Comparación entre la prostatectomía con preservación del espacio de Retzius y la prostatectomía radical estándar asistida por robot para el cáncer de próstata / Comparison of Retzius-Sparing versus Standard Robot-Assisted Radical Prostatectomy for Prostate Cancer

Introducción y objetivos Nuestro objetivo fue comparar los resultados oncológicos y funcionales del primer año tras la prostatectomía radical asistida por robot con preservación del espacio de Retzius (PRAR-PR) y la prostatectomía radical estándar asistida por robot (PRAR). Materiales y métodos Se incluyeron 88 pacientes sometidos a prostatectomía radical robótica entre 2018-2019. Se compararon los resultados oncológicos y funcionales de los pacientes que recibieron PRAR-PR o PRAR durante un periodo mínimo de seguimiento de un año. La potencia se evaluó con la puntuación del cuestionario de salud sexual del varón (Sexual Health Inventory for Men) y se definió como una erección con rigidez suficiente para la penetración. La continencia se definió como la ausencia de incontinencia urinaria sin compresa de seguridad. Los pacientes fueron evaluados en el primer mes y trimestralmente durante el resto del seguimiento. Resultados Se incluyeron 46 pacientes en el grupo PRAR-PR y 42 en el grupo PRAR. El tiempo de retirada del catéter fue menor en el grupo PRAR-PR (12 vs. 14 días, p=0,001). En la consulta del primer mes 41 pacientes (89%) eran continentes (no utilizaban compresas) en el grupo PRAR-PR, mientras que 25 pacientes (59%) eran continentes en el grupo PRAR (p=0,001). Los pacientes sometidos a PRAR-PR lograron una recuperación más rápida de la continencia urinaria (Log-rank, p=0,001). Tras un año de seguimiento 43 pacientes (93%) del grupo PRAR-PR y 38 (90%) del grupo PRAR eran continentes (p=0,6). Las tasas de potencia fueron del 38,7% en el grupo PRAR-PR y del 34,4% en el grupo PRAR (p=0,28). No se observaron recurrencias bioquímicas en ningún grupo. Conclusiones La PRAR-PR es una técnica factible, que proporciona un retorno temprano de la continencia. Sin embargo, las tasas globales de continencia fueron similares al final del periodo de un año de seguimiento (AU)

Introduction and objectives We aim to compare the first year oncological and functional outcomes of Retzius-sparing robot-assisted radical prostatectomy (Rs-RARP) and standard robot-assisted radical prostatectomy (RARP). Materials and methods Eighty-eight patients who underwent robotic radical prostatectomy between 2018-2019 were included. We compared the minimum one-year follow-up results of patients who underwent Rs-RARP or RARP in terms of oncological and functional outcomes. Potency was assessed with the Sexual Health Inventory for Men (SHIM) score and was defined as an erection sufficient for penetration. Continence was defined as the absence of urinary incontinence with no safety pad. Patients were evaluated in the first month of follow-up and subsequently, every three months. Results Forty-six patients in Rs-RARP and 42 patients in the RARP group were enrolled. Catheter removal time was shorter in the Rs-RARP group (12 vs. 14 days, P=.001). At the 1st month visit, 41 patients (%89) were continent (no pads) in the Rs-RARP group while 25 patients (%59) were continent in the RARP group (P=.001). Patients who underwent Rs-RARP achieved faster recovery of urinary continence (Log-rank, P=.001). After one year of follow-up, 43 patients (93%) in Rs-RARP group and 38 patients (90%) in RARP group were continent (P=.6). Potency rates were 38.7% in Rs-RARP and 34.4% in RARP group (P=.28). There were no cases of biochemical recurrence in any group. Conclusions Rs-RARP is a feasible technique, providing early return of continence. However, overall continence rates were similar at the end of the one-year follow-up (AU)

Comparison of Retzius-sparing versus standard robot-assisted radical prostatectomy for prostate cancer.

