Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol)

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate.     

Helicobacter pylori associated antigastric autoantibodies: role in Sjögren's syndrome gastritis

Background. Previous studies have shown that Helicobacter pylori seroprevalence in Sjögren's syndrome is comparable with that of the general population. However, the origin of the chronic gastropathy associated with this syndrome and the role of local autoimmunity – possibly triggered by bacterial infection – in its pathogenesis remain unclear. Materials and Methods. We initially determined the prevalence of IgG anti H. pylori in dyspeptic subjects with and without Sjögren's syndrome. In subsets of both groups we then determined anti CagA and human tissue‐tested anticanalicular/antifoveolar autoantibodies. We also compared activity, atrophy and Mucosa Associated Lymphoid Tissue (MALT) scores, as well as symptoms, before and after bacterial eradication. Results. Prevalence of H. pylori in Sjögren's syndrome patients was similar to controls: 31/54 (57%) vs. 93/150 (62%). Anti CagA prevalence was also similar in the two groups. Twenty weeks after H. pylori eradication, histological activity decreased in both groups, however, atrophy and MALT decreased significantly only in controls. Sixteen months after H. pylori eradication, 75% of Sjögren's syndrome patients still complained of dyspepsia compared with 13% of controls. Finally, antigastric autoantibodies were present in 29% of tested Sjögren's syndrome patients vs. 28% of controls. Conclusions. H. pylori infection was equally prevalent among dyspeptic Sjögren's syndrome patients and dyspeptic controls. Likewise, there were no differences regarding anti CagA prevalence or antigastric autoantibodies among the two groups. The persistence of symptoms as well as of the lymphocytic infiltration and atrophy after H. pylori eradication in Sjögren's syndrome may underlie the ‘endogenous’ and still unknown nature of the gastropathy in this condition.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.     

Hemodynamic and autonomic correlates of postexercise hypotension in patients with mild hypertension

We investigated the interplay of neural and hemodynamic mechanisms in postexercise hypotension (PEH) in hypertension. In 15 middle-aged patients with mild essential hypertension, we evaluated blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR), forearm (FVR) and calf vascular resistance (CVR), and autonomic function [by spectral analysis of R-R interval and BP variabilities and spontaneous baroreflex sensitivity (BRS)] before and after maximal exercise. Systolic and diastolic BP, TPR, and CVR were significantly reduced from baseline 60-90 min after exercise. CO, FVR, and HR were unchanged. The low-frequency (LF) component of BP variability increased significantly after exercise, whereas the LF component of R-R interval variability was unchanged. The overall change in BRS was not significant after exercise vs. baseline, although a significant, albeit small, BRS increase occurred in response to hypotensive stimuli. These findings indicate that in hypertensive patients, PEH is mediated mainly by a peripheral vasodilation, which may involve metabolic factors linked to postexercise hyperemia in the active limbs. The vasodilator effect appears to override a concomitant, reflex sympathetic activation selectively directed to the vasculature, possibly aimed to counter excessive BP decreases. The cardiac component of arterial baroreflex is reset during PEH, although the baroreflex mechanisms controlling heart period appear to retain the potential for greater opposition to hypotensive stimuli.     

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.     

Response to oxidative stress as a welfare parameter in swine

In pigs, the genetic selection for lean, large muscle blocks and fast growth has been linked to an increased prevalence of metabolic diseases such as porcine stress syndrome and mulberry heart disease. These diseases are associated with cardiovascular inadequacy, which may lead to oxidative stress. In the present study, reactive oxygen metabolites (ROMs) and the anti-oxidant power (OXY) in sera of different swine groups were investigated. The following groups were selected (each around 80 kg body weight): wild boars (WB), Cinta Senese (CS), and Landrace x Large White (LxLW), the latter as both specific pathogen-free (SPF) and intensively farmed animals. In addition, a group of LxLW agonic sows (AS) was also investigated; this group is known to be under oxidative stress. Two colorimetric micro-methods were used to measure ROMs and OXY; ROMs were expressed as mM H(2)O(2) and OXY as microM HOCl neutralised. Between groups, average ROM and OXY values were found to be significantly different by one-way ANOVA (P < 0.001). ROM levels were lower in WB (13.41 +/- 1.85) and CS (19.27 +/- 1.68), and highest in LxLW (42.00 +/- 1.36). OXY values ranged from 260.10 +/- 22.13 (WB) to 396.90 +/- 9.83 (LxLW). Only one swine group (the CS group) showed a significant, positive correlation between ROM and OXY values. The AS group even showed a negative correlation between ROM and OXY values. These results imply satisfactory environmental coping occurred only within the CS group. Results are discussed in the light of animal welfare legislation, food safety and consumers' protection.     

