Prevalence of Helicobacter pylori infection in male patients with osteoporosis and controls.

Cytokines that regulate bone turnover (tumor necrosis factor-alpha, interleukin-6, etc.) may influence the pathogenesis of skeleton disorders, such as osteoporosis. Since Helicobacter pylori infection increases the systemic levels of inflammatory cytokines, we investigated the possibility that this infection increases the risk of developing osteoporosis and affects the bone metabolism in a group of male patients with osteoporosis. We examined 80 osteoporotic male patients and 160 controls for serum antibodies to H. pylori and the CagA protein and determined, in patients alone, the most important biochemical and instrumental parameters of the disease. Fifty-one patients (63.7%) and 107 controls (66.8%) were seropositive for H. pylori infection (nonsignificant); 30 infected patients (58.8%) and 43 infected controls (40.1%) were positive for anti-CagA antibodies (P = 0.028; OR = 2.13). Levels of estradiol in infected CagA-positive patients were significantly lower than in infected CagA-negative patients (28.5 [SD = 10.18] vs. 39.5 [SD = 14.50] pg/ml; P = 0.002) and uninfected patients (35.2 [SD = 12.7] pg/ml; P = 0.028). Levels of urinary cross-laps(a marker of bone resorption) were increased in patients infected by CagA-positive strains compared to patients infected by CagA-negative strains (282.9 [SD = 103.8] vs. 210.5 [SD = 150.1]microg/mmol; P = 0.048) and uninfected patients (204.3 [SD = 130.1] microg/mmol; P = 0.016). Differences among uninfected and infected patients, independent of CagA status, were observed for other markers of bone turnover, but they did not reach statistical significance. Infection by CagA-positive H. pylori strains is more prevalent in men with osteoporosis, who show reduced systemic levels of estrogens and increased bone turnover. H. pylori infection by strains expressing CagA may therefore be considered a risk factor for osteoporisis in men.

Increased phospholipase activity in Helicobacter pylori strains isolated from patients with gastric carcinoma.

PURPOSE: Phospholipase activity, one of Helicobacter pylori pathogenicity factors, has not been investigated enough, so far, although it may induce a remarkable damage to the gastric mucosa. In the present work, we have compared the whole phospholipase activity of H. pylori strains isolated from patients with gastric carcinoma with that of strains isolated from dyspeptic patients without gastric carcinoma. METHODS: We measured the phospholipase activity of one distinct H. pylori colony isolated from each of 10 patients with gastric carcinoma and 10 controls, dyspeptic patients without endoscopic and histological signs of gastric carcinoma. We also determined the phospholipase activity of 20 additional strains isolated from different areas of neoplastic and non-neoplastic tissue of two patients with gastric carcinoma, the cagA and vacA positive G27 and 328 wild strains and their respective vacA and cagA negative isogenic mutants. The whole phospholipase activity of strains was determined by measuring the release of (14)C-labeled palmitic acid from the radioactive l-3-phosphatidylcholine, 1,2-di[1-(14)C]palmiloyl substrate; results were expressed in pmol of palmitic acid per mg of protein. RESULTS: H. pylori strains isolated from patients with gastric carcinoma had levels of phospholipase activity significantly higher than those of strains isolated from controls (99.37 [S.D. 40.45] versus 34.46 [S.D. 16.46], P<0.001). In patients with gastric carcinoma, the mean phospholipase activity of strains isolated from neoplastic tissue was similar to that of strains isolated from non-neoplastic tissues (123.02 [S.D. 44.36] and 115.77 [S.D. 81.48], respectively. Interruption of cagA gene caused a ca. 20% reduction of phospholipase activity (36.38 versus 45.22 of the wild strain); that of vacA caused no reduction of phospholipase activity (26.53 and 25.37 of the wild strain). CONCLUSIONS: The infection by H. pylori strains that produce high levels of phospholipase may increase the risk of developing gastric carcinoma. We hypothesise that indirect products of phospholipase activity, such as prostaglandins, leukotrienes and lysophospholipids, may mediate carcinogenesis.

Haemorheological and endothelial-dependent alterations in heart failure after ACE inhibitor, calcium antagonist and beta blocker.

