Luteolysis, progesterone, and pregnancy per insemination after modifying the standard 7-day Ovsynch program in Holstein-Friesian and Holstein cows.

Two experiments were conducted with Holstein-Friesian cows in the Republic of North Macedonia and with Holstein cows in Kansas. We hypothesized that 1 dose of PGF2α administered on d 8 (Ov-8×1) instead of d 7 (Ov-7×1) in an Ovsynch program [GnRH-1 (d 0)-7 d-PGF2α-56 h-GnRH-2-16 h-timed artificial insemination (AI)] would increase the proportion of cows with complete luteolysis compared with controls receiving a single dose on d 7. Cows were treated with Ov-7×1 or with Ov-8×1 in experiment 1 (n = 347), using only a single dose of PGF2α. In experiment 2 (n = 452), a third treatment was added (Ov-7×2), in which a second dose of PGF2α was administered on d 8. Progesterone was measured in blood samples collected before the first or only PGF2α administration and 72 h later before insemination. Complete luteolysis was defined as having occurred when progesterone was ≥1 ng/mL before PGF2α and ≤0.3 ng/mL 72 h later (time of AI). Follicles and luteal structures were mapped before GnRH-1 and PGF2α administrations. The results of experiment 1 demonstrated a greater percentage of multiparous cows in OV-8×1 having complete luteal regression compared with multiparous Ov-7×1 cows, whereas treatments were equally effective in primiparous cows, as reflected in the concentrations of progesterone before AI. Furthermore, pregnancy per AI did not differ between treatments. Results in experiment 2 revealed that 99.3% of cows in the Ov-7×2 treatment receiving the second dose of PGF2α had complete luteolysis, regardless of parity, compared with significantly fewer cows in the Ov-7×1 and Ov-8×1 treatments (91.2 and 90.6%, respectively). Neither concentrations of progesterone, which averaged <0.4 ng/mL at AI, nor pregnancy per AI differed among the 3 treatments. In both experiments, when status of luteal function before PGF2α treatment was examined [cows with no corpus luteum (CL) before GnRH-1 but which had formed a new CL in response to ovulation after GnRH-1; cows with an older CL (the same CL that was detected before GnRH-1); or cows with both a new and an older CL], treatments did not differ in causing complete luteolysis. Furthermore, complete luteolysis in experiment 2 did not differ regardless of whether cows had 1, 2, or 3 or more CL before PGF2α administration. Pregnancy per AI did not differ among treatments, indicating that any of the 3 treatments might produce similar pregnancy outcomes with the flexibility of applying either of the 7- or the 8-d treatments.

A rare case of double J stent migration in the kidney.

Double-J stents are among the basic and commonly used tools in urology. There are a lot of complications that can occur during DJ placement. We are presenting 62 years old woman, who was admitted in Urology Department with symptoms of left lumbar pain, irradiating towards inguinal area and hematuria. The computed tomography (CT) scan of abdomen and pelvis defined a propagated DJ stent in the left kidney. Lumenis Holmium laser VersaPulse 100 W was used for resection of the double J stent and after that it was removed from the kidney in pieces.

Physiologic responses to feeding rumen-protected glucose to lactating dairy cows.

It was hypothesized that rumen-protected glucose (RPG) in diets of dairy cows increases concentrations of insulin resulting in greater blood progesterone concentrations because elevated insulin decreases activity of liver enzymes inactivating steroid hormones. Timing of ovulation was synchronized among 64 postpartum Holstein cows using GnRH and PGF2α (Day 0 = ovulation). Cows were milked thrice daily and assigned randomly a basal diet supplemented with 0, 1, 2, or 4 kg of an RPG product in place of corn grain, top-dressed in the diet beginning on Day -3. Blood was collected pre- and post-prandial on Days 0, 2, and 4 to determine plasma glucose and insulin concentrations and daily from Days 2 through 12. Intake of crude protein and energy-soluble carbohydrates increased linearly with dose, whereas starch intake decreased linearly with dose. Neither daily milk yield nor dry matter intake (DMI), energy-corrected milk (ECM), somatic cell count, or percentages of milk fat, protein and lactose on Day 8 differed among dietary treatments. Neither pre- nor post-prandial changes in plasma glucose differed among treatments. In contrast, post-prandial glucose decreased from Days 0 through 4. A change in plasma insulin (post-prandial minus pre-prandial) was detected. Milk urea nitrogen increased linearly with RPG dose. Concentrations of progesterone were unaffected by RPG dose. It is concluded that insulin response to RPG was decreased relative to the control and RPG supplementation linearly increased crude protein intake and milk urea nitrogen with increasing dose, but did not affect concentrations of progesterone, milk yield, or dry matter intake.