INTRODUCTION AND OBJECTIVES: We aim to compare the first year oncological and functional outcomes of Retzius-sparing robot-assisted radical prostatectomy (Rs-RARP) and standard robot-assisted radical prostatectomy (RARP). MATERIALS AND METHODS: Eighty-eight patients who underwent robotic radical prostatectomy between 2018-2019 were included. We compared the minimum one-year follow-up results of patients who underwent Rs-RARP or RARP in terms of oncological and functional outcomes. Potency was assessed with the Sexual Health Inventory for Men (SHIM) score and was defined as an erection sufficient for penetration. Continence was defined as the absence of urinary incontinence with no safety pad. Patients were evaluated in the first month of follow-up and subsequently, every three months. RESULTS: Forty-six patients in Rs-RARP and 42 patients in the RARP group were enrolled. Catheter removal time was shorter in the Rs-RARP group (12 vs. 14 days, p = 0.001). At the 1st month visit, 41 patients (%89) were continent (no pads) in the Rs-RARP group while 25 patients (%59) were continent in the RARP group (p = 0.001). Patients who underwent Rs-RARP achieved faster recovery of urinary continence (Log-rank, p = 0.001). After one year of follow-up, 43 patients (93%) in Rs-RARP group and 38 patients (90%) in RARP group were continent (p = 0.6). Potency rates were 38.7% in Rs-RARP and 34.4% in RARP group (p = 0.28). There were no cases of biochemical recurrence in any group. CONCLUSIONS: Rs-RARP is a feasible technique, providing early return of continence. However, overall continence rates were similar at the end of the one-year follow-up.

Maturation of bovine oocytes under low culture temperature decreased glutathione peroxidase activity of both oocytes and blastocysts.

It is known that the basic variable in the cellular environment is temperature and low temperature decreases cellular metabolism rate. Also, low cellular metabolic activity reduces oxidative stress, resulting in low ROS production. The aim of this study was therefore to investigate the effect of 36.5°C (low) and 38.5°C (conventional) incubation temperatures during IVM on glutathione peroxidase activity of oocytes and blastocysts following fertilization. Bovine oocytes were matured in medium-199 for 22 hours at either 36.5°C or 38.5°C and they were subjected to in vitro fertilization (IVF). Putative zygotes were then transferred randomly into SOFaa embryo culture media with or without antioxidant (a mixture of GSH and SOD) until development to the blastocyst stage. Glutathione peroxidase enzyme (GSH-Px) activity was lower (p⟨0.05) in oocytes matured at low temperature than those of conventional temperature. Similarly, GSH-Px activity was lower (p⟨0.05) in blastocysts, which were obtained from oocytes matured at low temperature and cultured in antioxidants-supplemented embryo media. The GSH-Px activity of blastocysts, obtained from oocytes matured in low temperature, cultured in antioxidants-free embryo media was similar to blastocysts obtained from oocytes matured in conventional temperature, cultured in antioxidants-supplemented embryo media. The results of the present study show that decreasing the in vitro maturation temperature decreases antioxidant enzyme activity in both oocyte and blastocyst. Additionally, maturation of bovine oocytes at 36.5°C incubation temperature may provide an optimal thermal condition for the enzymatic antioxidant system of both oocytes and blastocyst.

Real-time assessment of the superovulatory effect of FSH and eCG with laparoscopy at different seasons in Akkaraman ewes.

Conventional methods for determining the reproductive performance of sheep bred either after estrus synchronization during the breeding season or after induction of estrus/ovulation during the non-breeding season take a long time and may give misleading results due to the effect of environmental factors. Laparoscopic observations allow real-time monitoring of ovarian activity around estrus or ovulation. This study was aimed at assessing the superovulatory effects of follicle-stimulating hormone (FSH) and equine chorionic gonadotropin (eCG) treatments by laparoscopy during breeding (September-November, n=12) and non-breeding (April-June, n=12) seasons in Akkaraman sheep. In both seasons, after CIDR withdrawal, the ewes were injected either with 600 IU eCG or 300 µl (20 mg/ml) FSH twice at 12 hour intervals. Plasma P4, E2 and LH concentrations were determined at the time of intra-vaginal CIDR insertion (day 0) and then at its withdrawal (day 12), followed by 3 and 6 days of eCG or FSH injections. After 3 (first observation) and 6 (second observation) days of hormone injections, laparoscopy was performed to record ovarian activity in both seasons. The eCG increased (p⟨0.05) the numbers of large follicles (first observation) and CL (first and second observations) in the breeding season compared to FSH treatment. CL, small-moderate and large follicle numbers of eCG treated ewes were higher (p⟨0.05) than those of FSH at both observations in the non-breeding season. In the breeding season, eCG treated ewes had higher (p⟨0.05) plasma P4 (3 and 6 days after hormones injections) and E2 (3 days after hormones injections) concentrations than those of FSH. In conclusion, the results of the present study indicate that treatment with eCG during the non-breeding season can support ovarian activity, and thus increase ovulation rate and plasma hormone concentrations around induced estrus/ovulation in Akkaraman ewes.