The aim of our study was to evaluate endothelium-dependent dilatation induced by an ACE-inhibitor, calcium antagonist and beta blocker in patients suffering from heart failure (NYHA class II and III). We studied 34 patients (19M, 15F, mean age 76.96+/-8.82) in pharmacological wash-out for at least one week, divided into 3 groups: Group A (15 patients, 9M and 6F) taking ramipril (5 mg/die); Group B (10 patients, 6M and 4F) taking amlodipine (10 mg/die), Group C: (9 patients, 4M and 5F) taking carvedilole (25 mg/die). The groups were homologous for NYHA class and instrumental echographic parameters (mean EF=22.5+/-6.7 and mean sAPP 38.4+/-8.7). At the beginning and after 3 weeks of therapy, we performed a clinical and instrumental assessment; we studied endothelial function by determination of L-arginine and L-citrulline (amino acids of the nitric oxide metabolic pathway), the L-citrulline/L-arginine ratio (an index of NOS activity) and VCAM-1 (endothelial dysfunction index); haemorheological parameters (blood viscosity, plasma fibrinogen and erythrocyte morphology); coagulative/fibrinolytic parameters (PT, aPTT, fibrinogen and PAI-1). The results show that L-citrulline and L-arginine increase, while VCAM-1 decreases. The L-citrulline/L-arginine ratio increases in a statistically significant way. This trend is maintained in each group. These results demonstrate that the drugs used induce an improvement of endothelium-dependent dilatation. In addition, there is progressive haemorheological and fibrinolytic improvement, with a reduction of PAI-1 and blood viscosity.

Spontaneous echo-contrast as an in vivo indicator of rheological imbalance in dilatative cardiomyopathy.

The aim of this study was to evaluate coagulative and hemorheologic assessment in patients with dilatative cardiomyopathy with or without spontaneous echo contrast (SEC). We studied 45 patients, 35 males and 10 females (mean age 72.1 +/- 9.2). We measured whole blood viscosity, plasmatic fibrinogen, prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer and red cell morphology with Zipursky-Forconi method. Transthoracic and transesophageal echocardiography was performed in all patients to evaluate the presence of SEC in left atrium. We divided all the patients into two groups: the 1st group of 20 patients with SEC and Atrial Fibrillation (AF) in 80% of cases, and the 2nd group of 25 patients without SEC and AF in 31%. Our results show that in patients with SEC there is a statistically significant increase of whole blood viscosity and plasma fibrinogen in comparison with patients without SEC. Red cell morphology in all patients demonstrates a reversed EMI. D-Dimer, was out of the normal range in about 1/3 of the patients in both groups. An analysis of our results points out that in patients with SEC and AF, with a major risk factor for cardioembolic stroke, we have alterations of hemorheologic assessment with an increase of whole blood viscosity and fibrinogen that seems to be caused by an increase of red cells aggregability favoured by fibrinogen. Our conclusions are that SEC in patients with dilatative cardiomyopathy and AF is an important in vivo indicator of hemorheologic imbalance and an important marker for cardioembolic risk stroke evaluation.

Blood viscosity and red cell morphology in subjects suffering from cirrhosis before and after treatment with S-adenosyl-L-methionine (SAM).

Alterations of fluidity of the hepatocytic membrane and of the transport related systems are the basis of the cholesteatic syndrome and favour the tissue accumulation of cytotoxic metabolites. S-Adenosyl-L-Methionine (SAM) is a natural molecule which acts as a giver of methylic groups and as an enzymatic activator in several enzymatic actions of transmethylase and of transulphuration and plays a key role in biochemical processes of hepatic cell. The aim of our study was to evaluate the effects of SAM on the restoration of the membrane fluidity and on the hepatic function in general. In studying the fluidity of the cell membrane we evaluated some hemorheological parameters (total blood viscosity and red cell morphology). Fluidity of the red cell membrane is one of the most important elements of red cell rheology. We studied 15 patients (Group A) suffering from micro- and macro-nodular cirrhosis verified through hepatic biopsy, with alcoholic or post-viral causes. We evaluated the values of: blood viscosity (with a cone-plate rheometer by Carri-med), haematocrit, plasma fibrinogen and the erythrocytic morphology at the optical microscope with the Zipursky-Forconi method before and after 7 days of therapy with SAM i.v.. Data were compared with those of a similar group (Group B) treated with traditional therapy only (hyposodic and hypoprotein diet supplemented with multivitamin preparations, vitamin K in particular, if necessary, and potassium sparing diuretics). We also measured biliary salts, alkaline phosphatase, transaminase and gamma-GT. In the first group we observed a statistically significant reduction of blood viscosity, haematocrit didn't change significantly; biliary salts reduced in a statistically significant way. Evaluation of red cell morphology showed in all cases a pathological percentage (>15%) of echinocytes and knizocytes which reduced to a mean of 5% after SAM therapy. We observed no further modifications of the other hemorheological parameters. Results demonstrate that SAM has a positive action on the fluidity of the membrane, as indicated by the improvement of haemorheological parameters and by the significant decrease of biliary salts, indicating the presence of cholesteasis.