Pathways of the dominant follicle after exposure to sub-luteal circulating progesterone concentrations are different in lactating dairy cows versus non-lactating heifers.

With the increased use of different synchronization programs in cattle, attention is given to the progesterone concentration during development of the ovulatory follicle. It has been shown that low peripheral progesterone concentrations during follicular development may lead to decreased fertility. To investigate the effect of low progesterone concentrations on the fate of the dominant follicle, a study was conducted where cycles of dairy cows and heifers were manipulated to induce the development of the first dominant follicle without progesterone (PLACEBO) or under sub-luteal progesterone concentrations from a progesterone releasing intravaginal device (PRID Delta(®)). After insertion of the devices, daily follow up was performed by transrectal ultrasonography to identify and measure follicular development and blood samples were taken to determine the circulating progesterone concentration. Follow up was continued until the ovulation of a follicle occurred. After ovulation, the fate of the first dominant follicle was identified as arrested, atretic or ovulatory. Arrest was defined as persistence of the dominant follicle followed by ovulation whereas atresia was defined as regression of the dominant follicle and subsequent growth and ovulation of a new follicle. During PLACEBO treatment, heifers ovulated earlier and smaller follicles in comparison to cows. During PRID Delta(®) treatment, heifers had greater progesterone concentrations compared to cows and arrest of the dominant follicle occurred more in cows in comparison to heifers. In cycles where the dominant follicle was arrested, the ovulatory follicle was larger in comparison to cycles where the dominant follicle was atretic.

[Indirect standard cardiotocography plus fetal blood sampling versus indirect quantitative cardiotocography--a randomized comparative study in intrapartum monitoring].

INTRODUCTION: In previous papers we proposed ways to improve the diagnostic potential of the "quantitative cardiotocography" computer method, which allowed us to introduce clinical practice guidelines. Using these guidelines we aim to evaluate the effectiveness of quantitative cardiotocography (qCTG) as compared to standard cardiotocography (CTG) and, if necessary fetal blood sampling (FBS). MATERIAL AND METHODS: The prospective study involves 220 pregnant women divided randomly into two groups. All 110 women in the control group were monitored by standard indirect cardiotocography Interpretation of CTG findings was performed according to the latest revision of FIGO classification. We performed FBS in all cases of abnormal or suspect CTG tracings and pH values below 7.20 were indicative for urgent delivery. If pH values were in the range between 7.20 and 7.25, another FBS was carried out after 30 minutes. If pH was above 7.25, FBS was repeated according to CTG evaluation by the attending doctor. All patients in the index group (110 women) were monitored by indirect quantitative cardiotocography (qCTG). Our clinical practice guidelines were used for the interpretation of CTG tracings. Obstetric behavior was strictly based on the recommendations in these guidelines. Outcome measures are: incidence of metabolic acidosis (defined by pH < 7.05 and BE > -12 mmol/l in the umbilical cord artery of the newborn), number of instrumental deliveries and sensitivity/specificity of each method (qCTG and CTG + FBS) in relation to fetal hypoxia (defined by pH < 7.20). RESULTS: In the CTG group, 4.4% of the newborns were affected by metabolic acidosisas opposed to 2.2% in the qCTG group (P > 0.05). The sensitivity/specificity rates for fetal hypoxia were 89/67% (control group) and 97/85% (index group). The number of operative deliveries was 27.25% and 16.35%, respectively (P < 0.05). CONCLUSION: The incidence of metabolic acidosis was comparable in both groups. Indirect quantitative cardiotocography shows much better specificity for fetal hypoxia which results in significantly lower rates of operative deliveries compared to standard indirect cardiotocography combined with fetal blood sampling.

