Genotypic and Phenotypic Variables Affect Meiotic Cell Cycle Progression, Tumor Ploidy, and Cancer-Associated Mortality in a brca2-Mutant Zebrafish Model.

Successful cell replication requires both cell cycle completion and accurate chromosomal segregation. The tumor suppressor BRCA2 is positioned to influence both of these outcomes, and thereby influence genomic integrity, during meiotic and mitotic cell cycles. Accordingly, mutations in BRCA2 induce chromosomal abnormalities and disrupt cell cycle progression in both germ cells and somatic cells. Despite these findings, aneuploidy is not more prevalent in BRCA2-associated versus non-BRCA2-associated human cancers. More puzzlingly, diploidy in BRCA2-associated cancers is a negative prognostic factor, unlike non-BRCA2-associated cancers and many other human cancers. We used a brca2-mutant/tp53-mutant cancer-prone zebrafish model to explore the impact of BRCA2 mutation on cell cycle progression, ploidy, and cancer-associated mortality by performing DNA content/cell cycle analysis on zebrafish germ cells, somatic cells, and cancer cells. First, we determined that combined brca2/tp53 mutations uniquely disrupt meiotic progression. Second, we determined that sex significantly influences ploidy outcome in zebrafish cancers. Third, we determined that brca2 mutation and female sex each significantly reduce survival time in cancer-bearing zebrafish. Finally, we provide evidence to support a link between BRCA2 mutation, tumor diploidy, and poor survival outcome. These outcomes underscore the utility of this model for studying BRCA2-associated genomic aberrations in normal and cancer cells.

SB-219383, a novel tyrosyl tRNA synthetase inhibitor from a Micromonospora sp. I. Fermentation, isolation and properties.

A novel, potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, designated SB-219383 has been isolated from Micromonospora sp. NCIMB 40684. The fermentation, isolation and some properties are described, whilst the structure determination is described in the succeeding paper). SB-219383 showed competitive, inhibitory activity against a Staphylococcus tyrosyl tRNA synthetase, with an IC50 of <1 nM, and exhibited weak in vitro activity against some Streptococcus sp.

Synthesis and activity of analogues of SB-219383: novel potent inhibitors of bacterial tyrosyl tRNA synthetase.

SB-219383 is a naturally occurring antibiotic, which acts by inhibition of tyrosyl tRNA synthetase. Semi-synthetic derivatives of SB-219383 were prepared with the objective of elucidating the key features required for inhibition of tyrosyl tRNA synthetase in order to improve the antibacterial activity. Some ester and amide derivatives as well as monocyclic analogues exhibited sub-nanomolar inhibitory activity against tyrosyl tRNA synthetase.

Academic espionage: dysfunctional aspects of the publish or perish ethic.

There are many dysfunctional manifestations relative to the tenure and promotion process. These are disruptive to academic life. Much of this is encouraged by the university because of their publish or perish ethic. Excellence in classroom teaching and success in the field of clinical and human endeavours are not highly valued in deliberations to grant tenure and advancement in academic rank. Research and publications are the major yardsticks upon which a faculty member is judged. This prevailing perspective poses a dilemma for many nursing faculty who have high clinical workloads and have not been socialized for academic survival. The pressures to publish and research can be achieved in a realistic and non-stressful way. Three aspects seem to be particularly relevant to facilitate this achievement; these are: anticipatory planning, balancing the workload, and understanding the interpersonal dimensions of collegiality.

Student unrest: a challenge for nurse educators.

Nursing students, like their college counterparts, are beginning to revolt against hierarchical authority and to claim a more active part in their educational planning. Nurse educators can no longer assume that their students will never revolt; they must face up to the challenge of developing strategies for proper management of student unrest. The authors propose some ways to deal with the complex issue of student unrest. These suggestions are made with emphasis on faculty-student relationship and the curriculum. Improved faculty-student relationships, proper utilization of student peer influence and student involvement in curriculum planning are some of the strategies proposed in this paper.

A conceptual approach to the development of motivational strategies.