Differences between predicted and observed sequences in Saccharomyces cerevisiae.

We recently studied the protein composition of a Saccharomyces cerevisiae wine yeast strain (K310) of enological interest. About 2,500 spots of 8-250 kDa observed molecular mass were resolved by two-dimensional gel electrophoresis. Experimental molecular masses and isoelectric points were calculated for most of them. Twenty-seven proteins were subjected to Edman microsequencing. N-terminal sequences of 12/27 proteins were determined, whereas internal sequences of 6/27 proteins were obtained following in situ proteolysis. Comparison between the experimental data and those reported in the SWISS-PROT database revealed some differences between genotypic and phenotypic sequences. These are indicative of the changes a protein can undergo with respect to the primary structure coded by the genomic DNA. Our results highlight the need to complement genomic analysis with detailed proteomics in order to refine the vast amount of information provided by DNA sequencing and to find an exact correlation between genome and proteome.

Evaluation of erythrocyte morphology as deformability index in patients suffering from vascular diseases, with or without diabetes mellitus: correlation with blood viscosity and intra-erythrocytic calcium.

The aim of our study was to evaluate the erythrocytic morphology in vascular patients, with or without diabetes, showing cell alterations correlated to blood viscosity and intra-erythrocytic calcium. We studied 108 subjects: 20 normal subjects, 58 vascular patients (25 suffering from CHD, 19 from CVD, 14 from POAD) and 30 non-insulin-dependent diabetes patients with vascular disease in metabolic compensation (13 CHD, 9 CVD, 8 POAD). Erythrocytic morphology, blood viscosity and intra-erythrocytic calcium were evaluated. Our results show that bowls, the most deformable red cells, decreased significantly in vascular patients and in POAD diabetics, while the discocytes, having a stiffer form, greatly increased in subjects suffering from ischemic disease and in POAD diabetics. The altered red cells (echinocytes and knizocytes) reached a statistical significance in CVD and POAD diabetics. Comparing the percentage of discocytes to intraerythrocytic calcium content in vasculopathic subjects, we obtained a significant correlation. No evidence of a relationship between discocytes and blood viscosity was found, even if blood viscosity significantly increased in patients affected by ischemic disease. These results suggest that ischemia decreases the deformability of red cells which is supported by the study of red cells morphology, by the erythrocytic morphology index (EMI), which becomes < 1, and by the evaluation of cytosolic calcium content.

Variations of erythrocyte morphology in different pathologies.

In our study we evaluated erythrocytic morphology in different pathologies which can modify flowing red cells. We followed the methodology proposed by Zipursky which allows a three-dimensional evaluation of the red cell and a classification according to the shapes observed through the optical microscope. We studied 150 subjects: 20 normal subjects, 58 patients suffering from vascular diseases, 40 affected by diabetes (type II) (10 without and 30 with vascular diseases), 22 patients with liver disease, 5 patients with monoclonal gammopathies and 5 dehydrated patients. Results show that in normal subjects bowls, which is the shape of the most deformable red cells, are more (55%) than discocytes (44%); the altered forms are only 1%. In vascular patients we noted a statistically significant increase of discocytes (60%). There are no significant differences between subjects affected by diabetes without vascular disease and normal subjects. In diabetics with vascular diseases there are more discocytes (57%) and some altered forms (3%). In patients suffering from chronic hepatitis a great increase (13%) in echinocytes and knizocytes was noticed, which suggests an alteration in the fluidity of the membrane. Our observations testify the importance of this simple methodology in focusing the morphological alterations which can be accounted for both by pathologies of the red cells and by changes in their metabolism.

Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1.

Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

Khellin, but not 8-methoxypsoralen, inhibits adenylyl cyclase system in HeLa cells.

Until recently, the therapeutic effects of furocoumarins and furochromones plus UV-A light were thought to be due to their ability to form photoadducts with DNA in the cell nuclei; now it appears that membrane effector systems may be involved as targets. Here we show that in HeLa cells khellin at 1 and 5 microM final concentration, in combination with UV-A light, inhibits NaF-stimulated adenylyl cyclase activity and Pertussis Toxin (PT)-catalyzed ADP-ribosylation of alpha-subunits of inhibitory guanine nucleotide regulatory proteins (Gi) and increases GTPase activity. In the same experimental conditions, 8-methoxypsoralen (8-MOP), either alone or plus UV-A, does not affect adenylyl cyclase and GTPase activities. Our results suggest that in HeLa cells, through an interaction with a receptor and the mediation of Gi proteins, the adenylyl cyclase system is a target for khellin but not for 8-MOP.