[Structure and function of the cardiotocographic score (CTG-score) calculated by the "quantitative cardiotocography" computer method. Determining the significance of its components for the accuracy of the estimates for the ph of the fetus].

INTRODUCTION: In the last three years "quantitative cardiotocography" has become the main method for fetal monitoring during late pregnancy and birth in Sheynovo hospital - Sofia, Bulgaria. Our previous studies presented opportunities for increasing the diagnostic potential of the methodology. In this paper we offer a new approach to further improve the accuracy of prognostic values for fetal pH during labor. This is achieved by analyzing the individual components of the CTG-score (microfluctuation - OSZ, basic fetal heart rate - FRQ and decelerations - DEC). Several groups of CTG-scores have been formed, according to the composition of the score and the correlation between forecast and actual results for the pH of the fetus. MATERIAL AND METHODS: For each of the stored 171 recordings we compared the CTG-score, produced prior to the delivery, with the pH measured in the umbilical artery (UA) before cutting the umbilical cord. As fetal pH forecast is based strictly on the CTG-score value, the difference between actual and prognostic results for the pH actually shows how accurate is the CTG score itself. We used standard deviation (Std. deviation) to assess this variability. RESULTS: We defined several groups of CTG-score based on its composition and the respective standard deviations. Each group includes CTG-scores with no significant statistical difference between the calculated standard deviations: CTG-score with low (composed of OSZ; Std. Dev. 0.065), satisfactory (composed of OSZ + FRQ and FRQ; Std. dev 0048 and 0044), high (composed of OSZ + DEC and DEC; Std. dev 0032 and 0027) and very high (composed of FRQ + DEC and OSZ + FRQ + DEC; Std. dev. 0019 and 0012) predictive value. We observed a substantial variety in the prognostic results, depending on which components of the CTG-score are involved in the evaluation of pH. CONCLUSION: The composition of the CTG-score seems to be crucial for the accuracy of the prognostic fetal pH values. In order to organize the gathered information it is necessary to develop clinical practice guidelines which would allow us to cohere the obstetric behavior with data readings from the quantitative cardiotocography method.

[Surgical treatment of hepatic echinococcosis--10-year experience].

INTRODUCTION: Especially debatable remains the problem concerning the volume of the surgical treatment of hepatic echinococcosis. At present it varies from radical typical and atypical liver resections, through closed conservative approaches, to minimally invasive methods like PAIR or laparoscopic echinococcectomy. AIM: The aim of the present investigation is to elucidate the problems, occurring during surgical treatment of hepatic echinococcosis and to offer adequate treatment-diagnostic algorithm. This retrospective study summarizes our 10-year experience in a number of debatable topics, concerning the surgical treatment of this socially significant disease. RESULTS: To fulfill the aim, we performed a retrospective clinical study for a period of 10 years. One-hundred-forty-seven patients had been admitted to hospital and underwent surgical treatment for hepatic echinococcosis during that period. One-hundred were males (58%) and 47 (32%)--females. The age of the patients included in the retrospective study varies between 6 and 80 years--(mean age 39.1 +/- 8.9). In 19 patients we found multiple echinococcosis of the liver (2 to 7 cysts). Two cysts--in 7 patients, 3 cysts--in 6 patients, 4 cysts in 1 patient, 5 cysts in 2 patients, 6 cysts in 1 patient and 7 cysts in 2 patients. The right hepatic lobe is three times more frequently engaged than the left one--106 patients with right-sided localization (72.1%) compared to 41 with left-sided (27.9%). Combined echinococcosis is found in 14 patients. Concomitant engagement of liver and spleen is present in 2 patients, peritoneal dissemination--in 7 patients and accompanying lung cyst--in 6 patients. Echinococcectomy with capitonage of the residual cavity is performed in 126 patients, echinococcectomy with external drainage in 4 patients, atypical liver resection in 8 patients, echinococcectomy via thoracofrenectomy approach in 6 patients and combined surgical interventions with spleen removal in 3 patients. In their majority the complications are not serious and life-threatening or with permanent consequences to the patient. Severe complications demanding active surgical intervention occur in approximately 4% of the treated patients. Our results are comparable with the ones of leading national and foreign centers and confirm the correctness of our treatment. The average hospital stay is 12 days. We have no lethal cases for the study period. CONCLUSION: Based on our experience, we consider that echinococcectomy with capitonage of the residual cavity and invagination of the fibrous rims is the method of choice for hepatic localization of the parasite. The above-mentioned surgical technique is characterized with low percentage of post-surgical complications, is well-tolerated from patients and relatively atraumatic and shows excellent long-term results. We consider more radical surgical methods, like atypical liver resections, appropriate in selected patients, ones with multiple echinococcosis and vast fibrous-altered areas of liver parenchyma. We find reasonable the obligatory adjuvant post-surgical treatment with Albendazole under parasitologist control, especially in cases of multiple and/or recurrent echinococcosis.

