cancer cell, and also laid foundation for further research and application of AAV vector.

Correlated flow cytometric analysis of H-ras p21 and DNA ploidy in acute myelogenous leukemia.

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment, DNA diploidy occurred in 18 cases including 13 p21 negative ones, and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

Correlative study on the expression of p53 and DNA ploidy in acute nonlymphocytic leukemia.

We used the flow cytometric immunoassay to study the correlation between the rumor-suppressor gene product p53- and the DNA ploidy in 30 de novo cases of acute nonlymphocytic leukemia (ANLL). The results showed that 15 cases were positive expression for p53. As compared with p53 negative (p53) cases, the patients with positive p53 (p53+) had higher percentage of bone marrow blasts and lower peripheral leukocyte and platelet counts, which had no influence on the complete remission rate. Before treatment, DNA diploidy was seen in 18 cases including 12 p53- cases, and DNA aneuploidy in 12 cases including 9 p53+. After therapy, aneuploidy could be transformed into diploidy. Patients with P53+ or having aneuploidy in complete remission were at risk for early relapse. We believe that p53 may be involved in the process of leukemogenesis and progression of ANLL.

Functional status of pancreatic islet in acute leukemia.

Using enzymatic assay and radioimmunoassay, we studied the functional status of pancreatic islet in 50 patients with acute leukemia. Oral glucose tolerance test and insulin and C peptide release were made in 40 patients before and after treatment. 14 patients who revealed diabetic curve and delayed insulin and C peptide release before treatment showed normal values in 6 after therapy. Five patients with impaired glucose tolerance and decreased insulin and C peptide release before treatment showed normalization of these parameters following therapy. Five patients with normal pretreatment values disclosed abnormal post-treatment results. The remaining 16 patients displayed normal results both before and after therapy. Anti-insulin antibodies were negative, and glucagon level was normal in all the 50 patients. The red cell insulin receptor binding rate analysed in 47 patients was significantly higher than in controls (P < 0.001). We considered that the disturbed glucose metabolism in acute leukemia was not uncommon mainly due to the dysfunction of pancreatic islet beta cells as a result of islet damage by leukemic cells, the effect of corticosteroid and chemotherapy and the preexisting diabetes. Impaired glucose metabolism had no influence on therapeutic effect.

Etoposide-based combination chemotherapy in malignant histiocytosis.

We investigated the responses of patients with malignant histiocytosis (MH) to the treatment of epotoside-based regimen. Of 11 evaluable cases, 9 (81.8%) achieved complete remission and 1 partial remission. 7 complete remission cases (77.7%) received only one therapeutic course. The side effects were mild and well-tolerated. We conclude that etoposide-containing regimen is highly effective and can be used as first-line treatment for MH and is worthy of further study.

Plasma ammonia in patients with acute leukemia.

Plasma ammonia level (PAL) was studied in 43 cases of acute leukemia (AL). PAL was 39.21 +/- 26.2 mumol/L in normal controls and 38.8 +/- 16.6 mumol/L in leukemic patients before chemotherapy. High PAL was found in 40 cases after chemotherapy. Six cases showed clinical manifestations due to severe hyperammonemia, including dizziness, lethargy, confusion, coma and mental changes of various degree, and there was also respiratory alkalosis. After ammonia-trapping therapy, 4 of the 5 patients recovered. The authors believe that high PAL is not uncommon after chemotherapy in leukemic patients. Respiratory alkalosis and unexplained mental and neurologic changes following intensive chemotherapy are useful clues for the diagnosis of hyperammonemia syndrome. Early diagnosis and treatment with ammonia-trapping may improve the rates of remission and survival.

Therapeutic and prognostic value of modal number of chromosomes at the blastic phase of Philadelphia-chromosome-positive chronic myeloid leukemia: comparison based on the same criteria between Nagasaki University and Roswell Park Memorial Institute.

