Prevalence of naturally-occurring strains of Beauveria bassiana in populations of coffee berry borer Hypothenemus hampei on Hawai'i Island, with observations on coffee plant-H. hampei-B. bassiana interactions.

Beauveria bassiana (Bb) strain GHA is a major component of an areawide pest management program for coffee berry borer (CBB) in Hawai'i. Recent studies have aimed to provide comprehensive assessments of the efficacy of the Bb-spray component of these programs for economic analyses; however, evaluations have been complicated by activity of naturally-occurring strains of this pathogen infecting CBB. Investigations were therefore undertaken to characterize these strains, assess their natural epizootic potential, and account for their contribution to CBB population suppression. A number of field sites were encountered with no history of significant use of commercial Bb-based biopesticides and where strain GHA was not detectable. Sampling of these sites was conducted early in the coffee season. Greatest activity of wild-type Bb strains was observed on high-elevation farms (>500 m), where 24-42% of foundress beetles in green coffee berries were infected. In contrast, infection rates did not exceed 4% on farms at low elevations (<300 m). Rates of 23-29% infection, comparable to those on high-elevation farms, were recorded in a stand of feral coffee at 293 m elevation, but the coffee was completely shaded and ventilation restricted by a dense overstory of vegetation. Despite high activity of naturally-occurring Bb at some sites (primarily sites at high elevations with humid, moderate-temperature environments and dense pest populations), these fungi did not prevent CBB from exceeding the economic threshold for commercial spray applications. Nevertheless, the high natural epizootic potential of these fungal strains suggests strong potential for development as microbial biocontrol agents.

Detection of rare nonsynonymous variants in TGFB1 in otosclerosis patients.

Otosclerosis is one of the most common forms of hearing loss in the European population. We have identified a SNP in the TGFB1 (transforming growth factor beta 1) gene that is associated with susceptibility to otosclerosis. The protective allele of this variant, with isoleucine at position 263 of the protein, is more biologically active than the risk allele, which has a threonine in this position. Because recent studies have shown that not only common, but also rare variants can be involved in complex diseases, we performed DNA sequence analysis of the exons and intron-exon boundaries of TGFB1 in 755 otosclerosis patients and 877 control samples. We found 3 different nonsynonymous variants (E29, A29 and I241) in four otosclerosis patients, but no such changes were found in controls. In silico analysis shows that these variations could influence TGF-beta1 function and activity. Taking into account that most rare missense alleles are thought to have a biological effect, the data suggest that multiple rare amino acid changing variants in TGF-beta1 may contribute to susceptibility to otosclerosis.

Plant Chimeras Used to Establish de novo Origin of Shoots.

When African violet leaf explants are cultured in vitro, buds and shoots develop directly from the upper leaf surfaces. Three developmentally different African violet chimeras were cultured, and in each case adventitious shoots that developed into plants had the parent chimera pattern. A multicellular origin of the adventitious buds accounts for these results.

Chainpur-like Chondrites: Primitive Precursors of Ordinary Chondrites?

Chainpur and similar, apparently primitive, chondritic meteorites may be precursors of ordinary chondrites; a variety of evidence supports this working hypothesis. In general, carbonaceous chondrites seem to be related collaterally to this genetic sequence rather than being direct ancestors of ordinary chondrites. Metamorphic processes may be responsible for fractionations of elements such as indium and iodine, and type-II carbonaceous chondrites seem to be more primitive than types I or IIIA.

Chemical composition of sawdust from lunar rock 12013 and comparison of a java tektite with the rock.

Abundances of 11 major and minor elements and 11 trace elements have been determined by instrumental neutron activation analysis of two Apollo 12013 rock fragments, a sample of rock 12013,17 sawdust, and a Java tektite (J2). Although the abundances of major elements in tektite J2 are similar to those of rock 12013, comparison of the minor and trace elements shows that no fragment or sawdust of rock 12013 that has been analyzed to date is chemically similar to tektite glass. Rock sawdust is representative of "whole rock" composition only if the amount of contamination from the sawing process is known. After appropriate correction for saw wire contamination, analyses of sawdust yield fairly accurate averaged elemental compositions of complex clastic lunar and other rocks.