["Quantitative cardiotocography"--clinical practice guideline].

INTRODUCTION: The "quantitative cardiotocography" method provides important information about the condition of the fetus during labor, non-invasively and in real time. Since the forecast pH-results are being updated every 5 (five) minutes, significant differences in the readings for pH can be observed. In certain cases this can hamper the evaluation of fetal condition and lead to contradictions. Such inconveniences can be easily avoided by using the arithmetic average value of the last 6 forecast results for pH, generated by the quantitative cardiotocography software. Another inconvenience of the "quantitative cardiotocography" method lies in the fact that the components involved in the formation of CTG-score seem to be unequal in terms of potential for evaluating the fetal condition. Based on that, we believe that clinical practice guideline is needed. We developed such guideline based on two main criteria: the arithmetic average value over the last 6 (six) pH estimates; which components of the CTG-score are involved in the formation of the pH forecast readings. MATERIAL AND METHODS: Our previous studies demonstrated that in 85% of all cases, the actual pH value of the newborn was in the range of -0037/+0046 from the average arithmetic value of the last six (6) pH estimates before the delivery. Based on this variability in the pH forecast results we defined three groups of findings--normal (pH 7.350 to 7.237), suspect (pH of 7.237 to 7.137) and abnormal (pH < 7.137). In another study, we differentiated several varieties of CTG-score (with very high, high, satisfactory and low predictive value), depending on the observed deviations between forecast and actual pH results. RESULTS: pH forecast results should always be assessed together with the composition of the CTG-score. This can be achieved by using the presented clinical guideline, which also contains recommendations for adopting specific obstetric behavior, based on the "quantitative cardiotocography" readings. CONCLUSION: In future studies we will investigate if it is possible the presented clinical practice guideline to replace fetal blood sampling for intrapartal assessment of the fetal condition. Further clinical studies will clarify whether the use of this clinical guideline could lead to a reduction in the incidence of metabolic acidosis in newborns or change the percentage of operative deliveries compared with the cases where classical indirect cardiotocography was used.

[A comparative study of the prognostic pH values of the fetus in utero, generated by the "quantitative cardiotocography" computer method, and the actual pH values measured immediately after delivery].

INTRODUCTION: The presented methodology for quantitative evaluation of the cardiotocographic (CTG) findings unconditionally provides essential opportunities for improving the diagnostic potential in modern obstetric practice. Current literature data concerning the clinical application of this method are scarce. Credible clinical trials are needed to determine what is the correlation between estimated and actual values of the studied variables. MATERIALS AND METHODS: A prospective study on 110 pregnant women was performed. All patients were monitored via indirect cardiotocography. The recordings were stored and analyzed by the computerized method for "quantitative cardiotocography". We compared the last prognostic fetal pH value, generated by the "quantitative cardiotocography" software during labor, with the actual pH measured from the umbilical artery (UA) of the newborn. RESULTS: For each of the stored CTG recordings we quantified the difference between the last forecast and the actual pH of the newborn. In 82% of these cases this difference was in range of -0081/+0074 from the projected results. However during the study we discovered that there is a significantly better correlation between the arithmetic average of the last 6 (six) predicted results and the actual pH of the newborn. In 85% of these cases the difference between forecast and actual pH values lies a in range of -0037/+0046. CONCLUSION: Using the arithmetic average of the last 6 (six) predicted results for pH leads to a significant increase in the clinical value of the "quantitative cardiotocography". More studied are needed if we are to find opportunities to further reduce the existing prediction variability.

