Modelling the seasonal impacts of a wastewater treatment plant on water quality in a Mediterranean stream using microbial indicators.

Faecal pollution modelling is a valuable tool to evaluate and improve water management strategies, especially in a context of water scarcity. The reduction dynamics of five faecal indicator organisms (E. coli, spores of sulphite-reducing clostridia, somatic coliphages, GA17 bacteriophages and a human-specific Bifidobacterium molecular marker) were assessed in an intermittent Mediterranean stream affected by a wastewater treatment plant (WWTP). Using Bayesian inverse modelling, the decay rates of each indicator were correlated with two environmental drivers (temperature and streamflow downstream of the WWTP) and the generated model was used to evaluate the self-depuration distance (SDD) of the stream. A consistent increase of 1-2 log10 in the concentration of all indicators was detected after the discharge of the WWTP effluent. The decay rates showed seasonal variation, reaching a maximum in the dry season, when SDDs were also shorter and the stream had a higher capacity to self-depurate. High seasonality was observed for all faecal indicators except for the spores of sulphite-reducing clostridia. The maximum SDD ranged from 3 km for the spores of sulphite-reducing clostridia during the dry season and 15 km for the human-specific Bifidobacterium molecular marker during the wet season. The SDD provides a single standardized metric that integrates and compares different contamination indicators. It could be extended to other Mediterranean drainage basins and has the potential to integrate changes in land use and catchment water balance, a feature that will be especially useful in the transient climate conditions expected in the coming years.

FIRST DAY SERUM CALCIUM AND PARATHYROID HORMONE LEVELS AS PREDICTIVE FACTORS FOR SAFE DISCHARGE AFTER THYROIDECTOMY.

CONTEXT: Permanent hypocalcemia is a rare but significant complication of thyroid surgery. OBJECTIVE: The aim of this study was to identify predictive factors of hypocalcemia and hypoparathyroidism after thyroidectomy. DESIGN: Study included 134 total patients submitted to thyroidectomy from two endocrine units (January 2015 - August 2016). METHODS: We measured total serum calcium (sCa) and intact PTH (iPTH) on postoperative day one and 1 month after surgery. RESULTS: 118 patients were women with F/M ratio of 7.3/1 and a mean age of 51.8 years. 64 patients were included in group A (iPTH <12 pg/mL) and 70 patients in group B (iPTH >12 pg/mL). sCa and hypocalcemia symptoms were correlated with iPTH, measured 24 hours after surgery. The cut-off value was for sCa 8.05 mg/dL with a sensitivity of 85.29% and a specificity of 88.0% and for iPTH 11.2 pg/mL, with a sensitivity of 82.3% and a specificity of 71.0%. SCa (< 8.05 mg/dL) was a predictive factor with a 99 (IC95%:12.86-761.58) and iPTH (<11.2 pg/mL) with a 10.77 higher risk (CI95%: 3.83-30.30) to be associated with symptoms. CONCLUSION: SCa and iPTH represent good predictive factors of early and safe hospital discharge and can predict the risk of prolonged and permanent hypoparathyroidism.

Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

BACKGROUND: Allergic reactions to ß-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. METHODS: We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. RESULTS: We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. CONCLUSIONS: These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response.

INCIDENTAL PARATHYROIDECTOMY DURING THYROID SURGERY - RISK, PREVENTION AND CONTROVERSIES; AN EVIDENCE-BASED REVIEW.

BACKGROUND: Postoperative hypocalcemia after thyroid surgery has a high prevalence ( 16-55% in different series). Incidental parathyroidectomy (IP) is a less discussed complication of thyroidectomy with consequences not properly defined. The aim of our study was to find incidence, risk factors and how to prevent IP. METHODS: Extensive search of English literature publications via PubMed was performed and 73 papers from 1980 to 2017 were analysed using the GRADE system/classification, quality of evidence was classified as "strong" when the result is highly unlikely to change existing recommendation and "weak" when opposite. RESULTS: Incidence of IP is 3.7-24.9%, while prevalence of permanent hypoparathyroidism is less frequent 6-12%. Direct relation between IP and hypoparathyroidism/hypocalcemia remains controversial. Female patients, ectopic parathyroids, small thyroids, Graves', malignancy, redo surgeries and total thyroidectomy favour IP. Routine visualization of parathyroids, new hemostatic devices, magnifying instruments and fluorescence can prevent incidental removal of parathyroids. Incidence of IP during videoassisted or robotic thyroidectomies was similar to open procedures. High volume, experienced and younger surgeons have lower complication rates (including hypoparathyroidism). CONCLUSIONS: Incidental parathyroidectomy is more frequent than we might have expected. It should be avoided and parathyroid glands should be kept in situ. Majority of studies are retrospective (low degree of evidence according to previous mentioned GRADE classification) and further meta-analysis or randomized control studies are welcome in order to define the impact of incidental removal of parathyroids on postoperative outcome.