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.     

Melatonin,melanogenesis, and hypoxic stress in the newt,Triturus carnifex

Groups of 6 specimens each of the newt Triturus carnifex were treated with melatonin to see if the hormone inhibited melanogenesis in the Kupffer cells of the liver (melanomacrophages), a process markedly stimulated by hypoxia. A dose of 500 microg/g in 27% ethanol, injected intraperitoneally, induced loss of consciousness and tetany of all the skeletal muscles, which on the contrary appeared relaxed in animals pre-anesthetised by immersion in chlorbutol at 0.2%. Anesthetised specimens injected with melatonin showed a significantly lower increase in hepatic pigmentation after acute hypoxia, a condition attained by sealing each specimen in a 620 mL respiratory chamber with water containing 1.1 ppm of oxygen for the time needed to consume it all (about two hours). If hypoxia is reached gradually, beginning with 8 ppm of oxygen (normoxic condition), the increase in hepatic pigmentation after melatonin injection does not differ significantly from that of non-hormone treated specimens: thus melatonin does not seem to play a direct part in controlling hepatic melanogenesis. Instead, the hormone induces significant increase in oxygen consumption, marked general steatosis of the liver and the almost total disappearance of glycogen. Intraperitoneal injection of 500 microg/g of melatonin in anesthetised animals exposed to the air (normoxic) also causes severe steatosis and an unexpected increase in the hepatic deposits of melanin, as after hypoxic treatment. A dose of 100 ng/g in 1% ethanol, ineffective when injected intraperitoneally, also induces these effects if injected directly into the arterial blood-stream through the conus arteriosus, thus avoiding the hepatic filter. The phenomena observed appear to be induced by a powerful endocrine mechanism that provokes metabolic hypoxia by consuming all the available ATP for synthesizing fat. A less intense form of steatosis can also be observed in animals subjected to hypoxia but without prior hormone treatment, indicating that a natural process triggered by hypoxic stress is pushed to the extreme by exogenous melatonin: the hormone changes the entire energy metabolism of the organism so that it can survive for a long time under adverse environmental conditions.     

Taurocholate feeding prevents CCl4-induced damage of large cholangiocytes through PI3-kinase-dependent mechanism

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to CCl(4)-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), CCl(4)-treated BDL rats, and CCl(4)-treated taurocholate-fed rats. In vitro, we tested whether CCl(4) induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The CCl(4)-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in CCl(4)-treated rats fed taurocholate. CCl(4)-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, CCl(4)-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on CCl(4) effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from CCl(4)-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies.     

Acute ischemia/hypoxia in rat hippocampal neurons activates nuclear ubiquitin and alters both chromatin and DNA

We investigated early alterations in rat neurons after experimental ischemic stress. Transient ischemia was generated by bilateral occlusion of the carotids after hypoxia. Data show a relevant increase of the nuclear level of ubiquitin 2 h post-stress as evaluated by immuno-cytolocalization. Ubiquitin returns to normal levels after 6 h. The increase in ischemic/hypoxic rats was localized preferentially in nuclei of hippocampal neurons, although some augmentation was also shown essentially in dendrites. The activation of ubiquitin system is related to a defective homeostasis and might trigger different degenerative processes. With respect to this, we observed chromatin alterations by densitometric analysis. The shown extensive DNA degeneration is consistent with the occurrence of necrotic phenomena at an early stage. However the parallel internucleosomal specific DNA fragmentation, strongly suggests that apoptotic events also occur. In any case both necrosis and apoptosis are likely to occur at same time, although apoptosis is less extensive, and two phenomena take place in different neural cells.