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This work presents a one-step synthesis methodology for preparing a hydrochar (HC) doped with TiO 2 (HC-TiO 2 ) for its application on the degradation of crystal violet (CV) using UV and visible radiation. Byrsonima crassifolia stones were used as precursors along with TiO 2 particles. The HC-TiO 2 sample was synthesized at 210 °C for 9 h using autogenous pressure. The photocatalyst was characterized to evaluate the TiO 2 dispersion, specific surface area, graphitization degree, and band-gap value. Finally, the degradation of CV was investigated by varying the operating conditions of the system, the reuse of the catalyst, and the degradation mechanism. The physicochemical characterization of the HC-TiO 2 composite showed good dispersion of TiO 2 in the carbonaceous particle. The presence of TiO 2 on the hydrochar surface yields a bandgap value of 1.17 eV, enhancing photocatalyst activation with visible radiation. The degradation results evidenced a synergistic effect with both types of radiation due to the hybridized π electrons in the sp 2 -hybridized structures in the HC surface. The degradation percentages were on average 20% higher using UV radiation than visible radiation under the following conditions: [CV] = 20 mg/L, 1 g/L of photocatalyst load, and pH = 7.0. The reusability experiments demonstrated the feasibility of reusing the HC-TiO 2 material up to 5 times with a similar photodegradation percentage. Finally, the results indicated that the HC-TiO 2 composite could be considered an efficient material for the photocatalytic treatment of water contaminated with CV., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jonathan M. Sanchez-Silva reports financial support was provided by National Council on Science and Technology., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Two haemoparasites, Theileria equi and Babesia caballi, cause equine piroplasmosis (EP), one of the most prevalent tick-borne diseases in horses. The main aim of the present study was to develop and evaluate a multinested PCR (mn-PCR) for simultaneous detection of the equine piroplasmids T. equi and B. caballi, by amplification of five genetic markers (18S rRNA, β-tubulin, cytB, EMA-1 and RAP-1). This novel assay detected a high prevalence of equine piroplasmids in 235 horse blood samples collected in Castilla-León and Extremadura, Spain. The overall prevalence of infection with equine piroplasmids by mn-PCR was 72.8% (171/235), with 66.0% (155/235) of the animals positive for T. equi and 29.4% (69/235) positive for B. caballi. The seroprevalence obtained by cELISA for the same set of samples was lower than the infection prevalence recorded by mn-PCR, for either of the two equine piroplasmids (62.6%) as well as for T. equi alone (61.7%) or B. caballi alone (3.8%). There was high agreement among the mn-PCR and cELISA assays for diagnosis of EP caused by T. equi (κ = 0.83) but not for B. caballi (κ = 0.06). A phylogenetic analysis based on the RAP-1 gene of B. caballi showed that the strains from Spain clustered with those from Israel., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Objective: Our objective was to describe the risk of hospital admission for virologically confirmed dengue (VCD) and the risk of clinically severe hospitalized VCD occurring up to 4 years after the first dose (years 1 to 4) in three randomized clinical trials comparing tetravalent dengue vaccine with placebo., Methods: The relative risks (RR) for hospitalized VCD from first dose to year 4 were estimated by year and age-group in individual and combined studies., Results: Overall, from Year 1 to Year 4, 233 and 228 participants had at least one episode of hospitalized VCD in the vaccinated (n = 22 603) and placebo (n = 11 301) groups, respectively (RR = 0.511, 95% CI 0.42-0.62). Among these, 48 and 47 cases, respectively, were classified as clinically severe. In children aged ≥9 years, 88 and 136 participants had at least one episode of hospitalized VCD in the vaccinated (n = 17 629) and placebo (n = 8821) groups, respectively (RR = 0.324; 95% CI 0.24-0.43). In vaccinated participants aged <9 years, particularly in those aged 2-5 years, there were more hospitalized VCD cases compared with the control participants in Year 3 but not in Year 4. The overall RR in those aged <9 years for Year 1 to Year 4 was 0.786 (95% CI 0.60-1.03), with a higher protective effect in the 6-8 year olds than in the 2-5 year olds., Conclusions: The overall benefit-risk remained positive in those aged ≥9 years up to year 4, although the protective effect was lower in years 3 and 4 than in years 1 and 2., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The presence of genetic changes is a hallmark of chronic lymphocytic leukemia (CLL). The most common cytogenetic abnormalities with independent prognostic significance in CLL are 13q14, ATM and TP53 deletions and trisomy 12. However, CLL displays a great genetic and biological heterogeneity. The aim of this study was to analyze the genomic imbalances in CLL cytogenetic subsets from both genomic and gene expression perspectives to identify new recurrent alterations., Patients and Methods: The genomic imbalances and expression levels of 67 patients were analyzed. The novel recurrent abnormalities detected with bacterial artificial chromosome array were confirmed by FISH and oligonucleotide microarrays. In all cases, gene expression profiling was assessed., Results: Copy number alterations were identified in 75% of cases. Overall, the results confirmed FISH studies for the regions frequently involved in CLL and also defined a new recurrent gain on chromosome 20q13.12, in 19% (13/67) of the CLL patients. Oligonucleotide expression correlated with the regions of loss or gain of genomic material, suggesting that the changes in gene expression are related to alterations in copy number., Conclusion: Our study demonstrates the presence of a recurrent gain in 20q13.12 associated with overexpression of the genes located in this region, in CLL cytogenetic subgroups.