A human FVIII inhibitor modulates FVIII surface electrostatics at a VWF-binding site distant from its epitope.

SUMMARY BACKGROUND: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab(BO2C11)) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. OBJECTIVES: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. METHODS: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab(BO2C11)-complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. RESULTS: Binding of Fab(BO2C11) to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. CONCLUSIONS: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.

[Quantitative cardiotocography].

UNLABELLED: Cardiotocography (CTG) is an up-to-date practical method for fetal surveillance during pregnancy and labor. More precise reading of the CTG data is possible with the use of some fetal acid-base parameters, taken from the umbilical artery (UA). Unfortunately, invasive procedures like fetal scalp blood sampling and/or cordocentesis are required to obtain these results. Latest computer technology enables us to create mathematical algorithms that uninvasively calculate prognostic values for fetal blood pH, based on interpretation of key CTG features. The method is called "Quantitative cardiotocography" and is implemented in a software solution for fetal surveillance in 2007. In this study the authors present their first clinical results using the method, which they recently introduced for the first time in Bulgaria. A total of 1356 CTG recordings with pH prediction were stored and analysed. These findings have been carefully taken into consideration in the context of some clinical conditions like preeclampsia, pre- and post-term delivery placental abruption, etc. Early results are showing fairly good correlation between computer-analysed abnormal CTG findings and presence of the above mentioned clinical conditions. CONCLUSIONS: Quantitative appraisal of CTG findings and acid-base parameters leads to much more precise interpretation of the CTG phenomena, which guarantees higher quality of fetal surveillance during pregnancy and labor.

pH-dependent stability of sperm whale myoglobin in water-guanidine hydrochloride solutions.

An experimental-theoretical approach for the elucidation of protein stability is proposed. The theoretical prediction of pH-dependent protein stability is based on the macroscopic electrostatic model for calculation of the pH-dependent electrostatic free energy of proteins. As a test of the method we have considered the pH-dependent stability of sperm whale metmyoglobin. Two theoretical methods for evaluation of the electrostatic free energy and p K values are applied: the finite-difference Poisson-Boltzmann method and the semiempirical approach based on the modified Tanford-Kirkwood theory. The theoretical results for electrostatic free energy of unfolding are compared with the experimental data for guanidine hydrochloride unfolding under equilibrium conditions over a wide pH range. Using the optical parameters of the Soret absorbance to monitor conformational equilibrium and Tanford's method to estimate the resulting data, it was found that the conformational free energy of unfolding of metmyoglobin is 16.3 kcal mol(-1) at neutral pH values. The total unfolding free energies were calculated on the basis of the theoretically predicted electrostatic unfolding free energies and the experimentally measured midpoints (pH(1/2)) of acidic and alkaline denaturation transitions. Experimental data for alkaline denaturation were used for the first time in theoretical analysis of the pH-dependent unfolding of myoglobin. The present results demonstrate that the simultaneous application of appropriate theoretical and experimental methods permits a more complete analysis of the pH-dependent and pH-independent properties and stability of globular proteins.

Spectrophotometric titration of ionisable groups in proteins: a theoretical study.