Prevalence of childhood ocular morbidity in a peri-urban setting in Bangladesh: a community-based study.

OBJECTIVES: To test a model of integrated pediatric eye care delivery and examine the prevalence and factors associated with childhood ocular morbidity in a peri-urban setting in Bangladesh. STUDY DESIGN: Cross-sectional, population-based study. METHODS: The study was conducted in three phases among children aged ≤15 years. Trained community health workers (CHWs) conducted awareness intervention and identified children with ocular problems. These children were then referred to the base hospital for examination and treatment by ophthalmologists. A pediatric ophthalmologist further examined the children with complicated eye diseases and ensured treatment at a tertiary public eye hospital. Awareness, referral patterns, and health-seeking behavior were also examined. All data were analyzed statistically using Statistical Package for Social Sciences. RESULTS: CHWs screened 33,549 eligible children and identified 1887 cases with ocular morbidity. The prevalence of ocular morbidity and childhood blindness were 5.63% (95% confidence interval [CI] = 5.27-6.16) and 0.060% (95% CI = 0.03-0.11), respectively. The most commonly observed ocular morbidities were refractive error (3.24%; 95% CI = 3.11-3.45), allergic eye conditions (1.2%; 95% = CI 0.74-1.27), and nasolacrimal duct obstruction (0.52%; 95% CI = 0.25-0.74). Blindness was more frequently seen in children aged <5 years than in those aged 5-15 years (χ2 = 7.25; P = 0.007). The causes of blindness were corneal opacity, congenital eye anomaly, cataract, retinopathy of prematurity, and retinoblastoma. The prevalence of ocular morbidity was higher among older children, boys, children with low parental education and income, and children from households dwelling in slums. CONCLUSIONS: This study demonstrated that in a setting where screening and treatment for vision problems remain low, ocular morbidity among children could be easily identified through well-designed community-based screening programs involving appropriately trained CHWs. Community mobilization, awareness, and early detection of childhood eye diseases, with effective referral mechanisms for accessing appropriate care, are crucially important to improve service delivery.

The effect of maternal nutrition level during the periconception period on fetal muscle development and plasma hormone concentrations in sheep.

The effect of maternal nutrition level during the periconception period on the muscle development of fetus and maternal-fetal plasma hormone concentrations in sheep were examined. Estrus was synchronized in 55 Karayaka ewes and were either fed ad libitum (well-fed, WF, n=23) or 0.5×maintenance (under-fed, UF, n=32) 6 days before and 7 days after mating. Non-pregnant ewes (WF, n=13; UF, n=24) and ewes carrying twins (WF, n=1) and female (WF, n=1; UF, n=3) fetuses were removed from the experiment. The singleton male fetuses from well-fed (n=8) and under-fed (n=5) ewes were collected on day 90 of gestation and placental characteristics, fetal BWs and dimensions, fetal organs and muscles weights were recorded. Maternal (on day 7 after mating) and fetal (on day 90 of pregnancy) blood samples were collected to analyze plasma hormone concentrations. Placental characteristics, BW and dimensions, organs and muscles weights of fetuses were not affected by maternal feed intake during the periconception period. Maternal nutrition level did not affect fiber numbers and the muscle cross-sectional area of the fetal longissimus dorsi (LD), semitendinosus (ST) muscles, but the cross-sectional area of the secondary fibers in the fetal LD and ST muscles from the UF ewes were higher than those from the WF ewes (P<0.05). Also, the ratio of secondary to primary fibers in the ST muscle were tended to be lower in the fetuses from the UF ewes (P=0.07). Maternal nutrition level during the periconception period did not cause any significant changes in fetal plasma insulin and maternal and fetal plasma IGF-I, cortisol, progesterone, free T3 and T4 concentrations. However, maternal cortisol concentrations were lower while insulin concentrations were higher in the WF ewes than those in the UF ewes (P<0.05). These results indicate that the reduced maternal feed intake during the periconception period may alter muscle fiber diameter without affecting fiber types, fetal weights and organ developments and plasma hormone concentrations in the fetus.