An automated screening approach for fluoroquinolone (FQ) antibiotics (norfloxacin and ciprofloxacin) in soil samples has been developed. The proposed approach consists on dynamic microwave-assisted extraction and subsequent real-time on-line monitoring of the analytes extracted; thus, the extraction is halted when complete leaching of the analytes has been reached (independently of the sample matrix), avoiding extraction times in excess. The end of the extraction allowed quantifying the total content of the analytes. The extraction was carried out using pure water as extractant and consisted of a number of extraction cycles (depending on the sample matrix) in which the sample was subjected to microwave irradiation while the direction of the extractant was changed in an iterative manner. The target analytes were fluorometrically monitored after derivatization with a terbium (Tb3+)/tri-n-octylphosphine oxide (TOPO)/cetylpyridinium chloride (CPCl)/acetate buffer solution. Optimum conditions for analytes extraction and formation of FQ-Tb3+-TOPO ternary complexes have been obtained using the experimental design methodology. The mean recoveries from soil samples spiked with 5 and 0.5 microg/g of each analyte were (95.2+/-4.16%) and (98+/-5.21%), respectively. The within-laboratory reproducibility and repeatability, expressed as relative standard deviation, were 7.29 and 5.80%, respectively. The approach only allows monitoring of the overall content of the species that yield fluorescent complexes with the derivatizing reagent, so the use of chromatography is mandatory for individual separation/quantification of the target compounds.

An overview of a new extraction technique called focused microwave-assisted Soxhlet extraction (FMASE) is here presented. This technique is based on the same principles as conventional Soxhlet extraction but using microwaves as auxiliary energy to accelerate the process. The different devices designed and constructed so far, their advantages and limitations as well as their main applications on environmental and food analysis are discussed in this article.

An approach based on continuous pressurized liquid-liquid extraction with 20% HNO(3)/1M KCl/10(-3)M EDTA as extractant has been developed for the extraction of metals from the oil resulting from recycled tires. A multivariate optimization of the main variables affecting the extraction process has been performed. The method has been applied to three oil samples with different contents in V, Ni, Zn, Fe and Cu. The target metals have been determined in both untreated and treated oil by flame atomic absorption spectrometry (FAAS) in order to obtain the extraction efficiency. Under the optimum conditions, recoveries higher than 90% have been obtained with a repeatability and within-laboratory reproducibility, expressed as relative standard deviations, ranging from 2.75 to 6.91 and 2.97 to 8.16%, respectively. For applications where the demetalization does not require being complete, a study of the demetalization degree achieved depending on the extraction temperature and extractant composition has also been performed. This study provides useful data about the cheapest working conditions to be used in order to obtain a given demetalization level.

