Antibacterial and anticancer activity of two NK-lysin-derived peptides from the Antarctic teleost Trematomus bernacchii.

The NK-lysin antimicrobial peptide, first identified in mammals, possesses both antibacterial and cytotoxic activity against cancer cell lines. Homologue peptides isolated from different fish species have been examined for their functional characteristics in the last few years. In this study, a NK-lysin transcript was identified in silico from the head kidney transcriptome of the Antarctic teleost Trematomus bernacchii. The corresponding amino acid sequence, slightly longer than NK-lysins of other fish species, contains six cysteine residues that in mammalian counterparts form three disulphide bridges. Real time-PCR analysis indicated its predominant expression in T. bernacchii immune-related organs and tissues, with greatest mRNA abundance detected in gills and spleen. Instead of focusing on the full T. bernacchii derived NK-lysin mature molecule, we selected a 27 amino acid residue peptide (named NKL-WT), corresponding to the potent antibiotic NK-2 sequence found in human NK-lysin. Moreover, we designed a mutant peptide (named NKL-MUT) in which two alanine residues substitute the two cysteines found in the NKL-WT. The two peptides were obtained by solid phase organic synthesis to investigate their functional features. NKL-WT and NKL-MUT displayed antibacterial activity against the human pathogenic bacterium Enterococcus faecalis and the ESKAPE pathogen Acinetobacter baumannii, respectively. Moreover, at the determined Minimum Inhibitory Concentration and Minimum Bactericidal Concentration values against these pathogens, both peptides showed high selectivity as they did not exhibit any haemolytic activity on erythrocytes or cytotoxic activity against mammalian primary cell lines. Finally, the NKL-MUT selectively triggers the killing of the melanoma cell line B16F10 by means of a pro-apoptotic pathway at a concentration range in which no effects were found in normal mammalian cell lines. In conclusion, the two peptides could be considered as promising candidates in the fight against antibiotic resistance and tumour proliferative action, and also be used as innovative adjuvants, either to decrease chemotherapy side effects or to enhance anticancer drug activity.

Volatile organic compounds as new biomarkers for colorectal cancer: a review.

Analysis of the volatile part of the metabolome (volatile organic compounds, VOC) present in the gas phase of excreted materials is a promising new screening tool for several cancers, including colorectal cancer (CRC). The VOC signature can reflect health status, like a 'fingerprint', and can be modified in several diseases. Technical difficulties still limit the widespread use of VOC analysis in the clinical setting, but this approach has already been applied successfully in the diagnosis of CRC. The present study reviews the available data on VOC present in the headspace (the gaseous constituents of a closed space above a liquid or solid) of blood, urine, faeces and breath as a potential screening tool for CRC. A systematic electronic literature search was conducted in PubMed, Scirus and Google using the following keywords: Metabolomic, Volatile Organic Compounds (VOC), Electronic-nose and Colorectal Cancer. Only articles published in English between 2000 and 2015 were selected and these were independently checked by two of the authors. Ten papers describing the reliability of VOC analysis in breath and faeces, blood and urine were selected; all indicated good reliability in detecting CRC. The use of different substrates and different analytical platforms has led to the identification of different patterns of VOC. The reliability of a metabolomic approach as a noninvasive biomarker for use in CRC screening is supported by this review despite several limitations due to the number of patients included in each study, the different analytical platforms and biological materials used and different VOC identified.

Transmission of Xylella fastidiosa to Grapevine by the Meadow Spittlebug.

