Management of severe head injury with brain exposure in three loggerhead sea turtles Caretta caretta.

The loggerhead Caretta caretta is the most common sea turtle in the Mediterranean. Currently, sea turtles are considered endangered, mainly due to the impact of human activities. Among traumatic lesions, those involving the skull, if complicated by brain exposure, are often life-threatening. In these cases, death could be the outcome of direct trauma of the cerebral tissue or of secondary meningoencephalitis. This uncontrolled study aims to evaluate the use of a plant-derived dressing (1 Primary Wound Dressing®) in 3 sea turtles with severe lesions of the skull exposing the brain. Following surgical curettage, the treatment protocol involved exclusive use of the plant-derived dressing applied on the wound surface as the primary dressing, daily for the first month and then every other day until the end of treatment. The wound and peri-wound skin were covered with a simple secondary dressing without any active compound (non-woven gauze with petroleum jelly). Data presented herein show an excellent healing process in all 3 cases and no side effects due to contact of the medication with the cerebral tissue.

An experience with paediatric burn wounds treated with a plant-derived wound therapeutic.

OBJECTIVE: To observe the efficacy of a plant-derived wound dressing (1 Primary Wound Dressing®), a mixture of hypericum and neem oil, in different types of paediatric burns. METHOD: A retrospective review was conducted over the complete healing course of 9 paediatric patients with a mean age of 8.17±3.35 (1-11 years), presenting mixed, partial or full-thickness burns. The treatment applied by the wound care specialist consisted of daily cleansing of the wound with a saline solution and application of 1 Primary Wound Dressing on the whole wound surface. There was no application of a secondary dressing. The time to heal, wound size, ease of handling, pain and complications were recorded. Procedural and background pain were observed in six of the patients older than 5 years (mean age 9.6±2.39, range 8-11 years). Due to the small number of patients examined during the period studied, it was not possible to perform statistical analyses. RESULTS: The mean wound size was 50.76±48.32cm2 (4.63-132.0cm2). A rapid induction of granulation tissue and re-epithelialisation was observed. Time to complete healing was 16.6±4.69 days (10-22 days). No complications related to wound infection was observed. The 6 patients older than five years reported a strong relief of pain, from an initial value of 7-8 out of 10 to 0 out of 10 within the first week of treatment. This remained at the 0 out of 10 level during the second and third weeks of treatment. CONCLUSION: This retrospective, non-controlled examination suggests that 1 Primary Wound Dressing could be an effective therapy for the treatment of burn wounds, with benefits including pain reduction and simplicity of use. Further evaluations with a larger population are required to document the effectiveness of this plant-derived wound dressing in a controlled fashion. DECLARATION OF INTEREST: There were no external sources of funding for this study. F. Carnevali is a researcher and co-inventor of 1 Primary Wound Dressing®.

Cytoplasmic DNA from petite colonies of Saccharomyces cerevisiae: a hypothesis on the nature of the mutation.

The density of the cytoplasmic DNA of two strains of "petite" mutants of yeast, obtained by treatment with acriflavin and with ultraviolet light, was examined in cesium chloride density-gradient centrifugation and in all cases appeared to be less than that of the wild type. A cytoplasmic respiratory-deficient strain, treated with additional acriflavin, can show a further shift of the position of the satellite band, always in the direction of reduction of density. Also, from the p(+) x p(-) cross, p(-) strains can be recovered in which the density of the satellite DNA is different from the density of the parent p(-) strain. This finding suggests the existence of recombination in cytoplasmic DNA moleciules.

XrpFI, an amphibian transcription factor composed of multiple polypeptides immunologically related to the GA-binding protein alpha and beta subunits, is differentially expressed during Xenopus laevis development.

XrpFI, first identified in the extract of Xenopus laevis oocyte nuclei, binds to a proximal sequence of the L14 ribosomal protein gene promoter. Its target sequence, 5'-TAACCGGAAGTTTGT-3', is required to fully activate the promoter, and the two G's of the central motif are essential for factor binding and transcriptional activation; our data also suggest that XrpFI may play a role in cap site positioning. The binding site of XrpFI is homologous to the sequence recognized by the family of ets genes. Antibodies specific for Ets-1 and Ets-2 proteins did not react with XrpFI, but those raised against the rat alpha and beta GA-binding proteins both supershifted the retarded bands formed by XrpFI. The Xenopus polypeptides related to GA-binding protein alpha interact with DNA both as monomers and as heterodimers associated with beta-related proteins. Oocyte nuclei contain multiple forms of alpha- and beta-related proteins: the alpha-like proteins remain throughout development, while the pattern of the beta species changes in the embryonic stages examined. beta-like proteins are undetectable in the cleavage period up to the neurula stage, but at later stages, when ribosomal protein genes are actively transcribed, two beta-related polypeptides reappear.

Analysis and stability of the constituents of St. John's wort oils prepared with different methods.

