Study of Pulmonary Hypertension in Patients Suffering from Chronic Obstructive Pulmonary Disease.

Pulmonary hypertension (PAH) represents a frequent complication in patients suffering from chronic obstructive pulmonary disease (COPD), but the impact of accelerated inflammatory status on the pulmonary vascular bed is still insufficiently studied. OBJECTIVES: The study of the PAH's prevalence in patients suffering from COPD, its severity compared with lung function and the correlation with certain clinical, biological and functional parameters. MATERIAL AND METHOD: The study was performed on a group of 64 patients, average age 53 years, 42 of whom were men (65,62%), suffering from COPD who were admitted to the Emergency County Hospital Craiova, on the II-nd Medical Clinic, within a period of 18 months. When assessing the patients their clinical state was stable, without acute exacerbations items. The control group included 61 patients suffering from other diseases without inflammatory background, who were hospitalized in clinic in the same period. All the patients included in the study were evaluated by: physical examination, thoracic radiological examination, spirometry, inflammatory syndrome, echocardiography Doppler and oximetry. RESULTS: The results of the study show a prevalence of 54.6 % PAH and a statistically significant impact of age, duration of the disease, Sa O2, inflammatory status, which was quantified in the study by ESR, serum levels of CRP and serum level of TNF alpha over the risk of developing COPD in patients suffering from PAH. CONCLUSIONS: The occurrence and the severity of PAH in patients suffering from COPD seems to be correlated with their age, duration of the disease, Sa O2 and serum levels of inflammatory markers.

Fatal Evolution in the Pneumonia Caused by Diesel Fuel Aspiration - Case Report.

Aspiration pneumonia in infants emerges as a result of deglutition disorders, congenital malformations and severe gastroesophageal reflux. Hydrocarbon pneumonitis is caused by the accidental ingestion and aspiration of hydrocarbons into the body. In children, it can be seen as a result of both the lack of monitoring and the tendency for the exploration of the environment. The accidental ingestion of a large quantity is quite rare due to the bad taste of hydrocarbons. Initially, the central nervous system is the one affected, followed by the respiratory system, causing the chemical pneumonia (through direct injury of the lung). Furthermore, we shall present the case of an 18 month infant with severe trauma as a result of the accidental ingestion of diesel, followed by vomiting and who was brought late to the hospital. The pneumothorax that emerged as a complication was remitted after the treatment, but the initially chemical and then mixed pneumonia through bacterial overinfection led to the infant's death after 12 days of medical attention in the Anesthesia and Intensive Care Unit. The histopathological examination of the analyzed lung fragments did not point out any lipid or foam cells that are characteristic to this type of pathology, but which are not mentioned by the majority of toxicology textbooks and those of pathological anatomy, through the low rate of mortality and numerous factors that can lead to negative false results.

Assessment of Oxidative Stress Genes SOD2 and SOD3 Polymorphisms Role in Human Colorectal Cancer.

PURPOSE: The aim of this study was to ascertain the oxidative stress genes SOD2 and SOD3 polymorphisms in patients with colorectal cancer and to assess the possible involvement of these polymorphisms that might increase the risk for patients to develop malignant intestinal tumors. MATERIAL AND METHODS: A total number of 306 subjects were divided into two groups (109 colorectal cancer patients as the study group and 197 normal healthy individuals as the control group).We genotyped two polymorphisms, SOD2 - 201A>G (rs4880) and SOD3 - 896C>G (rs1799895), by allelic discrimination, with TaqMan RT-PCR specific probes. RESULTS: No significant differences were found with either of the polymorphisms when comparing the association between them and an increased risk of developing colorectal tumors. CONCLUSION: In Romanian population, the risk of developing colorectal cancer is not increased by SOD2 and SOD3 polymorphisms.

Study of the Etiological Spectrum of Spontaneous Bacterial Peritonitis in a Group of Patients Suffering from Liver Cirrhosis.

INTRODUCTION: Spontaneous bacterial peritonitis (SBP) represents the most severe and common infectious complication in patients suffering from liver cirrhosis. The objectives of the study were the evaluation of the risk factors responsible for the occurrence of the spontaneous bacterial peritonitis in a group of patients suffering from liver cirrhosis, the identification of the bacterial spectrum and assessing the response to antibiotic therapy. Material and method - The studied group included 64 patients suffering from liver cirrhosis, with an episode of SBP, who were admitted to the IInd Medical Clinic of the County Hospital of Craiova, within a period of 24 months. The control group included 61 patients with liver cirrhosis with an episode of decompensation of liver disease. The diagnosis of liver cirrhosis was established by using clinical, biological and imagistic criteria, and the SBP's diagnosis was based on cytological and bacteriological analysis of the ascites fluid. Patients suffering from hepatocellular carcinoma, portal vein thrombosis and other infectious conditions were excluded. The anamnesis, the duration of the disease, the alcohol intake, the complete clinical examination, the clinical, biological and imagistic evaluation were monitored. Results and conclusions - The most frequent etiology of SBP is represented in 67% of the cases by Gram negative germs, and thus, the antibiotic therapy will be orientated against this etiological segment. In what antibiotic sensitivity is concerned, most of the germs were sensitive to third generation cephalosporins, quinolones, carbapenems and vancomycin.