There is little information available on Xylella fastidiosa transmission by spittlebugs (Hemiptera, Cercopoidea). This group of insect vectors may be of epidemiological relevance in certain diseases, so it is important to better understand the basic parameters of X. fastidiosa transmission by spittlebugs. We used grapevines as a host plant and the aphrophorid Philaenus spumarius as a vector to estimate the effect of plant access time on X. fastidiosa transmission to plants; in addition, bacterial population estimates in the heads of vectors were determined and correlated with plant infection status. Results show that transmission efficiency of X. fastidiosa by P. spumarius increased with plant access time, similarly to insect vectors in another family (Hemiptera, Cicadellidae). Furthermore, a positive correlation between pathogen populations in P. spumarius and transmission to plants was observed. Bacterial populations in insects were one to two orders of magnitude lower than those observed in leafhopper vectors, and population size peaked within 3 days of plant access period. These results suggest that P. spumarius has either a limited number of sites in the foregut that may be colonized, or that fluid dynamics in the mouthparts of these insects is different from that in leafhoppers. Altogether our results indicate that X. fastidiosa transmission by spittlebugs is similar to that by leafhoppers. In addition, the relationship between cell numbers in vectors and plant infection may have under-appreciated consequences to pathogen spread.

Exhaled volatile organic compounds identify patients with colorectal cancer.

BACKGROUND: An effective screening tool for colorectal cancer is still lacking. Analysis of the volatile organic compounds (VOCs) linked to cancer is a new frontier in cancer screening, as tumour growth involves several metabolic changes leading to the production of specific compounds that can be detected in exhaled breath. This study investigated whether patients with colorectal cancer have a specific VOC pattern compared with the healthy population. METHODS: Exhaled breath was collected in an inert bag (Tedlar(®) ) from patients with colorectal cancer and healthy controls (negative at colonoscopy), and processed offline by thermal-desorber gas chromatography-mass spectrometry to evaluate the VOC profile. During the trial phase VOCs of interest were identified and selected, and VOC patterns able to discriminate patients from controls were set up; in the validation phase their discriminant performance was tested on blinded samples. A probabilistic neural network (PNN) validated by the leave-one-out method was used to identify the pattern of VOCs that better discriminated between the two groups. RESULTS: Some 37 patients and 41 controls were included in the trial phase. Application of a PNN to a pattern of 15 compounds showed a discriminant performance with a sensitivity of 86 per cent, a specificity of 83 per cent and an accuracy of 85 per cent (area under the receiver operating characteristic (ROC) curve 0·852). The accuracy of PNN analysis was confirmed in the validation phase on a further 25 subjects; the model correctly assigned 19 patients, giving an overall accuracy of 76 per cent. CONCLUSION: The pattern of VOCs in patients with colorectal cancer was different from that in healthy controls. The PNN in this study was able to discriminate patients with colorectal cancer with an accuracy of over 75 per cent. Breath VOC analysis appears to have potential clinical application in colorectal cancer screening, although further studies are required to confirm its reliability in heterogeneous clinical settings.

[Hexadactylism of the four limbs: a case report]. / Esadattilia dei quattro arti: un insolito caso.

An unusual case of postaxial hexadactylism of the hands and feet in one female Caucasian neonate is described. The clinical picture was characterized by symmetrical duplication of the 5th finger in both hands and of the 4th finger in both feet. Malformations of the extremities both in the paternal and in the maternal family were reported. No other associated malformations have been found in the baby and her karyotype was normal. The performed analysis of the literature confirmed the peculiarity of the associated features of this case.

Induction of karyological damage by bleomycin in polyploidizating rabbit hepatocytes.

The effects of the antitumor drug bleomycin on polyploidizating hepatocytes of immature rabbits were investigated by combining cytochemical (static microfluorometry) and morphological (fluorescence and electron microscopy) approaches. In addition to a decelerating effect of the drug on the cell kinetics, there were several types of karyological damage (micronuclei, internuclear chromatin bridges, mitotic chromosome bridges), thus demonstrating a clastogenic activity of bleomycin on the DNA, Moreover, anomalous nuclear and cytoplasmic divisions suggest interference of the drug with other cellular structures (mainly the microtubule network) responsible for the cell kinetics.

Histochemical studies on the autonomic innervation of the femoral artery and vein.