Adeno-associated virus integration: virus versus vector.

Although a large percentage of the world population is seropositive for exposure to various strains of adeno-associated virus (AAV), a human parvovirus, AAV has never been identified as an etiologic agent of human disease. Most likely contributing to the pronounced lack of pathogenicity is the fact that AAV is a naturally defective virus that requires a helper virus for productive replication of its genome. Another unusual aspect of wild-type AAV biology is the ability of the virus to establish latent infection by preferential integration of its genome into a specific locus of human chromosome 19. Site-specific integration was a major impetus for the development of recombinant AAV vectors, which typically lack all AAV coding sequences. It was soon realized, however, that expression of at least one species of the virally encoded initiator proteins, Rep78 or Rep68, is necessary for targeted integration of AAV-derived DNA constructs to occur. This article will present a chronological outline of studies characterizing site-specific integration of wild-type AAV sequences and the quasi-random target site selection observed with recombinant AAV vectors.

Inhibition of PrKX, a novel protein kinase, and the cyclic AMP-dependent protein kinase PKA by the regulatory proteins of adeno-associated virus type 2.

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, Kis of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.

NT-proBNP and the diagnosis of left ventricular systolic dysfunction within two acute NHS trust catchment areas: the initial Teesside experience.

PURPOSE: To evaluate the predictive value of N-terminal pro B-type natiuretic peptide (NT-proBNP) reference cut-off values as diagnostic markers for left ventricular systolic dysfunction (LVSD). STUDY DESIGN: A retrospective study assessing the use of NT-proBNP in the diagnostic algorithm for the investigation of patients with suspected signs and symptoms of LVSD presenting to primary care. RESULTS: A generic NT-proBNP cut-off (150 ng/l) value has similar negative and positive predictive valves, specificity and sensitivity compared to age and sex specific cut-off values. CONCLUSION: When using NT-proBNP as a triage tool for screening patients with signs and symptoms suggestive of LVSD, a simple generic cut-off level is as effective as more complex age sex specific cut-off values.

Carbohydrate oxidation and storage in obese non-insulin-dependent diabetic patients. Effects of improving glycemic control.

We have determined total body carbohydrate and lipid oxidation rates in response to a standard breakfast in nine obese patients with non-insulin-dependent diabetes mellitus (NIDDM) and in seven age- and weight-matched controls. The patients with NIDDM were studied twice, once while in poor glycemic control (fasting blood glucose concentration 267 +/- 24 mg/dl, urinary glucose excretion 28.9 +/- 6.3 g/24 h) and again after modest glycemic improvement following 2 mo of fiber treatment (fasting blood glucose 227 +/- 19 mg/dl, urinary glucose excretion 10.7 +/- 1.9 g/24 h). Basal carbohydrate (CHO) oxidation rates were normal in patients with NIDDM before and after fiber treatment. However, in patients before fiber treatment the rise in CHO oxidation rates, the reciprocal fall in lipid oxidation rates, and the rise in serum insulin and C-peptide concentrations after the breakfast were all severely blunted. In addition, storage of ingested CHO was significantly reduced (from 55% to 32%, P less than 0.05). After fiber treatment, postbreakfast CHO oxidation rates had improved and were no longer significantly lower than control values. In contrast, CHO storage remained suppressed. We conclude that (1) basal CHO oxidation remained normal but that postbreakfast CHO oxidation was impaired in our obese patients with NIDDM. This impairment, however, appeared to be a relatively late event, occurring only during severely uncontrolled NIDDM. (2) Inability to dispose of CHO by storage appeared to be an earlier defect with a greater impact on glucose tolerance than the impairment of CHO oxidation.

Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HIV-1 RT complexed with 8-Cl TIBO (R86183, IC50 = 4.6 nM) and 9-Cl TIBO (R82913, IC50 = 33 nM) have been determined at 3.0 A resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (Tyrl81Cys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyrl81Cys HIV-1 RT in complex with 8-Cl TIBO (IC50 = 130 nM) determined at 3.2 A resolution. Averaging of the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (-165 degrees C) and cooled (-10 degrees C) crystals of the same complex was employed to improve the quality of electron density maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the beta5-beta6 connecting loop (residues 95 to 105) and the beta13-beta14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the beta12-beta13 hairpin or the "primer grip" is significantly displaced relative to the position in the structure of HIV-1 RT complexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA. When the structure of the Tyr181Cys mutant HIV-1 RT in complex with 8-Cl TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyr181Cys and wild-type HIV-1 RT and of the 8-Cl TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the beta6-beta10-beta9 sheet containing residue 181 are observed when the Tyr181Cys and wild-type complexes are compared, particularlty near residue Val179 of beta9. In the p51 subunit, the Cys181 side-chain is oriented in a similar direction to the Tyr181 side-chain in the wild-type complex. However, the electron density corresponding to the sulfur of the Cys181 side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-Cl TIBO or nearby amino acid residues. In the mutant complex, there are slight rearrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-Cl TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the corresponding wild-type complex, Tyr181 iin the p66 subunit has significant interactions with the bound inhibitor and the position of the Tyr181 side-chain is well defined in both subunits. Apparently the Tyr181 --> Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bou

In vitro proliferation of lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with dimethyl dioctadecyl ammonium bromide.

A blastogenesis assay employing lymphocytes from cyclophosphamide-pretreated mice immunized with antigen mixed with the immunopotentiating compound dimethyl dioctadecyl ammonium bromide is described. The model antigen used for determining the assay parameters was inactivated purified measles virus. The optimal time for removal of immunologically primed T cells was 7 days after immunization of mice pretreated 2 days previously with 200 mg of cyclophosphamide/kg. The peak lymphoproliferative response was found to occur after 3-5 days in culture, depending on the concentration of antigen used. Although fetal bovine serum and syngeneic mouse serum each worked well as a medium supplement, significantly higher specific and lower non-specific lymphoproliferation were obtained when the mouse serum was used. Most of the lymphocytes responding to antigen were of the Ly 1.2 phenotype. Specificity of the blastogenic response was shown by a lack of cross-reactivity among measles virus, herpes simplex virus type 1 and vesicular stomatitis virus antigens. This approach to a mouse blastogenesis assay involves an easy way to induce strong T cell priming in mice, while still providing an assay which has an ideal combination of low non-specific and high antigen-specific responses.

A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation.

This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis. A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h. We found the method of LPS administration for induction of acute pulmonary damage to be crucial. After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS. As for intravenous injection, it showed a baseline effect. This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop.

1,3-Dialkyl-3-acyltriazenes, a novel class of antineoplastic alkylating agents.

Aliphatic triazenes, such as 1,3-dimethyltriazene, are potent biological alkylating agents because they form alkyldiazonium ions. They are also subject to very rapid proteolytic decomposition, even at physiological pH. The acylated analogues 1,3-dialkyl-3-acyltrizenes are much more stable in aqueous solution, but they also give rise to alkyldiazonium ions. Four acylated 1,3-dimethyltriazenes, where the acyl groups were diethylphosphoryl (DMP), carbethoxy (DMC), acetyl (DMA), and N-methylcarbamoyl (DMM), were studied kinetically. Rate-pH profiles indicated that the acyl group had a profound effect on the mechanism of decomposition. The cytotoxic potential of all four compounds was studied in vitro by using the MTT-tetrazolium assay. The compounds had fair-to-good activity against some cell lines, particularly those deficient in methylation repair. In vivo assays of DMC and DMM against several tumor xenografts in nude mice showed promising activity for some cancers, particularly in the case of DMM. In vitro assays were also carried out on three 1-(2-chloroethyl)-3-methyl-3-acyltriazenes. The acyl groups were carbethoxy (CMC), acetyl (CMA), and N-methylcarbamoyl (CMM). The activity of these compounds largely paralleled that of bis(2-chloroethyl)-N-nitrosourea (BCNU), except for those cell lines which exhibited the Rem phenotype; triazenes were more active in those lines than BCNU. The in vivo activity of CMC, CMA, and CMM was tested in the P388 leukemia assay. All three were active but CMC and CMA proved to be rather toxic. CMM was well tolerated and was examined in several tumor xenografts in nude mice. Significant activity was found against MX-1 mammary carcinoma, against LX-1 small cell lung carcinoma, and particularly against LOX amelanotic melanoma, where complete cures were effected. The antineoplastic activity of the acyltriazenes is well-correlated with their chemical behavior.