The effect of maternal nutritional status during mid-gestation on placental characteristics in ewes.

The aim of this study was to determine the effects of maternal nutritional status during mid-gestation on placental characteristics in ewes. Time of estrus of 3-5 years old Karayaka breed ewes was synchronized and mating was monitored to determine the day 0 of gestation. The ewes had similar body weights (47.8±0.7kg) and loin eye muscle values (thickness; 20.9±1.0mm and fat thickness; 4.7±0.5mm) at mating. The ewes were allocated into two treatment groups at day 30 of gestation; under-fed (UF; n=12) and well-fed (WF; n=13) groups. The ewes in UF group were fed with a diet to provide 50% of their daily requirement from day 30 to day 80 of gestation and 100% of their daily requirement during the rest of the gestation period. The ewes in WF group were fed at least 100% of their daily requirement throughout gestation. The singleton bearing ewes in the UF group had a lesser (P<0.05) placental weight (354.1 compared with 378.3g), average cotyledon weight (1.50 compared with 1.82g) and lamb birth weight (3.8 compared with 4.2kg) than singleton bearing ewes in the WF group. There were positive correlations between placental weight and lamb birth weight (r=.469; P<0.05), placental weight and average cotyledon weight (r=.695; P<0.01), average cotyledon weight and lamb birth weight (r=.742; P<0.01) and placental efficiency and cotyledon density (r=.853; P<0.01) for ewes in WF group. Additionally, the pattern of weight gain/loss was different (P<0.05) between the two groups. Ewes in UF group lost body weight progressively from day 30 of gestation until day 80. The results of present study show that under-feeding of ewes during mid-gestation may cause an insufficient placental development and hence alter fetal development resulting in a reduced birth weight from singleton pregnancies.

X-ray imaging of differential vascular density in MMP-9-/-, PAR-1-/+, hyperhomocysteinemic (CBS-/+) and diabetic (Ins2-/+) mice.

Although protease activated receptor-1 (PAR-1) and matrix metalloproteinase-9 (MMP-9) play significant role in vascular remodelling in hyperhomocysteinemia (HHcy due to cystathionine beta synthase deficiency, CBS-/+) and diabetes, mechanism remains nebulous. We hypothesized that differential vascular density and remodelling in different vascular beds in HHcy and diabetes were responsible for an adaptive metabolic homeostasis during the pathogenesis. To test this hypothesis, vascular density in mice lacking PAR-1, MMP-9, CBS and Insulin-2 gene mutant (Ins2-/+, Akita) was measured and compared with wild type (WT, C57BL/6J) mice. The vascular density was detected by x-ray angiography using KODAK 4000 MM image station, using barium sulphate as contrasting agent. The % vascular density in the hearts of WT, CBS-/+ (HHcy), MMP-9-/-, PAR-1-/+ and Ins2-/+ (type-1 diabetes) was 100 ± 2.8, 85 ± 3.3, 90 ± 3.3, 95 ± 3.8 and 73 ± 1.7, respectively. The vascular density in CBS-/+ and Akita hearts decreased while it was increased in lungs of CBS-/+ and MMP-9-/-.There was decreased vascular density in liver and kidney of Akita mice. Vascular density in brain, kidney and mesentery was decreased in CBS-/+ mice. These findings support the notation that metabolic derangement in diabetes and HHcy causes the chronic decline and/or rarefaction in vascular density.

Homocysteine enriched diet leads to prolonged QT interval and reduced left ventricular performance in telemetric monitored mice.