A COMPARATIVE ANALYSIS OF THE INITIAL EAST EUROPEAN CENTER EXPERIENCE WITH A WESTERN HIGH-VOLUME CENTER FOR OPEN MINIMALLY INVASIVE PARATHYROIDECTOMY (OMIP) AS TREATMENT OF PRIMARY HYPERPARATHYROIDISM.

OBJECTIVE: To compare results of treatment of primary hyperparathyroidism (PHPT) in two teaching hospitals (eastern and western Europe) and to establish conclusions regarding quality of surgery for PHPT in Romania. METHODS: We reviewed two prospectively collected databases of patients submitted to open minimally invasive parathyroidectomy (OMIP) for symptomatic PHPT in two centers from Romania and the United Kingdom (UK). We included patients with biochemically proven PHPT and positive pre-operative localization studies. We excluded patients with negative localization studies, suspected multiglandular disease, concomitant thyroid disorders and chronic renal failure. RESULTS: 60 patients were included, 27 in group A (Romanian cohort) and 33 in group B (UK cohort). We noted significant differences between groups in pre-operative serum calcium and phosphorus levels (p<0.5). There were no differences between groups regarding the presence of symptoms; in group A we had significantly more patients with renal calculi history (p=0.02), digestive symptoms (p=0.006) and osteitis fibrosa cystica (p=0.01). Two patients from the UK group had lithium associated hyperparathyroidism and 2 patients had genetic disease. Intraoperative parathyroid hormone measurement (ioPTH) was available only for group B and frozen sections were selectively used in both groups. Both the adenoma size and weights were significantly higher in group A. The median operative time was significantly longer in Romanian group (p=0.001); in this group we noted the single conversion to traditional cervicotomy (3.7%) from all studied patients. In group A we noted two patients (7.4%) with failed parathyroidectomy and persistent PHPT; the cure rate was 92.5% for Romanian group and 97% for the UK group. CONCLUSIONS: OMIP can be performed safe with a high cure rate in "small" volume endocrine centres with results comparable to western experienced endocrine centres. Romanian patients presented with more severe PHPT with more frequent end-organ damage, due probably to late diagnosis.

Hypersensitivity reactions to ß-lactams: relevance of hapten-protein conjugates.

ß-Lactams (BL) are the drugs most frequently involved in allergic reactions. They are classified according to their chemical structure as penicillins, cephalosporins, monobactams, carbapenems, and clavams. All BL antibiotics have a BL ring that is fused to a 5-member or 6-member ring (except in monobactams) and has 1, 2 or 3 side chains (except in clavams). Differences in chemical structure mean that a wide range of BLs are recognized by the immune system, and patients may experience clinical reactions to one BL while tolerating others. Diagnosis is based on skin and in vitro testing, although both display low sensitivity, possibly because they are based on drugs or drug conjugates that are not optimally recognized by the immune system. BLs are haptens that need to bind to proteins covalently to elicit an immune response. These drugs have a high capacity to form covalent adducts with proteins through nucleophilic attack of amino groups in proteins on the BL ring. Allergenic determinants have been described for all BLs, although benzylpenicillin is the most widely studied. Moreover, formation of BL-protein adducts is selective, as we recently demonstrated for amoxicillin, which mainly modifies albumin, transferrin, and immunoglobulin heavy and light chains in human serum. Given the complexity of BL allergy, understanding the immunological mechanisms involved and optimization of diagnostic methods require multidisciplinary approaches that take into account the chemical structures of the drugs and the carrier molecules, as well as the patient immune response.

Minilaparotomy as surgical approach for aortoiliac occlusive disease -- single center initial experience.