Teachers in both general and professional education are well aware of the importance of motivation as the basis of effective teaching. Heidgerken maintains that a major problem that faces nursing educators is to select the right type of motivation and to know how to effect motivation. The authors propose a conceptual model for developing strategies to deal with motivational problems. This model is an application of Vroom's value/expectancy theory and Rotter's social learning theory, and is based on the premise that nursing faculty are in strategic positions to enhance student motivation for learning by manipulating variables that are believed to influence motivation. These suggested strategies include: enhancing student's need value by rewarding them with rewards that they value the most; increasing student's perceptions of a strong link between performance and rewards; providing students with consistent and clear role perceptions; improving student satisfaction towards learning.

[On the value of drill biopsy in diagnosis of breast lesions (author's transl)]. / Untersuchungen über den Wert der Drillbiopsie in der Mammadiagnostik.

During a period of 30 months 216 drill biopsies of palpable breast lesions were carried out. By using this method sufficient tissue material for a histological examination could be acquired in all cases. Out of 106 cases, in which the histological results could exactly be proved by following excision biopsy or mastectomy, 87% were correctly diagnosed by drill biopsy. The reason for failure in all of the 14 false drill biopsy diagnoses was due to sampling error. Comparing the diagnostic results of the first 15 month after establishing the method, with the second half of the investigation period, a considerable improvement from 80% to 94% of correct diagnoses could be established. This improvement was based on increasing experience and routine. By means of drill biopsy, cosmetically and diagnostically disturbing scar formation could be avoided. Drill biopsy can be carried out in local anaesthesia and on outpatient basis as well. It is also suitable for frozen-section technique.

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K4 = 1.7 +/- 0.9 microM) approximately 35-fold more tightly than to E (K1 = 50-100 microM), primarily due to a reduction in the Ile dissociation rate constant (k-1 approximately 100-150 s-1, cf. k-4 = 3 +/- 1.5 s-1). Similarly, ATP binds more tightly to E.Ile (K3 = approximately 70 microM) than to E (K2 = approximately 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k+5 = approximately 60 s-1) and the reverse rate constant (k-5 = approximately 150-200 s-1) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K6 = approximately 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (kcat,tr = approximately 0.35 s-1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology.

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.

The efficacy of topical benzocaine gel in providing anesthesia prior to cervical biopsy and endocervical curettage.

OBJECTIVES: Our goal was to determine the effectiveness of topical 20% benzocaine gel in providing anesthesia prior to cervical biopsy and endocervical curettage (ECC) and to identify specific patient characteristics that would predict which patients would be more likely to benefit from the application of benzocaine gel before cervical procedures. MATERIALS AND METHODS: Women requiring cervical biopsy and ECC completed a questionnaire that obtained demographical and pertinent gynecological information, perceived pain thresholds, and anxiety levels prior to the cervical biopsy procedures. Either benzocaine gel or a placebo was applied to the ectocervix and endocervical canal in a doubleblinded fashion. After each procedure, subjects reported the type, intensity, character, and duration of the sensation via visual analog scales (VAS) (0 = no sensation, 100 = maximum sensation). RESULTS: No difference was noted in the mean VAS scores reported by the placebo and benzocaine cohorts for the sensation felt at the time of cervical biopsy (30.1 SD ± 28.2 and 35.3 SD ± 24.3, respectively; p = .33). No significant difference in mean VAS scores was noted after ECC by the placebo cohort (53.0 SD ± 26.8) and the benzocaine cohort (41.0 SD ± 28.2; p = .09). Pain experienced with prior Papanicolaou (Pap) smears correlated significantly with the level of sensation noted during cervical biopsy (r = 0.395; p = .0001). The mean VAS scores for sensation experienced during cervical biopsy also were significantly greater among women who reported a history of dyspareunia (42.2 vs 27.7; p = .0059) and who reported a history of painful pelvic examinations (45.0 vs 29.8; p = .0125) than among women who did not report these painful experiences. CONCLUSIONS: Topical benzocaine gel was ineffective in reducing discomfort reported on cervical biopsy and ECC as compared with a placebo. Biopsy instrument sharpness may be a critically important factor that determines invoked pain. Discomfort associated with prior Pap smears, history of dyspareunia, and history of painful pelvic examinations correlated significantly with a greater perceived biopsy sensation. Prebiopsy recognition of these indicators may help clinicians to determine which women may be more likely to experience greater pain with cervical biopsy and enable them to intervene with other pain prevention measures.