BACKGROUND AND AIMS: Homocysteine (Hcy) is a sulfur-containing, non-protein amino acid produced in the metabolic pathway of methionine. Hyperhomocysteinemia is associated with cerebro- and cardiovascular disease in industrialized countries, mostly resulting from protein rich diet and sedentary life style. Matrix metalloproteinases are involved in cardiac remodeling, leading to degradation of intercellular junctions, cardiac connexins and basement membranes. The study was designed to investigate the relationship between Hcy, cardiac remodeling, cardiac performance, and rhythm disturbances in an animal model of hyperhomocysteinemia. We tested the hypothesis that induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 leads to connexin 40, connexin 43, connexin 45 expression changes contributing to decreased cardiac performance and disturbed atrioventricular conduction. METHODS AND RESULTS: Hcy was added to drinking water of male C57/BL6J mice to achieve moderate Hcy blood levels. ECG was monitored in conscious mice with a telemetric ECG device; echocardiography was used for assessment of left ventricular function. Immunoblotting was used to evaluate matrix metalloproteinase-2, matrix metalloproteinase-9, connexin 40, connexin 43, and connexin 45 expression in cardiac tissue. Animals fed Hcy showed significant prolongation of QRS, QTc, and PR intervals along with reduced left ventricular function. Western blotting showed increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45. CONCLUSION: Hcy has been identified as a nutritional factor contributing to cardiovascular disease. Cardiac remodeling induced by matrix metalloproteinase-2 and matrix metalloproteinase-9 and decreased expression of connexin 40, 43, and 45 appears to play a role in the pathomechanism of atrioventricular conduction delay and ventricular dilatation in hyperhomocysteinemia.

Homocysteine effects classical pathway of GPCR down regulation: Galpha(q/11), Galpha(12/13), G(i/o).

G protein-coupled receptors (GPCRs) are known to modulate intracellular effectors involved in cardiac function. We recently reported homocysteine (Hcy)-induced ERK-phosphorylation was suppressed by pertussis toxin (PTX), which suggested the involvement of GPCRs in initiating signal transduction. An activated GPCR undergoes down regulation via a known mechanism involving ERK, GRK2, beta-arrestin1: ERK activity increases; GRK2 activity increases; beta-arrestin1 is degraded. We hypothesized that Hcy treatment leads to GPCR activation and down regulation. Microvascular endothelial cells were treated with Hcy. Expression of phospho-ERK1 and phospho-GRK2 was determined using Western blot, standardized to ERK1, GRK2, and beta-actin. Hcy was shown to dephosphorylate GRK2, thereby enhancing the activity. The results provided further evidence that Hcy acts as an agonist to activate GPCRs, followed by their down regulation. Hcy was also shown to decrease the content of the following G proteins and other proteins: beta-arrestin1, Galpha(q/11), Galpha(12/13), G(i/o).

Targeted deletion of MMP-9 attenuates myocardial contractile dysfunction in heart failure.

Chronic volume overload (VO) on the left ventricle (LV) augments redox stress and activates matrix metalloproteinase (MMP) which causes the endocardial endothelial-myocyte (EM) disconnection leading to myocardial contractile dysfunction. VO-induced MMP-9 activation impairs cardiac functions, in part by endothelial endocardial apoptosis, but the role of MMP-9 on EM functions remains obscure. We conjecture that chronic VO activates MMP-9 and causes EM uncoupling. Arteriovenous fistula (AVF) was created in genetically identical wild type (WT) mice (FVB/NJ) and MMP-9 knockout mice (MMP-9KO, FVB.Cg-MMP9(tm1Tvu)/J). Sham-operated mice were used as controls. Before experimentation the phenotype analysis of MMP-9KO mice was carried out. In-gel-gelatin zymography for MMP-9 activation was performed on LV homogenates. The EM functions were determined on LV rings using tissue myobath. We report a decrease in MMP-9 activity in left ventricular myocardial extracts in MMP-9 deficient mice after AVF. The responses to drugs affecting cardiac functions (acetylcholine (Ach), nitroprusside and bradykinin) were attenuated in AVF mice suggesting the impairment of EM coupling. Interestingly, the EM functions were restored in the MMP-9 deficient mice after AVF. We suggest a direct cause-and-effect relationship between MMP-9 activation and EM uncoupling in LV myocardium after chronic VO and the possible involvement of MMP-9 in myocardial contractile performance.

Ciglitazone ameliorates homocysteine-mediated mitochondrial translocation and matrix metalloproteinase-9 activation in endothelial cells by inducing peroxisome proliferator activated receptor-gamma activity.

