S. gallolyticus Aortic Valve Endocarditis with Mitral Valve Leaflet Aneurysm.

S. gallolyticus is one of the pathogenic agents of endocarditis, and mitral valve aneurysm is a rare but potentially devastating complication. We present a case of S. gallolyticus aortic valve endocarditis with concomitant anterior mitral valve leaflet aneurysm. Patient underwent surgery before aneurysm perforation, and postoperative course was uneventful. Time of surgery is crucial to avoid severe complications due to aneurysm rupture.

Multiple hepatic and brain abscesses caused by Parvimonas micra: A case report and literature review.

Gram-positive anaerobic cocci (GPAC) are responsible for 30% of anaerobic infections. Parvimonas micra is an emergent pathogen that is part of the oral and gastrointestinal commensal flora, and its role in several infection processes has recently emerged thanks to the improvement of diagnostic techniques. P. micra bacteraemia is reported in immunocompromised patients and is often complicated by abscesses. Here, we present a case study of multiple hepatic and brain abscesses caused by P. micra bacteraemia in a patient with complicated diverticulitis.

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY.

BACKGROUND: The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila CRYPTOCHROME (CRY), a putative circadian photoreceptor, has previously been reported to interact with the clock protein TIMELESS (TIM) in a light-dependent manner. Although TIM dimerizes with PERIOD (PER), no association between CRY and PER has previously been revealed, and aspects of the light dependence of the TIM/CRY interaction are still unclear. RESULTS: Behavioral analysis of double mutants of per and cry suggested a genetic interaction between the two loci. To investigate whether this was reflected in a physical interaction, we employed a yeast-two-hybrid system that revealed a dimerization between PER and CRY. This was further supported by a coimmunoprecipitation assay in tissue culture cells. We also show that the light-dependent nuclear interactions of PER and TIM with CRY require the C terminus of CRY and may involve a trans-acting repressor. CONCLUSIONS: This study shows that, as in mammals, Drosophila CRY interacts with PER, and, as in plants, the C terminus of CRY is involved in mediating light responses. A model for the light dependence of CRY is discussed.

Photic entrainment of the circadian clock: from Drosophila to mammals.

Entrainment is as fundamental to an organism's circadian timing as are the molecular mechanisms involved in the functioning of the intracellular clock oscillator. In nature, one of the principle, although not the only, circadian entraining stimulus (Zeitgeber) is provided by the daily light--dark cycles. In animals, the visual processing apparatus alone is inadequate to accomplish the task of transducing circadian photic signals to the clockwork machinery. In fact, it is ever more appreciated by circadian biologists that organisms as divergent as plants and mammals have evolved a wonderfully complex array of partly redundant specializations which can guarantee the precise alignment of biological and environmental time. Research in circadian biology is cruising at such a rate that attempts to review the state of the art can only hope, at best, to provide a snapshot of the speeding cruiser from its wake. This paper will hopefully provide a reasonably sharp portrayal of what is at hand.

Efficient and heritable functional knock-out of an adult phenotype in Drosophila using a GAL4-driven hairpin RNA incorporating a heterologous spacer.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS-y-IR hairpin is expressed under the control of different promoter-GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Extra ocular photic entrainment in Drosophila melanogaster.