St. John's wort is a medicinal plant with a long history of use in traditional medicine all over Europe. Traditional preparations and in particular the infused oil from SJW flowers remains one of the most popular and curative topical remedy against ulcerations and burns. The presence of the characteristic polyprenylated acylphloroglucinol derivatives, namely hyperforin and analogs are instead related to the oil's therapeutic activity. Indeed, it is well known that hyperforin has a potent antibacterial activity. In this study we tried to rationalize the production system of the oily preparation in order to obtain the highest concentration and stability of phloroglucinols. Five different samples of SJW oils were evaluated by HPLC-DAD-MS analysis to verify the variability and stability of the constituents according to the following factors: different harvesting time, fresh or dried plant material, use of sunlight or heating systems during extraction. The stability of these oils during 1 year was also tested.

Topological modifications and template activation are induced in chimaeric plasmids by inserted sequences.

The effect of the insertion of foreign genes or gene systems in closed DNA domains has been investigated in vitro in purified systems. We observe that in chimaeric plasmids two apparently independent classes of modifications, (1) functional and (2) topological, do take place in defined instances. (1) Among the screened yeast gene systems, examples have been found of DNA sequences that upon insertion cause activation of in vitro transcription of distant genes. (2) Foreign DNA sequences may lead to new topological features of the harbouring plasmids; it is shown that more than one S1-sensitive secondary structure may be contemporaneously present on the same chimaeric plasmid. DNA superhelicity is a prerequisite of these modifications. The two classes of effects (1) functional and (2) topological are not a priori directly related one to the other but appear to be two independent consequences of the same cause: the insertion of foreign DNA sequences into closed DNA domains. These observations suggest a regulatory model of gene expression based on alternative topologies of closed DNA domains.

Negative regulation of axis formation and Wnt signaling in Xenopus embryos by the F-box/WD40 protein beta TrCP.

Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Electrocardiographic alterations in chronically Trypanosoma cruzi-infected persons exposed to cardiovascular factors.

METHODS: We analyzed the potential influence that associated risk factors (ARF), such as smoking, alcoholism, overweight, and hypertension, could have on the establishment of chronic chagasic cardiomyopathy (CC). The sample was comprised of 124 individuals, 69 males and 55 females (mean age +/- SD, 41 +/- 9.5 years), who were born in en demic areas of Northern Argentina and migrated further to Rosario City, an area where autochthonous cases of Chagas' disease have never been registered. Assessments included the following: clinical examination to discard previous cardiomyopathies; search for the presence of ARF according to standard criteria; specific serology; frontal chest X-ray, and 12-lead resting electrocardiogram (ECG). Subjects were classified on the basis of their serological status and presence of ARF into four groups: Tc+ARF+ T. cruzi-infected persons with ARF (n = 41); Tc-ARF+ seronegativity in presence of ARF (n = 27); Tc+ARF- individuals showing positive serology that lacked ARF (n = 27), and Tc-ARF- seronegative individuals having no ARF (n = 29). RESULTS: Except for a higher female/male ratio in groups presenting no ARF (p < 0.02), no statistical differences as to age, length of residence in endemicity areas (LR), and ARF distribution were recorded among groups. Forty-one persons presented abnormal ECG tracings, distributed thus: Tc+ARF+, 18/41; Tc-ARF+, 14/27, Tc+ARF-, 14/27, and Tc-ARF, 4/29 (p < 0.01, in relation to the latter group). Subjects from the Tc+ARF+, Tc-ARF+, and Tc+ARF- groups had 4.89-, 6.7-, and 6.7-fold increases, respectively, if having an abnormal ECG when compared with Tc-ARF- individuals. Comparisons on the frequency of abnormal ECG between seropositives carrying ARF or not yielded a non-significant odds ratio, be it estimated as crude, or after adjusting for sex, age, and LR in multivariate analysis. CONCLUSIONS: Presence of ARF was not associated with an increasing risk of cardiac affectation in chronically T. cruzi-infected persons, but resulted in chagasic-compatible ECG abnormalities in those seronegative individuals.

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle.

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.

Variations in the length of S-phase related to the time cells are blocked at the G1-S interface.

The inhibition of DNA synthesis with hydroxyurea or 5-fluorode-oxyuridine decreases the duration of S-phase of synchronously growing Chinese hamster cultures. - The observed drug effects are discussed in relation to an alteration of programmed DNA replication.

Activation of in vitro transcription and topology of closed DNA domains.

Activation of in vitro transcription of otherwise inert DNA sequences by purified yeast RNA polymerase II has been observed following the introduction in closed DNA domains of fragments of various origin. This enhancer-like effect on the in vitro transcriptional capacity is only detected in negatively supercoiled DNA domains and is characterized for each chimaeric plasmid by the superhelical density (- sigma) at which a sharp transition toward activation takes place. We have analyzed the topological state (as defined by localization and evaluation of the relative occurrence of secondary structures sensitive to S1 endonuclease) of the activated closed domains as a function of the conditions that determine the transcriptional enhancer effect, i.e. superhelical density, size, and nature of the components of the domains. We observe that variations in transcriptional capacity coincide with a defined pattern of secondary structures. These observations support a cause-effect relation between topology and regulation of transcription.