The activation of peroxisome proliferator activated receptor-gamma (PPARgamma) ameliorates the homocysteine (Hcy)-induced matrix metalloproteinase (MMP) by decreasing reactive oxygen species (ROS) production. However, the mechanism by which Hcy induces ROS generation and MMP activation is unclear. We hypothesize that Hcy increases NADH oxidase (Nox-4) and decreases thioredoxin (Trx). This leads to translocation of Nox-4 into the mitochondria and decrease in Trx. In addition, activation of PPARgamma ameliorates the translocation of Nox-4 into mitochondria and MMP-9 activation. Mouse aortic vascular endothelial cells (MVEC) were cultured in the presence or absence of 100 microM Hcy. The cells were pre-treated with ciglitazone (CZ, 150 microM). Activity of PPARgamma activity was measured by electrophoretic mobility shift assay (EMSA) and antibody super shift assay. In situ generation of ROS was measured using 2,7-dichlorofluorescin (DCF) as a probe. The expression of Nox-4 and Trx were measured by quantitative real-time polymerase chain reaction (Q-RT-PCR). The translocation of Nox-4 was measured by 2-D gel analysis. To determine the levels of Nox-4 and Trx, the mitochondria and cytosol were separated and Western blot analysis was preformed. The MMP-9 activity was measured by gelatin-zymography. The results suggested that CZ activated endothelial PPARgamma in the presence of Hcy. Production of ROS was ameliorated by PPARgamma activation. Expression of Nox-4 was increased, while production of Trx was decreased by Hcy. However, the treatment with CZ normalized the levels of Nox-4 and Trx. Nox-4 was translocated into mitochondria in Hcy-treated endothelial cells. This translocation was associated with decreased production of Trx in mitochondria. The treatment with CZ blocked this translocation and increased Trx levels in mitochondria. Hcy-mediated MMP-9 activity was decreased in cells pre-treated with CZ. These results suggest that Hcy increases NADH oxidase and decreases Trx by translocation of Nox-4 to mitochondria. The data show that indeed, activation of PPARgamma ameliorates the mitochondrial translocation of NOX-4 and MMP-9 activation.

Distributed quantum dense coding.

We introduce the notion of distributed quantum dense coding, i.e., the generalization of quantum dense coding to more than one sender and more than one receiver. We show that global operations (as compared to local operations) of the senders do not increase the information transfer capacity, in the case of a single receiver. For the case of two receivers, using local operations and classical communication, a nontrivial upper bound for the capacity is derived. We propose a general classification scheme of quantum states according to their usefulness for dense coding. In the bipartite case (for any dimensions), bound entanglement is not useful for this task.

Purification and preliminary X-ray studies on hen serotransferrin in apo- and holo-forms.

Serum transferrins are monomeric glycoproteins with a molecular mass of around 80 kDa, that transport iron to cells via receptor-mediated endocytosis. Although both serum transferrins (STfs) and ovotransferrins (OTfs) are derived from the same gene in aves, the ovotransferrins do not transport iron in vivo. Crystal structures of OTf have been solved, in contrast no three-dimensional structure of avian STf have been determined as yet. Here we report the purification, crystallization, and preliminary crystallographic studies of the hen STf both in apo- (iron free) and holo- (iron loaded) forms. The hen STf has been purified to homogeneity by hydrophobic interaction chromatography. Both the apo- and holo-forms were crystallized by hanging drop vapor diffusion method at 277 K. The apo-crystals diffract to a resolution of 3.0 A and belong to the space group P4(3)2(1)2 with unit cell parameters a=b=90.5 and c=177.9 A. The holo-crystals diffract to a resolution of 2.8 A and belong to space group P2(1) with a=72.8, b=59.6, c=88.2 A, and beta=95.7 degrees.

Crystal structure of the von Willebrand factor modulator botrocetin.

The binding of von Willebrand factor (vWF) to the platelet receptor, glycoprotein (GP) Ib-IX-V complex, has a key role in the initiation of thrombus formation and is regulated by interactions with extracellular matrix components under the influence of hemodynamic forces. To a certain extent, these effects can be mimicked in vitro by two nonphysiologic modulators, ristocetin and botrocetin. The latter, isolated from the venom of the snake Bothrops jararaca, is a 31-kDa heterodimeric protein that forms a soluble complex with vWF. As an initial step toward understanding the mechanisms that regulate vWF function, we have solved the crystal structure of botrocetin at 1.8 A resolution. Botrocetin exhibits homology with other snake proteins, but contains only one metal binding site as compared to two in Factor IX binding protein and Factor IX/X binding protein and none in flavocetin. A distinctive feature of botrocetin is the presence of a negatively charged surface that may play a role in the association with the vWF A1 domain.

