Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Time linkages between pollination onsets of different taxa over an 11-year period in Perugia,Central Italy

Times of pollination of different taxa in the atmosphere of Perugia (Central Italy) over an 11-year period (1982–1992) were recorded and analysed by means of a 7-day volumetric Hirst-type pollen trap. For some taxa, the pollination period varied from year to year from a chronological and/or quantitative point of view. Several taxa showed a linkage in their starting dates of pollination. Knowledge of this kind of linkage allows us to build a forecasting model.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

The impact of mitochondrial DNA on human lifespan: a view from studies on centenarians

The role of inherited and somatic mutations of mitochondrial DNA (mtDNA) in aging and longevity is complex and highly controversial, owing to its peculiar genetics, including the phenomenon of heteroplasmy. Most of the data on mtDNA and longevity have been obtained on humans and particularly on centenarians, i. e., people who escaped or delayed the major age-related pathologies and reached the extreme limit of human lifespan. In this review we summarize the most recent advances in this field that suggest a consistent role in human longevity of both germ-line inherited and somatically acquired mutations. The particular case of the association with longevity of the somatic C150T mutation is extensively discussed, challenging the tenet that mtDNA mutations are basically detrimental. We also stress several limitations of our present knowledge, regarding the difficulty in extrapolating to humans the results obtained in animal models, owing to a variety of biological differences, including the very limited genetic variability of mtDNA in the strains used in laboratory experiments. The use of high-throughput technologies and the extensive analysis, possibly at the single cell level, of different tissues and cell types derived from the same individual will help in disentangling the complexity of mtDNA in aging and longevity.     

NT Pro BNP Plasma Level and Atrial Volume Are Linked to the Severity of Liver Cirrhosis

Background and Aims

Plasma levels of NT-pro-BNP, a natriuretic peptide precursor, are raised in the presence of fluid retention of cardiac origin and can be used as markers of cardiac dysfunction. Recent studies showed high levels of NT pro BNP in patients with cirrhosis. We assessed NT pro-BNP and other parameters of cardiac dysfunction in patients with cirrhosis, with or without ascites, in order to determine whether the behaviour of NT pro BNP is linked to the stage of liver disease or to secondary cardiac dysfunction.

Methods

Fifty eight consecutive hospitalized patients mostly with viral or NAFLD-related cirrhosis were studied. All underwent abdominal ultrasound and upper GI endoscopy. Cardiac morpho-functional changes were evaluated by echocardiography and NT-pro-BNP plasma levels determined upon admission. Twenty-eight hypertensive patients, without evidence of liver disease served as controls.

Results

Fifty eight cirrhotic patients (72% men) with a median age of 62 years (11% with mild arterial hypertension and 31% with type 2 diabetes) had a normal renal function (mean creatinine 0.9 mg/dl, range 0.7–1.06). As compared to controls, cirrhotic patients had higher NT pro-BNP plasma levels (365.2±365.2 vs 70.8±70.6 pg/ml; p<0.001). Left atrial volume (LAV) (61.8±26.3 vs 43.5±14.1 ml; p = 0.001), and left ventricular ejection fraction (62.7±6.9 vs. 65.5±4%,; p = 0.05) were also altered in cirrhotic patients that in controls. Patients with F2-F3 oesophageal varices as compared to F0/F1, showed higher e'' velocity (0.91±0.23 vs 0.66±0.19 m/s, p<0.001), and accordingly a higher E/A ratio (1.21±0.46 vs 0.89±0.33 m/s., p = 0.006).

Conclusion

NT-pro-BNP plasma levels are increased proportionally to the stage of chronic liver disease. Advanced cirrhosis and high NT-pro-BNP levels are significantly associated to increased LAV and to signs of cardiac diastolic dysfunction. NT pro-BNP levels could hence be an useful prognostic indicators of early decompensation of cirrhosis.     

Head and Neck Veins of the Mouse. A Magnetic Resonance,Micro Computed Tomography and High Frequency Color Doppler Ultrasound Study

To characterize the anatomy of the venous outflow of the mouse brain using different imaging techniques. Ten C57/black male mice (age range: 7-8 weeks) were imaged with high-frequency Ultrasound, Magnetic Resonance Angiography and ex-vivo Microcomputed tomography of the head and neck. Under general anesthesia, Ultrasound of neck veins was performed with a 20MHz transducer; head and neck Magnetic Resonance Angiography data were collected on 9.4T or 7T scanners, and ex-vivo Microcomputed tomography angiography was obtained by filling the vessels with a radiopaque inert silicone rubber compound. All procedures were approved by the local ethical committee. The dorsal intracranial venous system is quite similar in mice and humans. Instead, the mouse Internal Jugular Veins are tiny vessels receiving the sigmoid sinuses and tributaries from cerebellum, occipital lobe and midbrain, while the majority of the cerebral blood, i.e. from the olfactory bulbs and fronto-parietal lobes, is apparently drained through skull base connections into the External Jugular Vein. Three main intra-extracranial anastomoses, absent in humans, are: 1) the petrosquamous sinus, draining into the posterior facial vein, 2) the veins of the olfactory bulb, draining into the superficial temporal vein through a foramen of the frontal bone 3) the cavernous sinus, draining in the External Jugular Vein through a foramen of the sphenoid bone. The anatomical structure of the mouse cranial venous outflow as depicted by Ultrasound, Microcomputed tomography and Magnetic Resonance Angiography is different from humans, with multiple connections between intra- and extra- cranial veins.     

Long-Term Fungal Inhibition by Pisum sativum Flour Hydrolysate during Storage of Wheat Flour Bread

In order to identify antifungal compounds from natural sources to be used as ingredients in the bakery industry, water/salt-soluble extracts (WSE) from different legume flour hydrolysates obtained by the use of a fungal protease were assayed against Penicillium roqueforti DPPMAF1. The agar diffusion assays allowed the selection of the pea (Pisum sativum) hydrolysate as the most active. As shown by the hyphal radial growth rate, the WSE had inhibitory activity towards several fungi isolated from bakeries. The MIC of the WSE was 9.0 mg/ml. Fungal inhibition was slightly affected by heating and variations in pH. The antifungal activity was attributed to three native proteins (pea defensins 1 and 2 and a nonspecific lipid transfer protein [nsLTP]) and a mixture of peptides released during hydrolysis. The three proteins have been reported previously as components of the defense system of the plant. Five peptides were purified from WSE and were identified as sequences encrypted in leginsulin A, vicilin, provicilin, and the nsLTP. To confirm antifungal activity, the peptides were chemically synthesized and tested. Freeze-dried WSE were used as ingredients in leavened baked goods. In particular, breads made by the addition of 1.6% (wt/wt) of the extract and fermented by baker''s yeast or sourdough were characterized for their main chemical, structural, and sensory features, packed in polyethylene bags, stored at room temperature, and compared to controls prepared without pea hydrolysate. Artificially inoculated slices of a bread containing the WSE did not show contamination by fungi until at least 21 days of storage and behaved like the bread prepared with calcium propionate (0.3%, wt/wt).     

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H₂S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H₂S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H₂S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l ‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l ‐cysteine/CSE/H₂S pathway is involved in melanoma progression.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

Double-strand break DNA repair genotype predictive of later mortality and cancer incidence in a cohort of non-smokers

We followed-up for mortality and cancer incidence 1088 healthy non-smokers from a population-based study, who were characterized for 22 variants in 16 genes involved in DNA repair pathways. Follow-up was 100% complete. The association between polymorphism and mortality or cancer incidence was analyzed using Cox Proportional Hazard regression models. Ninety-five subjects had died in a median follow-up time of 78 months (inter-quartile range 59-93 months). None of the genotypes was clearly associated with total mortality, except variants for two Double-Strand Break DNA repair genes, XRCC3 18067 C>T (rs#861539) and XRCC2 31479 G>A (rs#3218536). Adjusted hazard ratios were 2.25 (1.32-3.83) for the XRCC3 C/T genotype and 2.04 (1.00-4.13) for the T/T genotype (reference C/C), and 2.12 (1.14-3.97) for the XRCC2 G/A genotype (reference G/G). For total cancer mortality, the adjusted hazard ratios were 3.29 (1.23-7.82) for XRCC3 C/T, 2.84 (0.81-9.90) for XRCC3 T/T and 3.17 (1.21-8.30) for XRCC2 G/A. With combinations of three or more adverse alleles, the adjusted hazard ratio for all cause mortality was 17.29 (95% C.I. 8.13-36.74), and for all incident cancers the HR was 5.28 (95% C.I. 2.17-12.85). Observations from this prospective study suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly influence the risk of cancer and non-cancer disease, and can influence mortality.