Conventional Soxhlet extraction assisted in the cartridge by ultrasound has been developed and used to extract the total fat content from oleaginous seeds such as sunflower, rape and soybean seeds. The application of ultrasound to the sample cartridge enormously decreases the number of Soxhlet extraction cycles needed for quantitative extraction of the fat, thus reducing the extraction time at least to half the time needed by the conventional procedures. The results agree well with those obtained by conventional Soxhlet extraction and the ISO reference method, both in terms of efficiency and precision. The repeatability of the proposed approach, expressed as relative standard deviation, was 0.9%; the within-laboratory reproducibility was 1.3%. Qualitative analysis of the extracted fat showed that the application of ultrasound does not change the composition of the oil.

A screening method for linear alkylbenzene sulfonates (LAS) in sediments has been developed. Soxhlet extraction with water assisted by focused microwaves provides recoveries better (>90%) than obtained by conventional Soxhlet extraction (70-80%). Coupling of the extractor with an on-line preconcentration/derivatization/detection manifold through a flow injection (FI) interface allows a fully automated screening approach. A yes/no answer can be obtained in less than 2 h (for the whole analytical process), a short time compared with the at least 24 h of Soxhlet extraction (without final detection). Due to the use of water as leaching agent, the proposed method is environmentally friendly.

The three operational modes of pressurised liquid extraction (PLE) (namely, static, dynamic and static-dynamic) have been applied for the extraction of nitrated polycyclic aromatic hydrocarbons from both spiked and natural contaminated soils. A comparison of the three modes in terms of experimental set-up used, extraction time needed for total removal of the analytes and precision has been carried out. The use of a flow-injection manifold as interface between every pressurised extractor and a filtration-preconcentration system has allowed the partial automation of the proposed approaches. Efficiencies close to 100% have been provided by the three operational modes. However, the static-dynamic mode has been proved as the most suitable alternative providing the shortest extraction time (25 min) versus the static (30 min) and the dynamic (50-70 min) modes. Gas chromatography with MS-MS ion preparation mode has been used providing both high selectivity (no interferences were observed) and sensitivity (detection limits of low pg). The comparison of the proposed approaches with the reference method 3540 of the US Environmental Protection Agency (EPA) has shown that both methods provide similar efficiencies with an important shortening in the extraction time (25-70 min by PLE versus 24 h by the EPA method). The use of water as leaching agent has avoided the use of organic solvents providing an environmentally friendly method.

The application of a new focused microwave-assisted Soxhlet extractor for the extraction of polychlorinated biphenyls from differently aged soils is here presented. The new extractor overcomes the disadvantages of previous devices based on the same principle and enables a fully automated extraction of two samples simultaneously. The variables affecting the extraction step (namely, power of irradiation, irradiation time, extractant volume, extractant composition and number of extraction cycles) have been optimized using experimental design methodology. The optimized method has also been applied to a certified reference material (CRM910-050 "real" contaminated soil) for quality assurance validation. Quantification of the target compounds has been performed by GC with ion-trap MS. The mass spectrometer was operated in the electron-ionization mode, with selected-ion monitoring at m/z 152, 186, 292, 326 and 498. The results obtained have demonstrated that this approach is as efficient as conventional Soxhlet but with a drastic reduction of both extraction time (70 min vs. 24 h for the "real" contaminated soil) and organic solvent disposal, as 75-80% of the extractant is recycled.

An approach for the automated fast extraction of nitrated polycyclic aromatic hydrocarbons (nitroPAHs) from soil, using a focused microwave-assisted Soxhlet extractor, is proposed. The main factors affecting the extraction efficiency (namely: irradiation power, irradiation time, number of cycles and extractant volume) were optimised by using experimental design methodology. The reduction of the nitro-PAHs to amino-PAHs and the derivatisation of the reduced analytes with heptafluorobutyric anhydride was mandatory prior to the separation-determination step by gas chromatography--electron-capture detection. The proposed approach has allowed the extraction of these pollutants from spiked and "real" contaminated soils with extraction efficiencies similar to those provided by the US Environmental Protection Agency methods 3540-8091, but with a drastic reduction in both the extraction time and sample handling, and using less organic solvent, as 75-85% of it was recycled.