The ability to anticipate the daily changes occurring in the photic environment, by adjusting their physiology and behavior accordingly, should provide living organisms with a selective advantage. Organisms could thus sample the relevant entraining stimuli at early dawn and/or late dusk. At these times of the day the principal spectral components of sunlight reaching the Earth's surface consist of relatively low levels of irradiance in the blue and red parts of the visible spectrum. Biological circadian systems could entrain to twilight conditions by sampling either one or both of these components. Previous studies, based on the characterization of phase response curves, following brief exposures to relatively high intensity light of varying wavelength, demonstrated the particular sensitivity of the Drosophila circadian system to blue light. In this study, we addressed the capacity of the circadian system of Drosophila to respond to long periods of exposure to red light. Thus flies were initially exposed to 12 h : 12 h LD cycles with full spectrum white light. Following a 1-4 h phase shift, the flies were exposed to LD cycles with red light. Our results suggest that, under these experimental conditions, red light of wavelength between 650-700 nm, can function as an entraining stimulus. Furthermore, analysis of the circadian locomotor activity profiles in visually impaired flies, suggests that in wild type flies locomotor activity is triggered by the circadian clock at key times during the day. Once triggered, the whole cycle (i.e. onset, peak and offset) of locomotor activity occurring both at dawn and dusk can proceed autonomously. However, the occurrence of a lights-off signal (typically at dusk) before the autonomous cessation of locomotor activity, leads to a light-driven termination of such activity. In addition, so1 (eyeless) mutant flies show the presence of a single evening locomotor activity peak during the whole circadian day, suggesting that in wild type flies the morning and evening activity peaks may be under separate control.

Giant neuron pathway neurophysiological activity in per(0) mutants of Drosophila melanogaster.

In Drosophila melanogaster, the clock gene period (per) has a clearly defined role in the molecular machinery involved in generating free-running circadian rhythms. per mutations also influence rhythms in the Drosophila love song and in the ultradian timescale. The relationship between these two phenomena has so far escaped satisfactory explanation. Here we analyzed the neurophysiological activity of the giant fiber neural pathway in per(0) flies. Under constant light, and at relatively low stimulation frequencies (1-2 Hz), per(01) flies habituate significantly earlier than they do under 12 h light-dark cycles. The results suggest an involvement of per in phenomena of short-term neural plasticity.

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals.

The circadian clock in mammals.

The basic physiological and anatomical basis for circadian rhythms in mammalian behaviour and physiology is introduced. The pathways involved in photic entrainment of the circadian clock are discussed in relation of new findings that identify the molecules that are involved in signalling between the environment and the clock. The molecular basis of endogenous cycles is described in the mouse, and compared to the mechanism that is present in the fly. Finally we speculate on the relationship between circadian physiology and pain.

Different period gene repeats take 'turns' at fine-tuning the circadian clock.

The repetitive region of the circadian clock gene period in Drosophila pseudoobscura consists predominantly of a pentapeptide sequence whose consensus is NSGAD. In D. melanogaster, this region is replaced by a dipeptide Thr-Gly repeat, which plays a role in the thermal stability of the circadian phenotype. The Thr-Gly repeat has been shown to form a type II or III beta-turn, whose conformational monomer is (Thr-Gly)3. Here we report, using conformational analyses, that both an NSGAD pentapeptide, and a polymer of the same sequence, form type II beta-turns. Thus two peptide sequences, whose amino-acid composition is very different, nevertheless form the same secondary structure. The implications of these structures for clock function are discussed.

Conceptual translation of timeless reveals alternative initiating methionines in Drosophila.

We have sequenced genomic fragments which encode the N-terminus of the TIMELESS (TIM) clock protein in Drosophila simulans and D. yakuba. We observe that in these two species, the initiating methionine appears to lie downstream of the one proposed to encode the translational start inD.melanogaster, thereby truncating the N-terminus by 23 amino acids. We then sequenced the corresponding 5'fragment in a number of D. melanogaster individuals from different strains. We observed a polymorphism which strongly suggests that the originally proposed start site cannot be utilised in some individuals, and that these flies will initiate translation of TIM at the downstream ATG. Given the current interest in TIM regulation in D. melanogaster, it is important to correctly define the N-terminus in this species.

Cytogenetic and immunofluorescence analysis of benzo[a]pyrene-DNA adduct formation and chromosome damage in larval brain neuroblasts of Drosophila melanogaster.