A simple theoretical model is proposed for evaluation of the optical titration behaviour of tyrosyl and carboxyl residues in proteins. The pK values involved in the model are computed using the semi-empirical method. The titration curves are calculated using the values of the molar absorption differences for tyrosyl residues in the ultraviolet (UV) region at 245 and 295 nm, and for carboxyl residues in the infrared (IR) region at 1565 and 1707 cm(-1), respectively. The theoretical tyrosyl titration curves are compared with the experimental data for lysozyme, myoglobin and chymotrypsinogen (available in the literature). This approach provides a good tool for distinguishing between the ionisation and the conformational changes in the alkaline range. The quantitative evaluation of the change of molar extinction coefficients as a function of pH in the case of carboxyl titration for lysozyme, trypsin and cytochrome c shows a good agreement with the experimental titration data.

Protonation of the beta-lactam nitrogen is the trigger event in the catalytic action of class A beta-lactamases.

The pH dependence of the pK(a) values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 beta-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the beta-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the beta-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.

[Combined gynecological operations]. / Suchetani ginekologichni operatsii.

During the objective examination of patients admitted for operation, changes in the gynaecological status, corresponding to primary disease and demanding also operation, were determined. The authors studied clinical status of 33 women with the following primary diseases: myoma uteri, static anomalies of female genitalia and accompanying diseases like urinary stress incontinence, uterine cervical laceration and the accompanying static anomalies. Authors applied combined operative techniques, abdominal and vaginal (hysterectomy with colpouretherosuspension by Burch, vaginal hysterectomy with colpouretherosuspension by Ratz, myomectomy with colpouretherosuspension and ligamentopexy by McColl, etc.), in order to treat definitively the primary disease and the accompanying ones as well. The results showed that in appropriately selected cases and operative techniques, a good therapeutic outcome was achieved. In conclusion we may say that the operative approach should include simultaneously all pathological deviations of women genitalia.

[The late results of the surgical treatment of stress urinary incontinence]. / Kusni rezultati ot operativnoto lechenie na pikochnata stres inkontinentsiia.

The severe stress urinary incontinence is a condition, which traumatize women psychology. Amongst numerous well-known operative techniques for treatment of the stress urinary incontinence authors apply the following ones: suprapubic colpouretherosuspension by Burch, colpouretherosuspension by Ratz, sling operation with synthetic stuff--Ampoxen. The evaluation of late results from the applied operative approaches in 30 patients displaced a long term overcome of stress incontinence. The authors emphasized that during clinical follow up of all cases in the course of 3 years the best results were obtained after application of suprapubic and sling methods. Our conclusion is that the choice of operative approach have to be individual and is in dependence with every clinical case. The postoperative outcome depends on the exactness of the operation.

[Combined diagnostic methods for women in the pre- and postmenopause]. / Suchetani diagnostichni metodi pri zheni v pre- i postmenopauzata.

UNLABELLED: During one-year period, 20 women with pre- and post-menopausal problems were investigated. The diagnostic examination included transvaginal sonography (TS), hysteroscopy with separative abrasion and histopathological examination. The data from the three diagnostic methods were compared. RESULTS: In 6 patients the endometrial thickness (ET) was lower than 8 mm. The hysteroscopic finding (HF) comprised in endometrial atrophy, confirmed also histologically. ET was higher than 8 mm in 14 patients, and the HF revealed endometrial polyps and endometrial hyperplasia. The hystological examination confirmed cystic endometrial hyperplasia in 4 cases, endometrial secretary phase in 5 cases, endometrial polyp in 3 cases and endometrial carcinoma in 2 cases. The authors accepted that the combination of 3 diagnostic methods of examination, applied in the described consequence, gave the possibility for precise diagnostic investigation and adequate treatment.

X-ray structure at 1.76 A resolution of a polypeptide phospholipase A2 inhibitor.