PURPOSE: The purpose of this study was to emphasize the benefits and indications of performing minilaparotomy as surgical approach for occlusive aortoiliac disease. MATERIAL AND METHOD: From January 2011 to July 2012, a total of 23 patients (19 men and 4 women), with a median age of 60 years (range 49-75) diagnosed with aortoiliac occlusive disease(n=22) or abdominal aneurysm (n=1), were included in a retrospective non-randomized clinical study. Among these patients 11 underwent aortic bypass procedure by minila parotomy approach (ML group) and 12 patients by standard laparotomy(SL group). Demographic and clinical data, operative data, postoperative recovery data and complications were analysed according to these two groups of patients. Follow-up consisted of clinical examination and duplex scanning at 1, 3,6 and 12 months postoperatively. RESULTS: There were no significant differences between the minilaparotomy and standard laparotomy control groups concerning clinical and demographical data. Two surgical conversions to standard laparotomy were necessary (18.18%) in the ML group due to technical difficulties. The mean operative time was shorter in the ML group (124 ± 22 minutes) and the mean aortic clamping time was similar between the two groups.Major differences between the two lots were observed postoperatively;mean blood loss was more important in the SL group (550 ml) than in the ML group (350 ml) (statistical significance p=0.001, Student test). Patients who have undergone standard laparotomy required more fluids (10000 Â+-2000 ml) in comparison to the other group (6000 ± 1000 ml) p value=0.0001, while the duration of nasogastric suction and period before resuming a liquid diet was both shorter in the ML group (1.1 ± 0.5 days) than those from the SL group (2.5 ± 0.6 days) p value=0.001. The period spent in the ICU was significantly shorter for the ML lot of patients and the median hospitalization time was 5.6 days for patients in ML group,whereas in the SL group the median hospitalization time was 8.9 days (Student test - p value 0.01). We had no 30-day mortality in any of the groups included in the study. One patient from the ML group was readmitted in postoperative day 43 and re-operated on for a prosthetic limb graft thrombosis.Two patients were lost to follow-up and the mean follow-up was 9 ± 1.5 months. CONCLUSIONS: Minilaparotomy as surgical approach for aortic diseases is a feasible, safe procedure on selected patients.

Transthoracic versus Transhiatal esophagectomy: a permanent dilemma. our 15-year experience.

OBJECTIVE: Controversy still exists regarding the optimal surgical management of esophageal cancer. This study was performed to determine and compare early and late morbidity,mortality and overall survival after transthoracic (TTE) and transhiatal esophagectomies (THE). METHODS: Between 1997-2011, 100 patients underwent TTE or THE for squamous esophageal carcinoma (90 patients)and adenocarcinoma (10 patients). Assessed parameters included patient demographics, operative data, pathology results, postoperative morbidity and mortality and 1-3 year survival. RESULTS: Thoracic approach was preferred in cases of more advanced tumors, located in the upper and mid-third of the esophagus, in patients with a better cardiopulmonary status. Perioperative blood loss was significantly higher after transthoracic resections (p=0.0004) and these surgeries took significantly longer than transhiatal esophagectomies(p=0.02). We identified complications in 70.7% patients who under went TTE and in 59.3% patients with transhiatal approach. Respiratory complications were statistically significant in the TTE- group (p-0.0003). The 30-day mortality rates were 12.2% for patients in TTE group and 10.1% in THE patients group, respectively. The mortality ratefor the entire period of the study has been calculated at 84.4%.We have identified a survival rate after 1 year of 62.2%, after 2 years of 39.3% and after 3 years - 15.1%. CONCLUSIONS: According to the results of this study, both procedures appear to be acceptable depending on surgeon preference and appropriate patient selection.

Modeling conformational states of proteins with AlphaFold.

Many proteins exert their function by switching among different structures. Knowing the conformational ensembles affiliated with these states is critical to elucidate key mechanistic aspects that govern protein function. While experimental determination efforts are still bottlenecked by cost, time, and technical challenges, the machine-learning technology AlphaFold showed near experimental accuracy in predicting the three-dimensional structure of monomeric proteins. However, an AlphaFold ensemble of models usually represents a single conformational state with minimal structural heterogeneity. Consequently, several pipelines have been proposed to either expand the structural breadth of an ensemble or bias the prediction toward a desired conformational state. Here, we analyze how those pipelines work, what they can and cannot predict, and future directions.

Targeting in silico GPCR conformations with ultra-large library screening for hit discovery.

The use of deep machine learning (ML) in protein structure prediction has made it possible to easily access a large number of annotated conformations that can potentially compensate for missing experimental structures in structure-based drug discovery (SBDD). However, it is still unclear whether the accuracy of these predicted conformations is sufficient for screening chemical compounds that will effectively interact with a protein target for pharmacological purposes. In this opinion article, we examine the potential benefits and limitations of using state-annotated conformations for ultra-large library screening (ULLS) in light of the growing size of ultra-large libraries (ULLs). We believe that targeting different conformational states of common drug targets like G-protein-coupled receptors (GPCRs), which can regulate human physiology by switching between different conformations, can offer multiple advantages.

The Approach to Tetanic Hypocalcemia Caused by Vitamin D Deficiency.

Recently studies have shown that over half of infants, children and teenagers may be inadequately supplemented. A high prevalence of vitamin D deficiency in children has been observed worldwide, even in sunny countries. Regardless of the recommendations, vitamin D supplementation is sometimes underestimated, supporting the idea that for children in sunny country it is unnecessary. In the modern area of supplementation, tetany seems to be a problem of the past. Usually it occurs at 3 to 6 months of age, that's why diagnostic suspicion may be low after this age. We report a case of severe hypocalcemia presented with tetany, in an18-months-old child with severe vitamin D deficiency, because of not receiving vitamin D as supplementation. With underestimation of vitamin D supplementation, tetany may emerge again. Through our report we aim to highlight the aggressive approach to tetanic hypocalcemia in children with vitamin D deficiency and to sensitize a rigorous surveillance in order to ensure adequate vitamin D supplementation by pregnant, breastfeeding mothers, children and adolescents.

Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors.

Early diagnosis of PI3Kδ syndrome in a 2 years old girl with recurrent otitis and enlarged spleen.

Heterozygous gain of function mutations in the gene encoding p110δ subunit of PI3K have been recently associated with activated PI3K-δ syndrome (APDS), a novel combined immune deficiency characterized by recurrent sinopulmonary infections, lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. Here we report a dominant gain of function PIK3CD mutation (E1021K) in a patient presenting with recurrent otitis media, massive splenomegaly, and persistent EBV-viraemia. The immunological studies showed low IgA level, but normal IgM, IgG, and normal antibody response to diphtheria and tetanus toxoid vaccination. Analysis of B lymphocyte subsets revealed abnormal expansion of transitional B cells, and low percentage of switched CD27+IgD- and CD27+IgD+ memory B cells. Analysis of T cell compartment unveiled prevalence of terminally differentiated cells. This study suggests that PIK3CD gain of function mutations should be suspected despite incomplete phenotype in patients with early onset splenomegaly, persistent EBV viremia and abnormal B and T cell subsets despite normal IgG levels. Currently the optimal treatment is still debated, but prompt management can hopefully diminish incidence of severe long-lasting sequelae (i.e. bronchiectasis, ear and sinus damage).

PCA3 as a second-line biomarker in a prospective controlled randomized opportunistic prostate cancer screening programme. / PCA3 como biomarcador de segunda línea en un programa de screening oportunista prospectivo, aleatorizado y controlado.

OBJECTIVES: PCA3 performance as a single second line biomarker is compared to the European Randomised Study of Screening for Prostate Cancer risk calculator model 3 (ERSPC RC-3) in an opportunistic screening in prostate cancer (PCa). MATERIAL AND METHODS: 5,199 men, aged 40-75y, underwent prostate-specific antigen (PSA) screening and digital rectal examination (DRE). Men with a normal DRE and PSA ≥3ng/ml had a PCA3 test done. All men with PCA3 ≥35 underwent an initial biopsy (IBx) -12 cores-. Men with PCA3 <35 were randomized 1:1 to either IBx or observation. We compared them to those obtained with ERSPC RC-3. RESULTS: PCA3 test was performed on 838 men (16.1%). In PCA3(+) and PCA3(-) groups, global PCa detection rates were 40.9% and 14.7% with a median follow-up (FU) of 21.7 months (P<.001). In the PCA3(+) arm (n=301, 35.9%), PCa was identified in 115 men at IBx (38.2%). In the randomized arm, 256 underwent IBx and PCa was found in 46 (18.0%) (P<.001). The biopsy-sparing potential would have been 64.1% as opposed to 76.6% if we had used ERSPC RC-3. However, the estimated false negative cases for HGPCa would have been reduced by 37.1% (89 to 56 patients). Moreover, if we had applied PCA3-35 to avoid IBx, 14.7% PCa and 9.1% of clinical significant PCa patients would not have been diagnosed during this FU. CONCLUSIONS: When PCA3-35 is used as a second-line biomarker when PSA ≥3ng/ml and DRE is normal, IBx could be avoided in 12.5% less than if ERSPC RC-3 is used and would reduce the false negative cases by 36.2%. At a FU of 21.7 months, this dual protocol would miss 9.1% of clinically significant PCa, so strict FU is mandatory with established biopsy criteria based on PSA and DRE in cases with PCA3 <35.

Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.

Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels. Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone. Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells. Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin. Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene. The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1. Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene. Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin. Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis.

IL-2 deprivation triggers apoptosis in the murine T cell line TS1alphabeta, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1alphabeta cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1alphabeta cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

Novel aspects of Ras proteins biology: regulation and implications.

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.

[Essential thrombocythemia]. / La trombocitemia essenziale.

New diagnostic and therapeutic aspects of the essential thrombocythemia are summarized. A series of 14 patients with essential thrombocythemia is reported.

The stereochemical requirement for protein kinase C activation by 3-methyldiglycerides matches that found in naturally occurring tumor promoters aplysiatoxins.

Protein kinase C is stereospecifically activated by sn-1,2-(S)-diglycerides. A second chiral center was introduced into the diglycerides by preparing the 3-methyl derivatives. The activation of protein kinase C was also stereospecific with respect to the new chiral center established at the C3 position of the methylated diglycerides. The stereospecifically of protein kinase C directed towards the C2 and C3 positions of the diglycerides is matched in the analogous C29 and C30 stereocenters of the tumor promoting debromoaplysiatoxins. This finding strengthens the view that the structurally diverse tumor promotors contain the embedded diglyceride-like pharmacophore.