The innervation of the femoral artery and vein in the dog was studied using: a) glyoxylic acid fluorescence (for the histochemical localization of adrenergic nerve fibers); b) acetylcholinesterase (for the histochemical localization of cholinergic nerve fibers). -Andrenergic nerve fibers and related terminals in both vessels are confined to the adventitial-medial transitional zone and the outer layers of the media. Acetylcholinesterase containing nerve fibers are localized in the adventitial-medial transitional zone of the femoral artery. These findings may indicate the presence of a cholinergic vasodilatator system in the control of the circulation in the femoral artery.

Determination of L-glutamate and L-glutamine in pharmaceutical formulations by amperometric L-glutamate oxidase based enzyme sensors.

An amperometric biosensor for the direct determination of L-glutamate was developed by chemical bonding of L-glutamate oxidase (GAO) on a carboxylic Nylon membrane with polyazetidine prepolymer (PAP), and using a hydrogen peroxide electrode as indicating sensor. The biosensor is specific for L-glutamate and the peculiar analytical properties (linearity range, reproducibility, accuracy) were experimentally determined. Furthermore, the same basic biosensor was also modified to be used and characterized for the direct determination of L-glutamine. This L-glutamine biosensor was obtained by coimmobilizing, on two separate membranes, glutamic acid oxidase and glutaminase (GMN) on the same biosensor. The two sensors were then used for the determination of glutamate and L-glutamine contained in pharmaceutical formulations and the results were compared with those obtained by other analytical methods.

The action of cisplatinum (cis-DDP) on the postnatal growth of the rat liver. Cytokinetics and ultrastructural studies.

The early and late effects of cis-DDP treatment on liver cell kinetics were analyzed after its intraperitoneal injection into 17-day old rats. Frequency of binucleate hepatocytes, cellular DNA content distribution, 3H-thymidine labelling and ultrastructure of the nuclei were analyzed. Two days after treatment, a block of mononucleate hepatocytes in the S phase and in the G2 phase was demonstrated by the increase of intermediate 2c-4c and 4c DNA values in the absence of changes in 3H-thymidine labelling; 8c binucleate cells, which are essential for the formation of tetraploid mononucleate cells, were not found. In cell nuclei, large areas of more condensed chromatin appeared, perhaps providing further evidence for a G2 block. Seven days after treatment, there was a tendency to catch up with the normal situation, as shown by the unblocking of the S phase in mononucleate cells indicated by both cytophotometry and autoradiography. The presence of 8c binucleate cells and 4c-8c mononucleate cells indicates that 4c mononucleate hepatocytes are either diploid cells in the G2 phase or true tetraploid G1 cells. The decrease in the heterochromatin areas and the appearance of hypertrophic nucleoli demonstrate an increase in the metabolic activity of the nuclei. Thirty six days after treatment, the incidence of different DNA hepatocyte classes and the 3H-thymidine labelling were already similar in control and in treated rats.

Cholinergic nerves in blood vessels of the female reproductive system.

Cholinergic innervation of the blood vessels of the female reproductive system was investigated in human by the cholinesterase method. The following results were obtained: 1. The uterine and vaginal arteries are provided with a rich cholinergic innervation. 2. The ovarian, tubaric and ovarian-uterine artery are poorly innervated. 3. The ovarian and uterine vein are not provided with a cholinergic innervation.

Cytophotometric assay of cytochrome oxidase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities in human peroxidized spermatozoa.

Human spermatozoa contain appreciable amounts of intracellular glutathione, which has a protective function against peroxidative degradation of spermatozoal polyunsaturated fatty acids by the NADPH-dependent glutathione peroxidase/reductase enzymatic system. The glutathione system provides a basic defense against peroxidative damage, without which the superoxide dismutase system would dominate. Since oxidative damage is said to include enzyme leakage and changes in metabolism, cytochrome oxidase and lactate dehydrogenase activities were used as indicators of the energy metabolism in unwashed and washed human spermatozoa during lipid peroxidation. Lipid peroxidation was induced by aerobic incubation of sperms in the presence of sodium ascorbate and ferrous sulphate. In addition, since NADPH concentrations influence the concentration of reduced glutathione, we studied glucose-6-phosphate dehydrogenase activity as an indicator of pentose phosphate shunt activity, the main source of NADPH. Microdensitometric measurements of the three enzymes were made by a Vickers M85a scanning microdensitometer. We found that the lipid peroxidation process greatly affects the 3 enzymatic activities examined and that seminal plasma protects against the extensive deleterious effects of lipid peroxidation.