Prediction of binding affinities for TIBO inhibitors of HIV-1 reverse transcriptase using Monte Carlo simulations in a linear response method.

Monte Carlo (MC) simulations in combination with a linear response approach were used to estimate the free energies of binding for a series of 12 TIBO nonnucleoside inhibitors of HIV-1 reverse transcriptase. Separate correlations were made for the R6 and S6 absolute conformations of the inhibitors, as well as for the analogous N6-monoprotonated species. Models based upon the neutral unbound inhibitors produced overall better fits to experimental values than did those using the protonated unbound inhibitors, with only slight differences between the neutral R6 and S6 cases. The best results were obtained with a three-parameter linear response equation containing van der Waals (alpha), electrostatic (beta), and solvent accessible surface area (SASA, gamma) terms. The averaged (R6 and S6) rms error was approximately 0.88 kcal/mol for the observed range of 4.06 kcal/mol in inhibitor activities. The averaged values of alpha, beta, and gamma were -0.150, 0.114, and 0. 0286, respectively. Omission of the alpha term gave beta 0.152 and gamma 0.022 with a rms of 0.92. The unweighted van der Waals components were found to be highly attractive but failed to correlate well across the series of inhibitors. Contrastingly, while the electrostatic components are all repulsive, they show a direct correlation with inhibitor activity as measured by DeltaGbinding. The role of gamma is primarily to produce an overall negative binding energy, and it can effectively be replaced with a negative constant. During the MC simulations of the unbound solvated inhibitors, the R6 and S6 absolute conformations do not interconvert due to the formation of a favorable hydrogen bond to solvent. In the complex, however, interconversion of these conformations of the inhibitor is observed during the course of the simulations, a phenomenon which is apparently not observed in the crystalline state of the complex. Hydrogen bonding of the inhibitor to the backbone NH of K101 and the lack of such an interaction with the C=O of K101 or with solvent correlate with enhanced activity, as does the ability to assume a number of different orientations of the inhibitor dimethylallyl moiety with respect to residues Y181 and Y188 while retaining contact with W229. Overall, the use of a combination of MC simulation with a linear response method shows promise as a relatively rapid means of estimating inhibitor activities. This approach should be useful in the preliminary evaluation of potential modifications to known inhibitors to enhance activity.

T cell cross-reactivity among viruses of the paramyxoviridae.

Blastogenesis and delayed-type hypersensitivity assays were used to examine mouse T cell responses to five viruses representing the three genera of the Paramyxoviridae. Cross-reactive T cell responses were observed in a lympho-proliferative assay for measles, mumps, respiratory syncytial, canine distemper and parainfluenza type 3 virus. Confirmation of T cell cross-reactivity among measles, mumps and respiratory syncytial virus was obtained with a delayed-type hypersensitivity test. These results show that T cell cross-reactivity is common for Paramyxoviridae viruses, even though these viruses show virtually no inter-genus antibody cross-reactivity. The cross-reactivity among respiratory syncytial, measles and mumps virus at the T cell level may have implications for usage of the attenuated measles/mumps/rubella (MMR) vaccine. Respiratory syncytial virus is contacted by many children before they receive the MMR vaccine and T cells induced by respiratory syncytial virus may influence subsequent development of immunity to measles and/or mumps virus.

Tumors in F344 rats by oral administration of 1,3-diethyltriazene.