CT vs. MRCP in choledocholithiasis jaundice.

RATIONALE: Obstructive jaundice can raise problems to diagnostic imaging. The radiologist must choose the most appropriate examination that delivers the most important diagnostic information because the differences between a lithiasic obstruction and a tumoral one are vital. This information helps the surgeon speed up the process of decision-making, because the treatment may be very different in relation to the nature of the obstruction. OBJECTIVE: This study tries to demonstrate the diagnostic accuracy of computed tomography (CT) and magnetic resonance cholangiopancreatography (MRCP) in detecting the obstacle in the common bile duct (CBD) and the possibility of establishing the lithiasic nature of the obstruction. METHODS AND RESULTS: A retrospective analysis was analyzed during an interval of 18 months that included jaundice patients admitted in the General Surgery Department of "Coltea" Clinical Hospital. They were examined by CT scanning and by MRCP, being suspected of choledocholithiasis. 63 patients were included in the study, 34 females and 29 males. 33 CT scans and 30 MRCP exams were performed. DISCUSSION: CT scan is useful in detecting residual or iterative choledocholithiasis in patients after cholecystectomy, contrast enhanced CT (CECT), being able to differentiate between lithiasic and non-lithiasic obstruction. MRCP delivers important anatomic details of the biliary tree; it is superior to CT in diagnosing the hepatocholedochal lithiasis; MRCP tends to replace endoscopic retrograde cholangiopancreatography (ERCP)--the diagnostic "gold standard" reducing the number of unnecessary invasive diagnostic procedures.

Differential Diagnosis between Duchenne Muscular Dystrophy and Limb Girdle Muscular Dystrophy 2a.

Limb Girdle Muscular Dystrophy 2A (LGMD 2A) is the most common form of limb girdle muscular dystrophies caused by mutations in the calpain-3 gene (CAPN-3). The pattern of LGMD 2A can be clinically indistinguishable from that of Duchenne Muscular Dystrophy (DMD). We report a case of a 14-year-old boy which has the initial diagnosed as DMD at 6 years old, based on clinical features and very elevated serum creatine kinase levels. A muscle biopsy at the age of 10 showed atypical features which suggested a histiocytosis or neural damage. An MRI conducted 2 years later revealed fatty degeneration predominantly in the posterior region of the thigh and led the diagnosis to LGMD 2A, as well as the necessity to repeat the biopsy. Immunohistochemical analysis was normal for dystrophin, but the Western Blott showed a normal/borderline amount of calpain-3 in the muscle. We also performed a molecular analysis that identified a compound heterozygous mutation of the calpain 3 gene (CAPN 3). LGMD 2A was often misdiagnosed as DMD due to the similarities in clinical manifestations and technique limitations; the immunohistochemical examination, the magnetic resonance imaging examination and the molecular analysis are an essential tool for establishing a right diagnosis.

Diagnosis and treatment protocols of cutaneous melanoma: latest approach 2010.

Cutaneous melanoma is the most aggressive skin malignancies with increasing rate of incidence in the latest decades. New imaging technique plays an important role in melanoma management: dermoscopy and computer dermoscopy, ultrasound, MRI, CT, PET and PET/CT. Due to the dermoscopy and lesion diagnosis in early stages the increasing number of curative melanoma are registered. Sentinel lymph node biopsy became a compulsory phase for patients with tumor thickness > 1 mm. Serological biomarkers proved to be a necessary investigation for melanoma diagnosis, follow-up and treatment response. Current TNM melanoma staging is based on AJCC classification since 2001 witch includes new elements like histopathologic ulceration in stage I and II and lymph node micro- and macrometastases in stage III. Treatment protocols include surgical tumor excision with only 1-2 cm safety margins and radical lymphadenectomy is performed after positive sentinel lymph node biopsy. The adjuvant treatment in advanced stages including chemotherapy, unspecific immunotherapy and interferon offers poor results regarding free disease terms rate of survival. The advanced therapeutic procedure like golden nanospheres and gene therapy are recently studied and represent an alternative for future treatment of melanoma. Follow-up protocols have a great importance for detection of the melanoma recurrences and include clinical, serological and imaging evaluation. Despite all new knowledge and technological support the advanced stage melanoma management still remain an unsolved problem.

Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species.

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated from Antarctica has been purified and characterized. The gene encoding PAP has been cloned and sequenced and the derived amino acid sequence shows 66% identity with the mesophilic alkaline metalloprotease from Pseudomonas aeruginosa IFO 3455 (AP). Compared to the purified AP, PAP is three times more active at 20 degrees C, is very sensitive to chelating agents and is rapidly inactivated at 45 degrees C. The lower thermostability of PAP can tentatively be explained by a loss of a stabilizing Ca(2+), a decrease in the content of hydrophobic residues and a smaller aliphatic index.