Transitions in topological organization of supercoiled DNA domains as a potential regulatory mechanism.

We present an analysis of the influence of DNA superhelicity on the topology of chimeric plasmids. A correlation is found between topological variations and in vivo/in vitro functions. Data refer to the topological transitions observed in function of variations of the superhelical density in the three closed DNA domains that represent the three extreme examples, from the point of view of the topological organization, among many chimeric systems analyzed. The plasmids studied are ADR2-BS-pBR322 (the vector pBR322 + the yeast gene ADR2), ADR3-5c-yRp7 (containing yeast TRP1, a constitutive mutant of yeast ADR2, a fragment of 5 kilobases of unanalyzed chromosomal DNA and the vector pBR322), and p31 (pBR322 + a yeast DNA fragment encompassing the right moiety of the Ty1 element and its in vivo promoter). In the model systems analyzed, the topological transitions observed in the eukaryotic sequences (relevant in one case for activation of selective in vitro transcription (Ty), in another (ADR2) for the in vivo expression) take place in the range of superhelical densities predicted on theoretical ground (Vologodskii, A. V., Lukashin, A. V. Anshelevich, V. V. & Frank-Kamenetskii, M. D. (1979) Nucleic Acids Res. 6, 967-982).

HrpF, a human sequence-specific DNA-binding protein homologous to XrpFI, a Xenopus laevis oocyte transcription factor.

The identification in HeLa nuclei of a novel DNA-binding protein, designated HrpF, is presented. This factor recognizes and binds a sequence of the Xenopus laevis L14 ribosomal protein (r-p) gene promoter bound by the Xenopus r-p transcription factor I (XrpFI). We show here that XrpFI and HrpF share a conserved DNA-binding domain. We also present evidences suggesting that the two factors perform similar functions in the cell. We discuss the hypothesis that closely related factors might be involved in the control of rp-gene transcription in vertebrates.

Cloning, expression and functional role of XrpFI alpha and beta subunits in Xenopus laevis oocyte.

The cDNAs coding for the alpha and beta components of XrpFI, an activator of ribosomal protein genes transcription, were isolated. RNA analysis showed that transcripts of the beta genes are present at constant level throughout Xenopus development unlike the beta proteins which are undetectable in the early stages of embryogenesis when transcription is silent. On the contrary, alpha transcripts and proteins were found in all the embryo stages examined. The role of the XrpFI alpha and beta subunits in rp-gene transcription has been studied by mRNA microinjection into X.laevis oocytes. The presence of the beta subunit appears to be critical for the proper rp-gene promoter function.

Nuclear factors specifically bind to upstream sequences of a Xenopus laevis ribosomal protein gene promoter.

The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position.

In vitro transcription by purified yeast RNA polymerase II. Coarse promoter mapping on homologous cloned genes.

Clones of the yeast Tyl element and 2 microns plasmid have been selectively transcribed in vitro by partially or completely purified yeast RNA polymerase II. Electrophoretic analysis of whole and restricted ternary transcription complexes allows the localization of the in vitro actively transcribed regions of the analyzed genes. The DNA regions that actively promote in vitro transcription correspond to the nucleotide sequence that in the Tyl element encompasses the in vivo transcription initiation sites and that in the 2 micrometer plasmid encompasses the starting codons of two oppositely oriented potential protein coding frames. The transcription assay that we describe herein may be applied to analyze rapidly the in vitro transcription of clones genes, to localize transcription initiation sites on supercoiled templates and to evaluate the differential in vitro promoter strength in RNA polymerase II served genes. Data obtained with RNA polymerase II at two different stages of purification are presented in parallel. Studies with a completely purified enzyme should certainly be preferred although the use of a partially purified RNA polymerase II may be convenient and may reveal factors which affect specificity.

Differential effects of nalidixate on the cell growth of respiratory competent strains and cytoplasmic petite mutants of Saccharomyces cerevisiae.

Sodium nalidixate inhibited the cell growth and division of several respiratory competent strains of Saccharomyces cerevisiae. A number of cytoplasmic petite strains (both spontaneous and induced by ethidium bromide) were shown to be more resistant to sodium nalidixate than the wild-type strains from which they were derived. There was considerable variation in sensitivity of different petites derived from the same wild-type. Usually petite strains which were induced by ethidium bromide were more resistant than spontaneously arising petites. The susceptibility of a wild-type strain to nalidixate was found to be least when the mitochondrial respiratory system was maximally repressed. It was also noted that sodium nalidixate (100 mug/ml) induced petite mutants.

Large scale isolation of nuclei from oocytes of Xenopus laevis.

We describe an improvement on the procedure of Scalenghe et al. (F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa (1978) Chromosoma 66, 299-308) for the large scale isolation of nuclei from Xenopus laevis oocytes. The nuclear extract obtained was tested for its ability to transcribe a cloned Xenopus 5 S RNA gene and for the presence of nuclear factors interacting with a X. laevis ribosomal protein gene promoter. Efficiency of accurate transcription and of factor binding is comparable with that of an extract prepared from manually isolated nuclei.