A rapid analytical method for determination of organochlorine pesticide residues in sunflower seeds based on focused microwave-assisted Soxhlet extraction has been developed. The main factors affecting the extraction efficiency--namely microwave power, irradiation time, volume of extractant and number of cycles--were optimized by a two-level factorial fractional design. After extraction, a liquid-liquid extraction and a clean-up step including the use of Florisil macrocolumns were required prior to injection of the extracts into the chromatograph in order to isolate the pesticide residues from the lipid fraction of the original extract. The MS-MS ion preparation mode was selected due to the high sensitivity and selectivity it provides. Seed samples were used collected near a crop subjected to aerial pesticide application. Residues of hexachlorocyclohexane isomers and endosulfan were found in the seeds although they were not subjected to pesticide application, thus showing the spray-drift contamination. The validation of the proposed approach was carried out by comparison with the ISO 659-1988 reference extraction method obtaining similar, or even better efficiencies by the proposed approach.

The coupling of a static pressurized liquid extractor to a flow injection manifold has allowed real-time on-line fluorescence monitoring of the polycyclic aromatic hydrocarbons (PAHs) extracted from environmental solid samples, which can be used for either screening or semiquantitative purposes. Sodium dodecyl sulfate (SDS) was added to the water for favoring the extraction of the low-polar analytes. Different solid samples such as sandy soil, river sediments, trout and sardine spiked with the target PAHs were subjected to several 15-min static extraction cycles with SDS-water, at 200 degrees C. The results obtained demonstrated that fluorometric monitoring of static pressurized liquid extraction constitutes an approach as efficient as conventional Soxhlet for the extraction of PAHs from solid samples but with the following positive features: (a) drastic reduction of the extraction time as the extraction kinetics can be monitored and thus the end of the leaching step determined independently of the sample matrix; (b) use of water as extractant thus given place to an environmentally friendly method; and (c) coupling of static extraction to subsequent dynamic steps. The method has been applied to a certified reference material (CRM 524, BCR, industrial soil/organics) for quality assurance/validation. The total content of each analyte was determined by HPLC-fluorimetric detection.

Subcritical water extraction has been coupled with filtration, preconcentration and chromatographic analysis for the determination of acid herbicides in different types of soil. Two experimental designs were used for the optimization of the leaching step. The use of water as extractant in the continuous mode at a flow-rate of 1 ml/min and 85 degrees C was sufficient for quantitative extraction of the analytes. A static extraction time was unnecessary for reducing the extraction time to 1 h. A minicolumn containing C18-Hydra as sorbent proved an excellent material for the quantitative preconcentration of the herbicides prior to individual chromatographic separation. A flow-injection manifold was used as interface for coupling the four steps, thus allowing automation of the whole analytical process. Recoveries of the target analytes ranged between 94.2 and 113.1%, and repeatabilities, expressed as relative standard deviations, were between 0.61 and 6.83%.

A study of the feasibility of a focused microwave-assisted Soxhlet device for the extraction of N-methylcarbamates from soil has been performed. The main factors contributing to the extraction efficiency-namely microwave power, irradiation time and number of cycles--were optimized by means of a two-level full factorial design. The extracts were analysed by HPLC-post-column fluorescence derivatization-detection at excitation wavelength 340 nm and emission wavelength 445 nm. The method has allowed the extraction of carbamate pesticides from contaminated soil with quantitative recoveries, similar to those provided by the US Environmental Protection Agency method 8318, without degradation of the target compounds during the extraction and using less organic solvent, as 75-80% of the extractant was recycled.

An overview of the different extraction procedures of fat-soluble vitamins from human fluids, foods and pharmaceutical preparations is presented. Methods using organic solvent extraction (both liquid-liquid and solid-liquid extraction), supercritical fluid extraction and solid-phase extraction for the different types of both vitamins and matrices are discussed.