Xylanase from the psychrophilic yeast Cryptococcus adeliae.

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 +/- 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0 degrees-20 degrees C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

Cryptococcus adeliensis sp. nov., a xylanase producing basidiomycetous yeast from Antarctica.

Cryptococcus adeliensis sp. nov. (CBS 8351) is described based on phenotypic characteristics and molecular sequence analysis of the D1/D2 large subunit and internal transcribed spacer regions of the ribosomal DNA. Molecular comparisons include species closely related to Cryptococcus albidus and several species isolated from the Antarctic. C. adeliensis, which has a cold-adapted xylanase, was isolated from Terre Adelie, Antarctica. ATCC 34633, which has a mesophilic xylanase, was identified as Cryptococcus albidosimilis.

Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry.

The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.

Psychrophilic enzymes: a thermodynamic challenge.

Psychrophilic microorganisms, hosts of permanently cold habitats, produce enzymes which are adapted to work at low temperatures. When compared to their mesophilic counterparts, these enzymes display a higher catalytic efficiency over a temperature range of roughly 0-30 degrees C and a high thermosensitivity. The molecular characteristics of cold enzymes originating from Antarctic bacteria have been approached through protein modelling and X-ray crystallography. The deduced three-dimensional structures of cold alpha-amylase, beta-lactamase, lipase and subtilisin have been compared to their mesophilic homologs. It appears that the molecular adaptation resides in a weakening of the intramolecular interactions, and in some cases in an increase of the interaction with the solvent, leading to more flexible molecular edifices capable of performing catalysis at a lower energy cost.

In vivo mutational analysis of bacteriophage Mu operators.

In bacteria lysogenic for bacteriophage Mu, the phage repressor binds to a tripartite operator region, O1,O2,O3, to repress the lytic promoter pE, located in O2, and negatively autoregulate its own synthesis at the pCM promoter located in O3. We isolated and characterized operator mutations which lead to derepression of pE. Their location in the first and third repressor-consensus-binding sequences in O2 confirms the importance of these sites for repressor/operator interactions.

Uncoupling effects of diclofenac and aspirin in the perfused liver and isolated hepatic mitochondria of rat.

Gluconeogenesis, glycolysis and glycogenolysis were studied in rat perfused liver following the infusion of various concentrations of diclofenac and aspirin, two non-steroidal anti-inflammatory drugs (NSAIDs). Glucose synthesis was measured in livers isolated from 48-h fasted rats perfused with Krebs-Henseleit bicarbonate buffer containing L-lactate (2 mM) and pyruvate (0.1 mM) as precursors. Both diclofenac (0.01-0.1 mM) and aspirin (1-10 mM) had an inhibitory effect on gluconeogenesis (GNG). The inhibition was dose-dependent and reversible. For the estimation of glycogenolysis and glycolysis, the rates of glucose release and of lactate and pyruvate production were measured in livers of well-fed rats perfused with substrate-free buffer. Infusion of diclofenac (0.1 mM) or aspirin (5 mM) strongly stimulated glycogenolysis and glycolysis (GGL/GL). In general, an increased oxygen consumption by the liver tissue was also noted in both types of experiments, as deduced from the continuous monitoring of oxygen concentration changes in the effluent. Such a pattern of response can be attributed to the uncoupling effects of the two drugs on oxidative phosphorylation. Measurements of respiration rates and membrane potential in isolated liver mitochondria submitted to various concentrations of diclofenac and aspirin confirms this assumption. Thus, 0.01 to 0.2 mM diclofenac stimulates state-4 respiration and slightly inhibits state 3, decreasing the respiratory control ratio, while the membrane potential is decreased or collapsed (depending on the drug concentration). Similar effects are recorded for aspirin at higher concentrations (0.2-5 mM), even though state 3 is not affected in this case. Arguments are presented that the concentrations of the drugs used largely correspond to the pharmacological doses employed in antipyretic and anti-inflammatory treatments. Therefore, a greater consideration should be given to the uncoupling effect, at least from the toxicological viewpoint.

Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.

UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus.

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.

Microalbuminuria following exercise, an early sign of diabetic nephropathy.

Microalbuminuria after physical exercise is characteristic for the preclinical stage of diabetic nephropathy, permitting an early diagnosis of this severe complication of diabetes mellitus. In 24 patients with type I diabetes, 11 women and 13 men free of clinical and biological signs of renal damage, the presence of microalbuminuria has been investigated by radial immunodiffusion in the urines collected between 3 and 7 hrs in the morning, as well as after a 20-min rest following a moderate exercise of 75 watts during 20 min at the ergometric bicycle. The patients were distributed into four groups, according to the age of disease, and microalbuminuria was expressed in micrograms/min. Compared with the mean values recorded in normal subjects by the same method (6.8 micrograms/min), the level of microalbuminuria has been significantly higher in the cases evaluating since more than two years, in keeping with the time elapsed after onset, and particularly following exercise. No relationship could be established between the quality of metabolic control of diabetes and the levels of microalbuminuria after exercise.

Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A.

The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.