Ribonucleotide reductases: divergent evolution of an ancient enzyme

Ribonucleotide reductases (RNRs) are uniquely responsible for converting nucleotides to deoxynucleotides in all dividing cells. The three known classes of RNRs operate through a free radical mechanism but differ in the way in which the protein radical is generated. Class I enzymes depend on oxygen for radical generation, class II uses adenosylcobalamin, and the anaerobic class III requires S-adenosylmethionine and an iron–sulfur cluster. Despite their metabolic prominence, the evolutionary origin and relationships between these enzymes remain elusive. This gap in RNR knowledge can, to a major extent, be attributed to the fact that different RNR classes exhibit greatly diverged polypeptide chains, rendering homology assessments inconclusive. Evolutionary studies of RNRs conducted until now have focused on comparison of the amino acid sequence of the proteins, without considering how they fold into space. The present study is an attempt to understand the evolutionary history of RNRs taking into account their three-dimensional structure. We first infer the structural alignment by superposing the equivalent stretches of the three-dimensional structures of representatives of each family. We then use the structural alignment to guide the alignment of all publicly available RNR sequences. Our results support the hypothesis that the three RNR classes diverged from a common ancestor currently represented by the anaerobic class III. Also, lateral transfer appears to have played a significant role in the evolution of this protein family.     

Comparison of the efficacy of 1% flutrimazole cream twice a day with 1% flutrimazole cream once a day for the treatment of superficial dermatophytoses

Different clinical studies have demonstrated flutrimazole's efficacy in the treatment of superficial dermatomycoses when administered either twice daily or once daily for four weeks. The aim of the present study was to compare both dosing schedules for the treatment of superficial dermatomycoses. In this randomized, controlled, double blind study, we included 84 patients suffering superficial dermatophytosis (confirmed by microscopic examination (KOH) and culture) susceptible for topical monotherapy. Forty-one patients received flutrimazole 1% twice daily (TD) and forty-three once daily (OD) for four weeks. The efficacy of treatment was evaluated by clinical and mycological criteria at the end of treatment (D28) and after four weeks without treatment (D58). Clinical and mycological cure rates on D28 were 50% with TD and 65% with OD treatment. Only considering clinical evaluation, clinical cure rates on D28 were 63% (TD) and 70% (OD). Also, clinical and mycological cure rates on D56 were 65% with TD and 72% with OD treatment. Only considering clinical evaluation, clinical cure rates on D56 were of 68% (TD) and 72% (OD). The overall tolerability was similar in both treatment groups. The efficacy assessment at the end of treatment (D28) and four weeks after treatment discontinuation (D56) showed that the OD treatment is not inferior to the TD treatment, with similar clinical and mycological cure rates and clinical cure rates in both cases. The OD administration of flutrimazole cream 1% is the most appropriate dosing schedule as it provides the same efficacy, it improves patient's compliance and the cost per day of treatment.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

Assessing the Semelparity Hypothesis: Egg-guarding and Fecundity in the Malaysian Treehopper Pyrgauchenia tristaniopsis

According to the semelparity hypothesis, iteroparous insects should provide either no maternal care or less care than related semelparous species. We present field data on reproductive output and maternal care in the Southeast Asian treehopper Pyrgauchenia tristaniopsis (Mt. Kinabalu, Borneo) relevant to a preliminary assessment of the hypothesis. In a mark‐recapture experiment, more females than expected under semelparity were found to have oviposited a second clutch (37%). Female longevity was a of 75 d. Both these estimates were highly conservative. Oviposition was successive resulting in a of 46 eggs per clutch. Females provided care for eggs only, occasionally scraping their legs along the sides of the clutch apparently attempting to deter Brachygrammatella sp. egg parasitoids (Trichogrammatidae). Females straddled their clutch for a of 27 d, i.e. until 8 d after the beginning of first instar hatching. First instars hatched successively over a period of 11 d. When a female deserted her clutch, it contained about 37% yet unhatched eggs. Egg‐guarding effectively reduced egg mortality: the earlier a female was experimentally removed from her clutch the higher the egg mortality. Displacement experiments demonstrated that egg‐guarding is a behaviour actively maintained despite disturbances and specifically directed towards the egg clutch but not to the feeding site. We interpret our findings as being in accordance with the weaker claim of the semelparity hypothesis, i.e. the iteroparous P. tristaniopsis provided less maternal care than semelparous membracid species. Continued female feeding is discussed as a mechanism to display some level of care despite iteroparity.     