Splenic marginal zone lymphoma (SMZL) has recently been recognized in the World Health Organization classification of hematological diseases as distinct type of non-Hodgkin's lymphoma. In contrast to the well-established chromosomal changes associated with other B-cell non-Hodgkin's lymphoma, few genetic alterations have been found associated with SMZL. The aim of our study was to analyze by comparative genomic hybridization (CGH) the chromosomal imbalances in 29 patients with SMZL and to correlate these findings with clinical and biological characteristics and patient outcome. In 21 cases, cytogenetic studies were simultaneously performed. Most of the patients (83%) displayed genomic imbalances. A total of 111 DNA copy number changes were detected with a median of four abnormalities per case (range, 1 to 12). Gains (n = 92) were more frequent than losses (n = 16), while three high-level amplifications (3q26-q29, 5p11-p15, and 17q22-q25) were observed. The most frequent gains involved 3q (31%), 5q (28%), 12q and 20q (24% each), 9q (21%), and 4q (17%). Losses were observed in 7q (14%) and 17p (10%). SMZL patients with genetic losses had a shorter survival than the remaining SMZL patients (P < 0.05). In summary, chromosomal imbalances in regions 3q, 4q, 5q, 7q, 9q, 12q, and 20q have been detected by CGH in SMZL. Patients with SMZL displaying genetic losses by CGH had a short survival.

We have found two murein hydrolases (LytB and LytC) tightly bound to the cell envelope that have completely changed the domain building plan previously reported for the murein hydrolases of Streptococcus pneumoniae. The active center of LytB and LytC is located at the C-terminal, whereas the binding domain is at the N-terminal. LytC has been characterized as the first lysozyme of S. pneumoniae and behaves as an autolysin at 30 degrees C. LytB appears as the main hydrolase responsible for cell separation since inactivation of lytB leads to the formation of long chains of more than 100 cells. These findings indicate that genetic adaptation of mobile domains is extremely efficient in pneumococcus.

Background: Atypical chronic myeloid leukemia (aCML) is an infrequent chronic myeloproliferative disorder characterized by leukocytosis, absence of Philadelphia chromosome or BCR-ABL rearrangement, and marked myeloid dysplasia. Some cases have an absolute monocytosis but can be distinguished from chronic myelomonocytic leukemia (CMML) by the presence of a higher percentage (> 15%) of circulating immature granulocytes., Patients and Methods: In a series of 11 patients with a diagnosis of aCML according to the FAB proposals we have analyzed the most relevant clinical, hematological and cytogenetic characteristics., Results: The median age was 65 years (16-84). All but one case showed, at time of diagnosis, leukocytosis (median WBC was 36 x 10(9)/l), 55% had moderate anemia and 36% had thrombocytopenia. Most cases had marked dysplasia, particularly in the granulocytic lineage (82% of the cases), and all cases showed bone marrow red hypoplasia. Cytogenetic abnormalities were present in 9 out of the 11 patients. Trisomy 8 was observed in three cases and other clonal chromosomal abnormalities included deletions of 5q, 13q, 17p, 12q, and 11q as well as a t(6;8)(p23;q22) translocation. Fluorescence in situ hybridization (FISH) studies failed to demonstrate ETV-6 gene involvement. The median survival time from diagnosis was only 14 months (range 3-56 months)., Conclusions: These data suggest that aCML is a rare disease which is characterized by leukocytosis, with dysgranulopoiesis, BM erythroid hypoplasia, chromosomal, though not recurrent, abnormalities and poor prognosis.

We evaluate the acute hemodynamic and neurohormonal effects of losartan in 15 patients with symptomatic chronic heart failure (CHF), mean age 72+/-8 years, which were classified in two subgroups: (A) Patients with left ventricular ejection fraction (LVEF)< or =0.35 (n = 7); (B) subjects with LVEF>0.35 (n = 8). Sympathetic reactivity (blood pressure, heart rate and plasma norepinephrine) and plasma endothelin-1 (ET-1) were evaluated by a cold pressor test (CPT). Single doses of losartan (50 mg p.o.) lowered delta DBP in both subgroups (A, 8+/-9 to 0+/-5 mm Hg, P<0.05; B, 10+/-6 to 3+/-4 mm Hg, P<0.05) and attenuated the rise of HR in patients with mild (4+/-6 to -1+/-2 bpm, P<0.05) but not with severe (4+/-5 to 2+/-5 bpm, n.s.) impairment of left ventricular function. Losartan blunted the response (delta) of PNE during CPT (A, 142+/-131 to 10+/-74 pg/ml, P<0.05; B, 129+/-72 to 1+/-144 pg/ml, P<0.01). A significant rise in plasma ET-1 was observed during CPT in patients from subgroup B (0.64+/-0.40 to 0.81+/-0.40 fmol/ml, P<0.05) but not in patients with LVEF< or =0.35 (1.79+/-0.44 to 1.51+/-0.66 fmol/ml, n.s.). Losartan attenuated the rise in ET-1 during CPT in patients with LVEF>0.35 (delta ET-1 0.17+/-0.86 to 0.03+/-0.11 fmol/ml, P<0.05), with no significant changes in subgroup A. Acute effects of losartan were characterized by a more favorable hemodynamic and neurohumoral response in patients with chronic heart failure and preserved systolic ventricular function related to subjects with lower ejection fractions.