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics.

The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.J complex (K*j = 50 pM). To overcome tight-binding artifacts when K*j << [E], K*j values were estimated from PPi/ATP exchange where [S] >> Km, thus raising K*j,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E.I was identical to that when E.Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.

Inhibitors of bacterial tyrosyl tRNA synthetase: synthesis of four stereoisomeric analogues of the natural product SB-219383.

Synthetic analogues of the microbial metabolite SB-219383 have been synthesised with defined stereochemistry. Densely functionalised hydroxylamine containing amino acids were prepared by the addition of a glycine anion equivalent to sugar-derived cyclic nitrones. One of four stereoisomeric dipeptides incorporating these novel amino acids was found to be a potent and selective inhibitor of bacterial tyrosyl tRNA synthetase, suggesting analogous stereochemistry of the natural product.

Aminoalkyl adenylate and aminoacyl sulfamate intermediate analogues differing greatly in affinity for their cognate Staphylococcus aureus aminoacyl tRNA synthetases.

Aminoalkyl adenylates and aminoacyl sulfamates derived from arginine, histidine and threonine, have been prepared and tested as inhibitors of their cognate Staphylococcus aureus aminoacyl tRNA synthetases. The arginyl derivatives were both potent nanomolar inhibitors of the Class I arginyl tRNA synthetase whereas for the Class II histidyl and threonyl tRNA synthetases, the acyl sulfamates were potent inhibitors but the adenylates had very little affinity.

Inhibitors of bacterial tyrosyl tRNA synthetase: synthesis of carbocyclic analogues of the natural product SB-219383.

Carbocyclic analogues of the microbial metabolite SB-219383 have been synthesised and evaluated as inhibitors of bacterial tyrosyl tRNA synthetase. One compound showed highly potent and selective nanomolar inhibition.

Identification, cloning, and expression of a functional phenylalanyl-tRNA synthetase (pheRS) from Staphylococcus aureus.

Phenylalanyl-tRNA synthetase (pheRS) is unique among aminoacyl tRNA synthetases in that it is a heterotetrameric enzyme composed of two alpha-subunits and two larger beta-subunits. In prokaryotes, the alpha- and beta-subunits of pheRS are encoded by the genes pheS and pheT, respectively. In this report we describe the isolation of a DNA fragment (3.52 kb) containing the pheS and pheT genes from a Staphylococcus aureus (WCUH29) genomic DNA library. Both genes, found as a part of transcriptional operon, were predicted to encode polypeptides which showed strong primary and structural similarity to prokaryotic phenylalanyl-tRNA synthetase alpha- and beta- subunits. We describe the high-level overexpression and purification of recombinant S. aureus pheRS using pheS and pheT genes as part of an artificial operon in Escherichia coli. For comparative analysis we also report a procedure for the purification of native pheRS from S. aureus (Oxford Strain) and demonstrate that Michaelis-Menten parameters for both recombinant and native enzyme, at least for phenylalanine tRNA aminoacylation are comparable.

Synthetic analogues of SB-219383. Novel C-glycosyl peptides as inhibitors of tyrosyl tRNA synthetase.

Novel inhibitors of bacterial tyrosyl tRNA synthetase have been synthesised in which the cyclic hydroxylamine moiety of SB-219383 is replaced by C-pyranosyl derivatives. Potent and selective inhibition of bacterial tyrosyl tRNA synthetase was obtained.

Potent synthetic inhibitors of tyrosyl tRNA synthetase derived from C-pyranosyl analogues of SB-219383.

Novel pyranosyl analogues of SB-219383 have been synthesised to elucidate the structure-activity relationships around the pyran ring. Analogues with highly potent stereoselective and bacterioselective inhibition of bacterial tyrosyl tRNA synthetase have been identified. A major reduction in the overall polarity of the molecule can be tolerated without loss of the nanomolar level of inhibition.