Listeria species escape from the phagosomes of interleukin-4-deactivated human macrophages independent of listeriolysin

Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts. Human macrophages are able to phagocytose and digest L. monocytogenes but IL-4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown. In the present study, we examined various listeria species and strains including wild-type and deletion mutants in human macrophages pretreated with IL-4. To analyse the IL-4-mediated deactivation process, we combined quantitative infection assays with various morphologic methods. IL-4 facilitates survival and escape of the pathogenic L. monocytogenes wild-type strain 10403S from the macrophage phagosomes. In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP-L2161 was killed and did not escape from the phagolysosomes. However, after macrophage deactivation with IL-4 DP-L2161 survived and escaped from the phagosomes. This was also the case, but to a lesser extent, even for the naturally avirulent L. innocua. As detected by confocal laser-scanning fluorescence microscopy and electron microscopy, IL-4 permitted the escape of all listeria species tested, including DP-L2161 and L. innocua from the phagosomal compartment of the macrophages. We conclude that escape from the phagosome and survival of the listeria species tested in IL-4-deactivated human macrophages is independent of the virulence factor listeriolysin.     

Fatty acid variations in symbiotic dinoflagellates from Okinawan corals

The fatty acid composition of polar lipids and triacylglycerols was determined in different morphophysiological types of symbiotic dinoflagellates (SD) isolated from the hydrocoral Millepora intricata and the scleractinian corals Pocillopora damicornis, Seriatopora caliendrum, Seriatopora hystrix and Stylophora pistillata from a fringing reef of Sesoko Island, Okinawa, Japan. The distribution of the fatty acids among the morphophysiologically distinct types of SD reported in these corals makes it possible to readily distinguish one type of SD from the other. Moreover, differences were found both in polar lipids and triacylglycerols. The polar lipids of SD from M. intricata showed a very distinctive fatty acid profile. A combination of large proportions of 18:4 (n-3), 18:5 (n-3), 22:5 (n-6), and 22:6 (n-3) and negligible amounts of 20:4 (n-6), and 20:5 (n-3) in SD from M. intricata was particularly noteworthy. The fatty acid profiles of SD from P. damicornis and SD isolated from S. caliendrum and S. hystrix differed in the proportion of 18:4 (n-3) and 22:6 (n-3). It is suggested that fatty acids might provide useful information on possible taxonomic differences among symbiotic dinoflagellates. It is assumed that biochemical differences can reflect the genetic diversity of the morphophysiological types of SD associated with several species of hermatypic corals from this region.     

Cholic acid-carboplatin compounds (CarboChAPt) as models for specific drug delivery: synthesis of novel carboplatin analogous derivatives and comparison of the cytotoxic properties with corresponding cisplatin compounds

This report continues our work on new compounds which consist of three functional parts--a transport fragment, a spacer and a biologically active 'drug' component. Here cholic acid functions as the transport fragment, linked via an alkyl spacer to a carboplatin analog, representing the drug (carbo-ChAPt-Fig. 1). We describe the synthesis and characterization of the series of complexes [Pt(Cyclobutane-1,1-dicarboxylato)(diamine)], [diamine=CholCOO(CH(2))(n)CH(CH(2)NH(2))(2) and THP(CH(2))(n)CH-(CH(2)NH(2))(2), n=4, 6, 8, 11]. The compounds were characterized by elemental analysis and NMR-measurements. Cytostatic activity data are given. In general, the cytostatic activity is similar to that of the parent compound and is strongly influenced by the length of the alkyl chain spacer separating the drug and transport fragments, the ones with long chain spacers being more toxic than the parent complexes. Preliminary investigations indicate the ability of the ChAPt to break resistance of tumor cells against common platinum tumor drugs, e.g. cisplatin. They are effective even on cell lines that have developed resistance to other drugs such as cis- and carboplatin. They are more cytotoxic so they are potentially effective at lower dose concentrations. The mode of cell death was examined by trypan-blue exclusion test and DNA gelelectrophoresis. Typical fragmentation of DNA was observed and the cells were still able to exclude trypan-blue.     

Minimally invasive silicon probe for electrical impedance measurements in small animals

It is commonly accepted that electrical impedance provides relevant information about the physiological condition of living tissues. Currently, impedance measurements are performed with relatively large electrodes not suitable for studies in small animals due to their poor spatial resolution and to the damage that they cause to the tissue. A minimally invasive needle shaped probe for electrical impedance measurements of living tissues is presented in this paper. This micro-probe consists of four square platinum electrodes (300 microm x 300 microm) on a silicon substrate (9 mm x 0.6 mm x 0.5 mm) and has been fabricated by using standard Si microelectronic techniques. The electrodes are not equally spaced in order to optimise the signal strength and the spatial resolution. Characterisation data obtained indicate that these probes provide high spatial resolution (measurement radius <4 mm) with a useful wide frequency band going from 100 Hz to 100 kHz. A series of in vivo experiments in rat kidneys subjected to ischemia was performed to demonstrate the feasibility of the probes and the measurement system. The impedance modulus and phase were measured at 1 kHz since this frequency is sufficiently low to permit the study of the extracellular medium. The extracellular pH and K+ were also simultaneously measured by using commercial miniaturised Ion Selective Electrodes. The induced ischemia period (45 min) resulted in significant changes of all measured parameters (Delta/Z/ approximately 65%; DeltapH approximately 0.8; DeltaK+ approximately 30 mM).