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve the selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinity interaction between the poly-His tail and a single chelate moiety seems to be the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each ml of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor).

Mantle cell lymphomas (MCLs) are characterized by 11q13 chromosomal translocations and cyclin D1 overexpression. The secondary genetic and molecular events involved in the progression of these tumors are not well known. In this study, we have analyzed 45 MCLs (32 typical and 13 blastoid variants) by comparative genomic hybridization (CGH). To identify the possible genes included in the abnormal chromosome regions, selected cases were analyzed for P53, P16(INK4a), RB, C-MYC, N-MYC, BCL2, BCL6, CDK4, and BMI-1 gene alterations. The most frequent imbalances detected by CGH were gains of chromosomes 3q (49%), 7p (27%), 8q (22%), 12q (20%), 18q (18%), and 9q34 (16%) and losses of chromosomes 13 (44%), 6q (27%), 1p (24%), 11q14-q23 (22%), 10p14-p15 (18%), 17p (16%), and 9p (16%). High-level DNA amplifications were identified in 11 different regions of the genome, predominantly in 3q27-q29 (13%), 18q23 (9%), and Xq28 (7%). The CGH analysis allowed the identification of regional consensus areas in most of the frequently involved chromosomes. Chromosome gains (P =. 02) and losses (P =.01) and DNA amplifications (P =.015) were significantly higher in blastoid variants. The significant differences between blastoid and typical tumors were gains of 3q, 7p, and 12q, and losses of 17p. CGH losses of 17p correlated with P53 gene deletions and mutations. Similarly, gains of 12q and high-level DNA amplifications of 10p12-p13 were associated with CDK4 and BMI-1 gene amplifications, respectively. One of 2 cases with 8q24 amplification showed C-MYC amplification by Southern blot. Alterations in 2p, 3q, 13, and 18q were not associated with N-MYC, BCL6, RB, or BCL2 alterations, respectively, suggesting that other genes may be the targets of these genetic abnormalities in MCLs. Increased number of gains (0 v 1-4 v >4 gains per case) (P =.002), gains of 3q (P =.02), gains of 12q (P =.03), and losses of 9p (P =. 003) were significantly associated with a shorter survival of the patients. These results indicate that an increased number of chromosome imbalances are associated with blastoid variants of MCLs and may have prognostic significance.

This study was designed to test the hypothesis that the properties of novel 1,4-dihydropyridine PCA50941 could favor the recovery of cardiogenic shock. Coronary blood flow (CBF), measured with an electromagnetic flow probe placed on the left circumflex coronary artery, systemic arterial pressure and heart rate were recorded in 24 anesthetized goats; left ventricular pressure and dP/dt were also recorded in 19 of these goats. Under control conditions, intracoronary injections in 5 goats of PCA50941 (10-120 microg) caused smaller reductions of CBF than those of Bay K 8644 (0.3-10 microg) (the reduction of CBF by 120 microg PCA50941 was 25% and that by 10 microg Bay K 8644 was 43%), and i.v. infusions in 4 goats of PCA50941 (10-300 microg/min) did not modify CBF nor the other hemodynamic variables recorded, whereas i.v infusion of Bay K 8644 (10-30 microg/min) reduced CBF by 20% and increased arterial pressure, left ventricular pressure and dP/dt. During control conditions and endothelin-induced cardiogenic shock, respectively, the values for 15 goats were: for CBF, 33+/-4 vs. 16+/-4 ml/min; for mean arterial pressure, 88+/-4 vs. 60+/-5 mm Hg; for left ventricular systolic pressure, 102+/-5 vs. 75+/-4 mm Hg; for dP/dt, 1453+/-147 vs. 925+/-101 mm Hg/s (all P<0.05), and for heart rate, 77+/-6 vs. 81+/-6 beats/min (P>0.05). Intravenous infusion of PCA50941 (100 microg/min) reversed the hemodynamic variables from the shock state to control values within 20 min in 5 of 6 animals, whereas i.v. administration of Bay K 8644 (10-30 microg/min) was not effective in 4 of 5 animals, and the vehicle (DMSO) was not effective in none of 4 animals in reversing the hemodynamic shock state. Therefore, it is suggested that PCA50941, a novel 1,4-dihydropyridine, has a cardiovascular profile that might be suitable for treating cardiogenic shock states.