Microphotometric study on cytochrome oxidase and lactate dehydrogenase activities in mouse spermatozoa during maturation and in vivo and in vitro capacitation.

In Eutherian (mammalian) spermatozoa, maturation and capacitation are associated to modifications of the metabolic activities. In order to demonstrate such variations, a quantitative cytochemical study was carried out on cytochrome oxidase and L-lactate dehydrogenase activities in mouse spermatozoa collected from the male and female genital tracts and at different times of the in vitro capacitation. Microdensitometric measurements were made on a Vickers M85 integrator microdensitometer at lambda = 480 +/- 5 nm and lambda = 585 +/- 5 nm wavelengths for the cytochrome oxidase and LDH activities, respectively. The cytochrome oxidase activity first decreases and then increases significantly both during maturation and during capacitation in vivo and in vitro. The LDH activity decreases significantly and gradually in the male and female genital tracts as well as in the course of in vitro capacitation where, however, an enhancement in the anaerobic glycolysis occurs.

Microphotometric study of glucose-6-phosphate dehydrogenase activity in epididymal spermatozoa during spontaneous lipid peroxidation.

Mammalian spermatozoa are highly sensitive to lipid peroxidation and the glutathione peroxidase/reductase system provides an effective defense against oxidative damage to different degree in different species. Rabbit spermatozoa rely on superoxide dismutase as the primary enzymatic defense against lipid peroxidation and contain only low detectable endogenous glutathione reductase activity while in mouse spermatozoa the glutathione system is the major protective enzyme against cell damage by autoxidation. We describe a cytochemical quantitative assay for glucose-6-phosphate dehydrogenase activity in rabbit and mouse spermatozoa undergoing spontaneous lipid peroxidation during in vitro incubation. Microdensitometric measurements were made by a Vickers M85 a scanning microdensitometer at lambda 585 nm wavelength. Our findings suggest that in mouse spermatozoa, the enhanced glutathione reductase and peroxidase activities induced by the spontaneous lipid peroxidation increases NADPH production from the pentose phosphate shunt, while in rabbit spermatozoa, NADPH production is much lower.

Quantitative cytochemical study of some enzymatic activities in preovulatory bovine oocytes after in vitro maturation.

In the present study quantitative cytochemical assays were used to measure some enzymatic activities in situ in bovine meiotically immature oocytes and oocytes matured in vitro, since the special metabolic activity of the growing oocytes may be a pivotal factor in stabilizing the meiotically arrested oocytes. Modifications of this particular metabolism might destabilize the arrested meiosis. Preovulatory oocytes, mostly at the germinal vesicle stage, were obtained by puncturing follicles ranging from 2 to 6 mm in diameter with a hypodermic needle. A group of collected oocytes was incubated in maturation medium CRML 1066 to obtain metaphase II oocytes. Succinate, lactate and glucose-6-phosphate dehydrogenase activities in just collected meiotically immature and in vitro matured oocytes were assayed cytochemically. Microdensitometric measurements were made with a Vickers M85a scanning microdensitometer. Our findings show that: 1) succinate dehydrogenase activity was significantly increased in matured oocytes; 2) lactate dehydrogenase activity was present and very strong in immature oocytes but was detectable in only about 50% of matured oocytes, with significantly lower integrated optical density values; 3) glucose-6-phosphate dehydrogenase activity was very high in immature oocytes but significantly decreased after in vitro maturation; 4) there was no linear correlation between the integrated optical densities of the three enzymatic activities and the diameters of the oocytes. We suggest that the ability to utilize glucose may appear earlier in bovine oocytes than in other species and takes place at the time of maturation.