In a comparison of 47 patients with Philadelphia-chromosome (Ph)-positive chronic myeloid leukemia (CML) in the Nagasaki University School of Medicine and 64 patients with the same disease in the Roswell Park Memorial Institute, the correlation between the modal number of chromosomes and the therapeutic response and/or survival after the onset of the blastic phase (BP) was evaluated. The patients were divided into four groups on the basis of the modal number of chromosomes of the cells in the bone marrow: those with hypodiploidy (group 1), those with pseudodiploidy carrying a Ph chromosome (group 2), those with 47 chromosomes (group 3), and those with 48 or more chromosomes (group 4). The results revealed similar trends in the two institutes. Namely, the therapeutic response and the survival after the onset of the BP in groups 1 and 4 were more unfavorable and shorter than those in groups 2 and 3, although the former (group 2) had a better prognosis than the latter (group 3). Thus, the statistical analysis revealed that the numerical chromosome findings at the BP are useful parameters for assessing the therapeutic response and survival after the onset of the BP of CML.

Therapeutic and prognostic value of chromosomal AP classification at the blastic phase of Ph-positive chronic myeloid leukemia: comparison of data from Nagasaki University, Japan and Roswell Park Memorial Institute, U.S.A.

To assess parameters of therapeutic response and survival after the onset of the blastic phase (BP) in 47 patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML), a number of clinical hematologic, and cytogenetic data at the BP were evaluated. Among the eleven parameters examined, only the chromosomal findings correlated with the therapeutic response and survival after onset of the BP. The patients were divided into three groups on the basis of the chromosome findings in the bone marrow: one, with only a Ph (PP), another with two different clones, i.e., one clone with Ph only and another with additional karyotypic changes (AP), and a third group with only abnormal clones containing karyotypic abnormalities in addition to the Ph (AA). The number of patients with AA was 12, 18 with AP, and 17 with PP. The results were as follows: (1) The percentage of patients with a good therapeutic response was 25% (3/12) in AA, 22% (4/18) in AP, and 76% (13/17) in PP; (2) The median survival after the onset of the BP was 1.5 mo for AA, 2.4 mo for AP, and 7.3 mo for PP. Statistically, there was a significant difference between PP and the other two groups (p < 0.05). These data were reevaluated and compared to those of 64 patients with Ph-positive CML in Roswell Park Memorial Institute (RPMI) who had been reported earlier in 1983.

Cytogenetic studies of human T-cell leukemia virus type I carriers. A family study.

Recently, the chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL), and this anomaly has also been confirmed in the preleukemic state of adult T-cell leukemia (pre-ATL) patients. Because the cytogenetic abnormality at the stage of human T-cell leukemia virus type I (HTLV-I) carrier remains uncertain, we performed cytogenetic studies of lymphocytes stimulated with phytohemagglutinin in three HTLV-I carriers and three non-HTLV-I carriers in an ATL family. As a result, in three HTLV-I carriers, four of 311 cells examined (1.3%) had chromosome 14q11 anomaly. However, in three non-HTLV-I carriers, none of 260 cells examined had chromosome 14q11 anomaly. These results suggest that chromosome 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.

Significance of FCM-DNA measurement in detecting minimal residual disease in leukemia.

Using flow cytometry (FCM), we measured the DNA content of bone marrow from 25 patients with acute leukemia (AL) in complete remission (CR) and followed them up for 0.5 to 1 year. Five of 13 patients in early relapse had normal DNA index (DI), while the remaining 8 showed DNA aneuploidy 1.5 to 5 months (mean 2.6 months) before relapse. The relapse rate was higher in CR patients with DNA aneuploidy (88.9%) than in those with normal DI (31.3%, P less than 0.05). Intensive chemotherapy was helpful to prolong CR duration in CR patients with DNA aneuploidy. DNA measurement by FCM was considered a reliable method for detecting minimal residual disease (MRD), and the appearance of DNA aneuploidy might predict a relapse of CR.

16;21 translocation in acute nonlymphocytic leukemia with abnormal eosinophils: a unique subtype.

Two patients with acute nonlymphocytic leukemia (ANLL) and t(16;21)(p11;q22) were studied. The patients exhibited such clinical and hematological pictures, characterized by M2 and M4 with eosinophilia (FAB classification), as relatively matured leukemic cells, low neutrophil alkaline phosphatase activity, abnormal eosinophils and a high count of monocytic cells in the bone marrow. The prognosis was poor in both patients. From these data, the chromosomal abnormality of t(16;21)(p11;q22) seems to be specifically associated with a unique subtype of ANLL.