The hpa cluster of Escherichia coli W ATCC 11105 encodes the enzymes involved in the catabolism of 4-hydroxyphenylacetate (4-HPA). The catabolic genes are organized in two operons, the hpaBC operon, which produces the hydroxylase activity, and the meta operon, which encodes the enzymes that cleave the aromatic ring and allows its further metabolism. Using a monocopy or multicopy lacZ reporter system, we have demonstrated that the hydroxylase genes are transcribed from the PBC promoter which is positively regulated by the hpaA gene product. HpaA is activated by 4-HPA, 3-HPA, or phenylacetate and represents a novel member of the AraC/XylS family of regulators that recognizes aromatic effectors. The -35 box of the PBC promoter clearly deviates from the consensus sigma 70 promoters of E. coli, but upstream of this box we observed two direct repeats, a common characteristic of promoters regulated by the AraC family of proteins. The hpaA gene, which appears to form a transcriptional unit with the putative hpaX transport gene, is also expressed from an alternative promoter that is located within the hpaX structural sequence. On the basis of these findings, we propose a working model for the regulation of the unique aromatic catabolic pathway thus far characterized at a molecular level in E. coli. This regulatory circuit opens a new scenario for the development of novel microbiological tools for environmental purposes.

In contrast with the general thought that penicillin G acylases (PGAs) were only able to hydrolyse amides or esters of higly hydrophobic acids, we have demonstrated that the PGA from Kluyvera citrophila catalysed the hydrolysis of 4-nitrophenyl esters of acetic, propionic, butyric and valeric acids. Values of kcat. and kcat./Km were greatest for the first compound and less than values for benzylpenicillin by factors of 30 and 7, respectively. 4-Nitrophenyl acetate was hydrolysed faster than 2-nitrophenyl acetate but slower than phenyl acetate. The pH dependence of the reaction was similar to that of benzylpenicillin. Several experiments showed that hydrolysis of 4-nitrophenyl acetate was not catalysed by contaminating esterase activity. The implications for the structural basis of substrate binding are discussed. These substrates open, for the first time, a way to investigate the kinetic parameters of PGA at the presteady-state and provides a new perspective about the role of PGA in nature.

We report a very unusual case of a patient with recurrent massive PEs eight years after mediastinal radiotherapy for HD, in which evidence of lymphomatous recurrence could not be demonstrated. The diagnosis of PE as a complication of radiation is presumptive, and other disorders causing PE must be excluded. This condition requires symptomatic treatment and a close follow-up of the patient.

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.

A new lytic enzyme isolated from Streptococcus pneumoniae has been purified to electrophoretical homogeneity. The enzyme, showing a Mr of 64000, has been characterized as an endo-beta-1,4-N-acetylglucosaminidase that requires choline in the teichoic acid of the cell wall substrate for catalytic activity. In vivo experiments demonstrate that the glucosaminidase behaves as an autolytic enzyme.

The mutant gene lyt-4 of the autolysin-defective mutant R6ly4-4 of Streptococcus pneumoniae, which synthesized a temperature-sensitive autolytic enzyme, has been cloned in Escherichia coli. The nucleotide defect of the lyt-4 mutation has been characterized as a CG to TA transition. This transition causes the appearance of a glutamic acid instead of a glycine in the amino acid sequence of the autolysin, altering the hydropathic profile of the protein. This alteration might explain the observed thermosensitivity of the mutated autolytic enzyme. The present work represents the first molecular characterization of a mutation in the structural gene of a bacterial autolysin.