A group of 20 male F344 rats was given by gavage a solution of 2.5 mg of 1,3-diethyltriazene in 0.2 ml corn oil twice a week for 20 weeks. Diethyltriazene is very unstable in hydroxylic solvents, but when administered in oil by gavage it induced tumors in almost all of the treated animals, 16 of which had neoplasms of the forestomach; 13 of which were carcinomas. In addition 9 rats had adenomas of the nasal mucosa, two had alveolar-bronchiolar adenomas of the lung, one had a carcinoma of the esophagus and one an adenocarcinoma of the ileum. Half of the rats had died by week 56 of the experiment, the first at week 40 and the last at week 73. The induction of tumors at sites distant from the stomach indicate that diethyltriazene is more stable in the body than would be predicted from its chemical behavior. Although diethyltriazene is a protected alkyldiazonium ion, its carcinogenic effects in rats differ from ethylnitrosoureas, which are also ethylating agents.

An unexpected pathway for the metabolic degradation of 1,3-dialkyl-3-acyltriazenes.

In the presence of NADPH, rat liver microsomes catalyzed the degradation of a series of 1,3-dialkyl-3-acyltriazenes, and the extent of the reaction was correlated with compound lipophilicity. In the case of two methylcarbamoyltriazenes, 1-(2-chloroethyl)-3-benzyl-3- (methylcarbamoyl)triazene (CBzM) and 1-(2-chloroethyl)-3-methyl-3-(methylcarbamoyl)triazene (CMM), microsomal metabolites were isolated. Identification of the CBzM metabolites as 1-(2-chloroethyl)-3-benzyl-3-(hydroxymethylcarbamoyl)triazene and 1-(2-chloroethyl-3-benzyl-3-carbamoyltriazine, and the CMM metabolite as 1-(2-chloroethyl)-3-methyl-3-(hydroxymethylcarbamoyl)triazene indicated that the first metabolic step involves hydroxylation of the methylcarbamoyl substituent. Detailed studies of the metabolism of CBzM indicated that the Km for the reaction was 84 microM, and that metabolism was more efficient if microsomes were prepared from male than from female rats. During prolonged incubation, the metabolites of CBzM were also degraded. The degradation of CBzM and its metabolites was inhibited by SKF-525A and metyrapone, suggesting the involvement of a cytochrome P450 isozyme, and supporting the hypothesis that the process is oxidative rather than hydrolytic in both cases. Metabolic oxidation represents an alternative pathway to chemical or enzymatic hydrolysis for the in vivo decomposition of (methylcarbamoyl)triazenes. This mechanism may ultimately explain the antitumor efficacy and low acute toxicity of selected compounds.

Transbronchoscopic versus surgical resection of tracheobronchial granular cell myoblastomas. Suggested approach based on follow-up of all treated cases.

The case of a 31-year-old patient with a granular cell myoblastoma causing significant obstruction of the distal trachea is presented. A review of the literature suggests that size may help in deciding whether bronchoscopic removal or surgical resection should be performed. Fifty-four percent of the tumors removed bronchoscopically whose follow-up was described showed recurrent disease. All tumors removed bronchoscopically whose diameter was 1 cm or greater recurred. The correlation of full-thickness involvement of the tracheal wall with increasing tumor size appears to explain the failure of bronchoscopic treatment of these tumors. A suggested surgical approach to these rare tumors is proposed.

Mebendazole and insulin secretion from isolated rat islets.

In a preliminary communication we reported that mebendazole, a vermicide, decreased plasma glucose and free fatty acid concentrations and increased plasma C peptide concentrations in both type II diabetic patients. Therefore, we suggested that mebendazole was an insulin secretagogue. However, these were uncontrolled studies, and improved metabolic control in these patients due to spontaneous remission rather than drug-induced insulin secretion was a possibility. To investigate the direct effect of mebendazole on insulin secretion we used intact islets isolated from normal rat pancreata. Mebendazole in concentrations as low as 10 to 20 mumol/L caused a twofold to threefold increase in acute-phase insulin release from isolated perifused rat islets. This heightened insulin release occurred in the presence of glucose-stimulated insulin secretion.