Tests to localize free proteolytic enzymes in vivo by 14C-cysteine-aprotinin.

Aprotinin, a polyvalent protease inhibitor from bovine organs, has been labelled with 14C-cysteine in 6 M urea to produce a radioactive conjugate, without effect on the inhibitor activity. 138x10(3) dpm of radioactive product, containing 5 mg of protein (20.000 kal. inhibitor units) in 5 ml of Locke's solution were perfused in anesthetized rabbits via ascending aorta. Then the anesthetized rabbit was killed and specimens of some organs were admitted to autoradiographic analysis. Small intestine, ischiatic nerve and testis have been the most rich radiolabelled organs (+++). In kidney, stomach, large intestine, liver and eye the radiolabelled aprotinin was found in fair amounts (++); while in pancreas, spleen, spinal cord, brain, cava vein and aorta of rabbit the 14C-cysteine-Aprotinin was practically undetectable by autoradiography.

Effects of deep freezing on the energy metabolism of bovine spermatozoa during in vitro capacitation: A cytochemical approach.

In order to study the effects of deep freezing on the energy metabolism of bovine spermatozoa, a cytochemical quantitative study was carried out by a microdensitometric method on cytochrome oxidase and lactate dehydrogenase (LDH) activities. These were evaluated in situ on individual frozen-thawed bull spermatozoa collected at different times during in vitro capacitation. The results showed that in bull spermatozoa both the initiation of motility and capacity to fertilize eggs were associated with the anaerobic rather than aerobic glycolysis. The freezing-thawing processes and storage in liquid nitrogen induced a general enhancement of both the enzymatic activities examined. The high ionic strength treatment gave rise to a significant but reversible decrease in both the cytochrome oxidase and LDH activities in the fresh as well as in the frozen-stored sperm. The findings, based on cytochemical observations of energy metabolism of spermatozoa and evaluated during in vitro capacitation, suggest that the respiration and the anaerobic glycolysis of spermatozoa seem to be slightly impaired by the freezing-thawing and storage processes.

[Spondylo-costal dysostosis. Description of a case and review of the literature]. / Disostosi spondilo-costale. Descrizione di un caso e revisione della letteratura.

The Authors describe a case of spondylocostal dysostosis. The infant, born from consanguineous Caucasian parents, showed at birth marked shortening of the trunk and neck. Radiographic examination revealed severe congenital anomalies of the entire spine, associated with secondary deformities of the ribs. A review of literature's cases, with differential diagnosis and pathogenetic hypothesis, is also reported.

[HLA typing in a neonate with posttransfusional graft vs host disease (PT-GVHD)]. / Tipizzazione HLA in un neonato affetto da "graft versus host disease" post trasfusionale (PT-GVHD).

The authors report a case of post-transfusion-graft-versus-host disease (PT-GVHD) in a premature infant after parental blood donation. This disease seems to be due to the transfusion of immunocompetent T lymphocytes into an immunodeficient recipient or into an immunocompetent host who shares an HLA haplotype with HLA-homozygous blood donors (i.e. relatives or members of inbred populations) and who is therefore unable to reject the graft cells. The results of HLA typing of the patient and his family demonstrated that the infant was identical with his father for HLA class II antigens (DR, DQ) and, concerning HLA class I, he had in common the remaining paternal haplotype (A29, B44). Prevention of this disease, performed by gamma irradiation of blood components before transfusion, appears to be effective in most cases.

[Neonatal scrotal hemorrhage caused by post-traumatic hemoperitoneum]. / Emorragia scrotale neonatale da emoperitoneo post-traumatico.

The Authors describe a case of neonatal scrotal hematoma due to post-traumatic intra-abdominal hemorrhage. Differential diagnosis with testicular torsion and other causes of neonatal acute scrotum is considered and discussed.