Recently we have evaluated the relationship between benzo[a]-pyrene(BaP)-DNA adducts, determined by 32P-postlabelling, and clone frequencies in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Following that study we proceeded to characterise further the mechanism of induction of genetic damage in vivo by BaP in Drosophila by cytogenetic analysis of larval brain neuroblasts. Third stage larvae were treated with 4 and 10 mM BaP for 24, 48 or 72 h. In all cases, the larvae were killed 72 h after the beginning of treatment, entailing 48, 24 or 0 h post-treatment recovery in BaP-free medium, respectively. At the end of the treatment the following data were collected: (i) the types and levels of chromosome aberrations in neuroblast metaphase and anaphase nuclei; (ii) the distribution and level of BaP-DNA adducts, revealed by indirect immunofluorescence in neuroblast nuclei using an anti-(BaP-DNA) antibody. The results indicate that BaP induces chromosome breaks, deletions and exchanges in this system. In particular, chromosome exchanges decrease as the post-treatment recovery time increases, and the dynamics of breaks and deletions appear to be inversely related to those of the exchanges. This suggests that exchanges may require few preconditions to occur and are thus expressed soon after treatment. Chromosome breaks and deletions could require multiple single events before the actual damage is expressed (even some cell divisions away from the end of treatment). The immunofluorescence analysis suggests that BaP-DNA adducts are more abundant in the heterochromatin of the neuroblast nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloral hydrate is recombinogenic in the wing spot test in Drosophila melanogaster.

In order to characterise the response of the wing spot test in Drosophila melanogaster to the effects of compounds with known aneugenic properties, experiments were performed with chloral hydrate (CH). Following chronic exposure of 72-h-old larvae to rising concentrations of CH, significant increases in the frequency of small (1-2 cells) single spots were observed. Comparison of results obtained in parallel from the wings of marker-trans-heterozygous individuals and individuals heterozygous for one of two different balancer chromosomes suggests that practically all the single clones originated from recombinational events. Twin clone frequencies were, however, only weakly affected. These results are discussed with reference to the literature regarding the effects of CH in different experimental systems and to the characteristics of Drosophila as a tester organism.

Relationship between benzo(a)pyrene-DNA adducts and somatic mutation and recombination in Drosophila melanogaster.

The evaluation of the relationship between the dose to DNA of a mutagen/carcinogen and in vivo somatic cell mutagenesis may provide information on the mechanisms leading to induced mutational events. This can be achieved, for example, by coupling test systems that permit the detection of somatic mutation and recombination on the basis of phenotypic changes in cuticular structures of Drosophila melanogaster, with methods for the quantitation of carcinogen-DNA adducts such as the 32P-postlabeling technique. In this article, we evaluate the quantitative relationship between BaP-DNA adduct formation, determined by 32P-postlabeling, and the induction of mutant cells in the wing marker version of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. The total single clones in the trans-heterozygous mwh/flr3 flies show a linear relationship with the BaP-DNA adduct levels, suggesting a single hit mechanism for the genetic damage giving rise to this type of clones. In contrast, the twin clones (which are of recombinational origin) display a linear-quadratic relationship with the adduct levels, suggesting that multiple hits may be involved in generating these clones. The total single clones in the mwh/TM3, Ser flies (in which mitotic recombination is suppressed) show a logarithmic relationship with the adduct levels. The discussion of these data in terms of the pathways that may be involved in the repair of the BaP-DNA adducts leads to the suggestion that in Drosophila melanogaster the repair of Bap metabolite-DNA adducts in somatic cells may proceed, in large part, via post-replicative recombinational repair.

[The urinary mutagenicity test in monitoring exposure to aromatic polycyclic hydrocarbons in workers in the aluminum industry]. / Test di mutangenicità urinaria nel monitoraggio dell'esposizione ad idrocarburi policiclici aromatici di lavoratori dell'industria di alluminio.