The high resolution crystal structure of a natural PLA2 inhibitor has been determined by Patterson search methods. In the heterodimeric, neurotoxic complex, vipoxin, isolated from the venom of Bulgarian viper, PLA2 inhibitor represents the non-toxic subunit. The model was refined to a crystallographic R-factor of 15.5% for data between 6 and 1.76 A resolution. The packing of the inhibitor in the crystal reveals close contacts between the molecules, which are symmetry-related by the 2-fold axes of the lattice. These pairs associate as a crystallographic dimer, stabilized by a set of interactions, including van der Waals contacts between residues from symmetry-related pairs, denoted as the recognition site and the recognition surface. Residues Ph3, Trp31 and Tyr119 represent the recognition site of inhibitor which possibly fits to the hydrophobic wall of the target PLA2. The topology of the inhibitor represents the PLA2 type of folding: three long helices and a beta-hairpin. Superposition of the structure of the inhibitor shows an almost complete overlap with different mammalian and viper PLA2 in the backbone and in the position of the sidechains of the residues that belong to the active centre and the hydrophobic wall. A "lock and key" mechanism of recognition of its native PLA2 in gland cells and other toxic PLA2 in vitro has been suggested. The mechanism includes complementary "head to tail" interactions between the recognition site of the inhibitor and a recognition surface located on the hydrophobic wall of the target PLA2. Having a high spatial homology with the PLA2 family of enzymes but opposing their action, the inhibitor from vipoxin presents an example of a divergent evolution of an ancient PLA2. The presence of a space for binding calcium in the inhibitor is believed to be a rudiment and proof of a common origin with PLA2.

Local electrostatic potentials in pyridoxal phosphate labelled horse heart cytochrome c.

The present work shows the application of an optical label pyridoxal phosphate (PLP) for the experimental determination of local electrostatic potentials in singly substituted cytochromes c modified by pyridoxal phosphate at Lys 79 (PLP-Lys-79-cyt.c) or at Lys 86 (PLP-Lys-86-cyt.c). PLP has also been used to calculate the pKa values of all ionizable groups and the electrostatic potentials in the modified proteins and to analyse their properties. The experimental pKa values for the pyridine nitrogen and phenolic hydroxyl of the bound label were obtained from pH-dependent absorbance and fluorescence measurements, as follows: in PLP-Lys-79-cyt.c for pyridine nitrogen 4.5 (absorbance) and 5.1 (fluorescence), for phenolic hydroxyl 8.6 (absorbance) and 8.3 (fluorescence); in PLP-Lys-86-cyt.c for pyridine nitrogen 4.7 (absorbance) and 5.8 (fluorescence), for phenolic hydroxyl 8.3 (absorbance) and 8.5 (fluorescence). The differences between absorbance and fluorescence data are related to differences in the behaviour of the bound label in the ground and excited electronic states and to intermolecular charge-charge interactions. Molecular modelling was used to generate the atomic co-ordinates of the PLP-modified horse heart cytochrome c necessary for the theoretical calculations of the pKa values and electrostatic potentials.

Thermal unfolding of the DNA-binding protein Sso7d from the hyperthermophile Sulfolobus solfataricus.

Thermal unfolding of the small hyperthermophilic DNA-binding protein Sso7d was studied by circular dichroism spectroscopy and differential scanning calorimetry. The unfolding transition can be described by a reversible two state process. Maximum stability was observed in the region between pH 4.5 and 7.0 where Sso7d unfolds with a melting temperature between 370.8 to 371.9 K and an unfolding enthalpy between 62.9 and 65.4 kcal/mol. The heat capacity differences between the native and the heat denatured states obtained by differential scanning calorimetry (620 cal/(molK)) and circular dichroism spectroscopy (580 cal/(mol K)) resulted in comparable values. The thermodynamic reason for the high melting temperature of Sso7d is the shallow stability curve with a broad free energy maximum, corresponding to the relatively small heat capacity change which was obtained. The calculated stability curve shows that Sso7d has, despite of its high melting temperature, an only moderate intrinsic stability, which reaches its maximum (approximately 7 kcal/mol) at 282 K. Sso7d is particularly poorly stabilized (approximately 1 kcal/mol) at the maximum physiological growth temperature of Sulfolobus solfataricus. Sso7d has furthermore untypically low specific enthalpy (0.99 kcal/(mol residue)) and entropy (2.99 cal/(mol K)) values at convergence temperatures. No significant differences in thermal stability of the partially methylated Sso7d from Sulfolobus solfataricus and the cloned non-methylated form of the protein expressed in Escherichia coli were observed.