[An epidemiological study on blood viscosity and intraerythrocytic calcium in a group of high-school students selected according to their family histories]. / Indagine epidemiologica sulla viscosità ematica e sul calcio intraeritrocitario in un gruppo di studenti liceali selezionati in base all'anamnesi familiare.

An epidemiological study was performed in a group of secondary school students selected according to their family history to assess whether changes exist in blood viscosity and intraerythrocytic calcium levels in young healthy subjects with positive family histories of arterial hypertension or cerebral and cardiac ischemic vasculopathies, compared to a control group with a negative family history of these disorders. A population of 130 secondary school students without any pathologies were subdivided into 4 groups: 1) with a positive family history of ischemic cardiopathy (ICP); with a positive family history of cerebral ictus; 3) with a family history of arterial hypertension; 4) a negative family history of these diseases. Total blood viscosity, hematocrit, plasma fibrinogen and intraerythrocytic calcium was evaluated in all groups. The results show that these parameters were within the normal range, as was to be expected in healthy subjects. Blood viscosity was also normal in all groups; intraerythrocytic calcium levels were slightly higher in groups with histories of cardiovascular disease and in particular there was an increased percentage of cases with values above the threshold level. Higher fibrinogen levels were also recorded, but always within the normal range, in the group with a positive history of ICP. The epidemiological study is important to assess whether a family pattern of cardiovascular disease can also influence such independent risk parameters as blood viscosity and intraerythrocyte calcium, owing to the possible greater frequency of development of cardiovascular disease.

Photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen.

The photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen (AcPso), proposed for the photochemotherapy of some skin diseases, were investigated. The photoreaction of AcPso with DNA is weaker in the presence of air than in a nitrogen atmosphere, in terms of total photobinding and DNA cross-linking; when UVA irradiation is performed in air, AcPso behaves as a monofunctional reagent. The quenching effect of oxygen is related to the high capacity of AcPso to produce singlet oxygen. Furthermore, it is demonstrated that AcPso photoadducts are better producers of singlet oxygen than free AcPso in solution. Using DNA sequencing methodology, two modes of DNA photosensitization by AcPso are shown, these lead to the formation of photoadducts mainly at T residues (and at C to a lesser extent) and to photo-oxidized G residues probably via singlet oxygen. Chemical or enzymatic cleavage were used as probes in these experiments. A rapid assay for the detection of the photodynamic effect of a photosensitizer on DNA, involving oxygen, is also described. Finally, the cytotoxicity and genotoxicity of AcPso on E. coli WP2 cells appear to be related to its ability to form photoadducts, in particular cross-links, rather than to its capacity to produce singlet oxygen.

Photosensitization of DNA of defined sequence by furochromones, khellin and visnagin.

The sequence specificity in the in vitro DNA photobinding of khellin and visnagin, two naturally occurring furochromones proposed for chemotherapy of vitiligo, was investigated by using DNA sequencing methodology. The 3'-5' exonuclease associated with the T4 DNA polymerase served as a tool for determining photoadducts distribution on DNA fragments of the lac I gene of Escherichia coli. The photoadduct distribution of psoralen is also studied for comparison. Upon UVA irradiation, visnagin mainly forms monoadducts with thymine and to a lower extent with cytosine. Alternating (A-T)n sequences are hot spots for visnagin photoaddition. This is a property shared with furocoumarins. TTT sites are also quite reactive to visnagin, as they are to methylated angelicins. In contrast, with psoralen derivatives, there is no preferential photobinding in 5'-TpA sites, and 5'-ApT sites react as well. Furthermore, many sites such as T in the GC context, and C in any context, react, although weakly. The visnagin photoadduct distribution resembles very much the photoadduct distribution of methylated angelicins as described by Miolo et al. The photoreaction of these two series of compounds is less sequence dependent than the photobinding of psoralen derivatives as described by Sage and Moustacchi and by Boyer et al. The sequence specificity in khellin-DNA photobinding is the same as for visnagin, even though it forms much fewer photoadducts. The absence of photo-oxidation of DNA after treatment with visnagin or khellin plus UVA suggests that furochromones do not present any photodynamic effect on DNA.