The sensitivity of 3 urinary mutagenicity tests was assayed: the plate test, the fluctuation test and the micropreincubation test, in order to assess their possible use in monitoring human exposure to polycyclic aromatic hydrocarbons (PAH). Urine samples from workers of an anode production plant exposed to coal tar and from psoriatic patients undergoing treatment with coal-tar ointments were tested for mutagenic activity on strain TA98 Salmonella typhimurium, in the presence of the microsome fraction and deconjugating enzymes. Parallelly, the urinary concentration of PAH metabolites or one of their trace metabolites, 1-hydroxypyrene, was determined. Increased levels of PAH metabolites were observed in the urine of anode production workers after a work shift compared with controls. Results of the plate test and the fluctuation test performed on urine of exposed subjects, both smokers and nonsmokers, showed mutagenicity values similar to the controls. Much higher 1-hydroxypyrene concentrations were found in the urine of psoriatic patients treated with coal tar than in post-shift urine of anode production workers. The urine of the former was also mutagenic in the 3 mutagenicity tests used. The minimum mean dose of PAH metabolites was calculated, expressed as quantity of 1-hydroxypyrene, that would give a mutagenic response in the 3 tests: the micropreincubation test was found to be about 100 times more sensitive than the plate test and about 30 times more sensitive than the fluctuation test. The theoretical minimum urinary concentration of 1-hydroxypyrene detectable by each test was determined: the micropreincubation test was 15 times more sensitive than the plate test and 7 times more sensitive than the fluctuation test.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of benzo[a]pyrene-diol-epoxide-DNA adducts in white blood cells of psoriatic patients treated with coal tar.

An enzyme-linked immunosorbent assay (ELISA) was used to detect BPDE-DNA adducts in white blood cells of 23 psoriatic patients undergoing clinical coal tar therapy. Ten of these patients were reanalyzed 2-5 months after the end of the coal tar treatments. The results show that the mean adduct level during the treatment period was 0.26 +/- 0.16 fmole BPDE/micrograms DNA (7.7 +/- 4.9 adducts/10(8) nucleotides), while 2-5 months later the mean adduct level had decreased significantly (P less than 0.005) to 0.11 +/- 0.08 fmole BPDE/micrograms DNA (3.3 +/- 2.4 adducts/10(8) nucleotides). No relationship could be ascertained between the level of exposure and the amount of BPDE-DNA adducts. In addition, no difference in the level of DNA adducts was found between smoking and non-smoking patients.

Direct involvement of human gastric mucosa in the activation of alimentary pro-carcinogens.

Exposure to N-nitroso-compounds and aromatic amines, xenobiotics which require an activation in order to exert their genotoxic potential, is causally associated with gastric cancer. We evaluated the capacity of microsome-containing fractions from human gastric mucosa to activate two model carcinogenic compounds. A 9,000 x g supernatant (S9) was obtained from gastric mucosal specimens and, for comparison, from human liver and aroclor-induced and non induced rat liver. The capacity of the S9 to activate N-nitrosopyrrolidine (NPY) and 2-aminofluorene (2AF) to mutagenic metabolites was tested in the Ames/Salmonella reversion assay, while dimethylnitrosamine (DMN) and aminopyrine (AP) demethylation activities were spectrophotometrically evaluated by using an enzymatic assay of the amount of formaldehyde released following the enzymatic demethylation of the corresponding substrates. Results indicate that human gastric S9 fractions may activate 2AF to a genotoxic derivative and are characterized by DMN and AP demethylase activities higher (p < 0.05) than those of human liver, when expressed in mg/protein (p < 0.05). Although the parameters evaluated can only be considered as a partial measure of the general activating capacity toward dietary and environmental procarcinogens, these results suggest that human gastric mucosa may be directly involved in the metabolic activation of these compounds to mutagenic/carcinogenic species.

The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster.

The genotoxicity of a chelating agent, the trisodium salt of nitrilotriacetic acid (NTA), was assessed in a somatic mutation and recombination test (SMART) in Drosophila melanogaster employing the wing hair markers mwh and flr3. The experiments were performed in parallel in two different laboratories (Padua, Italy and Schwerzenbach, Switzerland). The effectively absorbed doses of NTA, which was administered by feeding to larvae, were determined by a sensitive method employing [3H]leucine which allowed individual consumption levels to be measured. The particular pattern of clone induction produced by this compound suggests that NTA is active in inducing mitotic recombination and possibly aneuploidy in somatic cells of Drosophila. This is discussed in relation to the data present in the literature regarding the genotoxicity of NTA in a variety of experimental systems.