Distinguishability of Bell states.

More than two multipartite orthogonal states cannot always be discriminated if only local operations and classical communication (LOCC) are allowed. We show that four Bell states cannot be discriminated by LOCC, even probabilistically, using the separability properties of a four-party unlockable bound entangled state. Using an existing inequality among the measures of entanglement, we show that any three Bell states cannot be discriminated with certainty by LOCC. Exploiting the inequality, we calculate the distillable entanglement of a certain class of 4 multiply sign in circle 4 mixed states.

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction.

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.

Identification of maturation-inducing steroid in a freshwater perch Anabas testudineus and differential responses of intact follicles and denuded oocytes to cyclic AMP in oocyte maturation.

Postvitellogenic follicles of freshwater perch Anabas testudineus incubated with [(3)H]pregnenolone as exogenous precursor produced several metabolites, including 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and 5 beta-pregnane-3 alpha, 17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P). These were identified by chromatography, microchemical reactions, and crystallization to constant specific activity. Following stimulation with fish (perch) pituitary extract (FPE) there was significant high production of DHP and 5 beta-3 alpha,17 alpha,20 beta-P, concomitant with a high percentage of germinal vesicle breakdown (GVBD). Inhibitor of steroidogenesis (trilostane) and inhibitors of protein synthesis (cycloheximide and actinomycin-D) completely blocked FPE-induced pregnenolone metabolism and oocyte maturation. The effectiveness of various C(21) steroids in inducing GVBD was examined. Results indicate that DHP was the most potent inducer of GVBD than other structurally related C(21) steroids. In intact follicles, FPE-stimulated production of DHP was shown to be mediated through the adenylate cyclase-cAMP pathway. Addition of IBMX or forskolin, which increases the endogenous cAMP level, as well as directly supplementing dbcAMP to the incubation medium, had no inhibitory effect on DHP-induced GVBD in the intact follicles. But all these agents were shown to inhibit GVBD in fully denuded oocytes. This study provides evidence that DHP, produced by postvitellogenic follicles through the adenylate cyclase-cAMP pathway, is the maturation-inducing steroid in freshwater perch and that the role played by cAMP in the induction of GVBD in intact follicles is different from that in the denuded oocytes. J. Exp. Zool. 287:294-303, 2000.

Cryocrystallography of a Kunitz-type serine protease inhibitor: the 90 K structure of winged bean chymotrypsin inhibitor (WCI) at 2.13 A resolution.

The crystal structure of a Kunitz-type double-headed alpha--chymotrypsin inhibitor from winged bean seeds has been refined at 2.13 A resolution using data collected from cryo-cooled (90 K) crystals which belong to the hexagonal space group P6(1)22 with unit-cell parameters a = b = 60.84, c = 207.91 A. The volume of the unit cell is reduced by 5.3% on cooling. The refinement converged to an R value of 20.0% (R(free) = 25.8%) for 11100 unique reflections and the model shows good stereochemistry, with r.m.s. deviations from ideal values for bond lengths and bond angles of 0.011 A and 1.4 degrees, respectively. The structural architecture of the protein consists of 12 antiparallel beta-strands joined in the form of a characteristic beta-trefoil fold, with the two reactive-site regions, Asn38-Leu43 and Gln63-Phe68, situated on two external loops. Although the overall protein fold is the same as that of the room-temperature model, some conformational changes are observed in the loop regions and in the side chains of a few surface residues. A total of 176 ordered water molecules and five sulfate ions are included in the model.

Refined crystal structure (2.3 A) of a double-headed winged bean alpha-chymotrypsin inhibitor and location of its second reactive site.

The crystal structure of a double-headed alpha-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 A resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6(1)22 with cell parameters a = b = 61.8 A and c = 212.8 A. The final model has a good stereochemistry and a root mean square deviation of 0.011 A and 1.14 degrees from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 A led to an understanding of the mechanism of inhibition of the protein against alpha-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve anti-parallel beta-strands with connecting loops arranged in a characteristic beta-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones.