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Sliding clamps are loaded onto DNA by ATP-driven clamp loader complexes. The structure of the E. coli clamp loader in a nucleotide-free state has been determined previously. We now report crystal structures of a truncated form of the isolated gamma-ATPase subunit, gamma(1-243), of the E. coli clamp loader, in nucleotide-free and bound forms. The gamma subunit adopts a defined conformation when empty, in which the nucleotide binding site is blocked. The binding of either ATPgammaS or ADP, which are shown to bind with equal affinity to gamma(1-243), induces a change in the relative orientation of the two domains such that nucleotides can be accommodated. This change would break one of the gamma:gamma interfaces seen in the empty clamp loader complex, and may represent one step in the activation process.

Binding of cystatin-type inhibitors to papain-like exopeptidases cannot be explained by the stefin B-papain complex. The crystal structure of human stefin A bound to an aminopeptidase, porcine cathepsin H, has been determined in monoclinic and orthorhombic crystal forms at 2.8A and 2.4A resolutions, respectively. The asymmetric unit of each form contains four complexes. The structures are similar to the stefin B-papain complex, but with a few distinct differences. On binding, the N-terminal residues of stefin A adopt the form of a hook, which pushes away cathepsin H mini-chain residues and distorts the structure of the short four residue insertion (Lys155A-Asp155D) unique to cathepsin H. Comparison with the structure of isolated cathepsin H shows that the rims of the cathepsin H structure are slightly displaced (up to 1A) from their position in the free enzyme. Furthermore, comparison with the stefin B-papain complex showed that molecules of stefin A bind about 0.8A deeper into the active site cleft of cathepsin H than stefin B into papain. The approach of stefin A to cathepsin H induces structural changes along the interaction surface of both molecules, whereas no such changes were observed in the stefin B-papain complex. Carboxymethylation of papain seems to have prevented the formation of the genuine binding geometry between a papain-like enzyme and a cystatin-type inhibitor as we observe it in the structure presented here.

Objective: Our purpose was to determine maternal and foetal outcome in patients undergoing surgery for a pelvic mass in pregnancy., Study Design: Maternal and foetal records (outcomes) of eight cases of adnexal masses associated with intrauterine pregnancy that required laparotomy or aspiration or that were diagnosed incidentally at the time of caesarean section were reviewed. The review was performed on patients who were seen with an adnexal mass in pregnancy from January 1994 to February 2001. We included patients with simple or complex masses > or = 6 cm that were persistent on ultrasonographic evaluation and patients with adnexal masses with complications (torsion, haemorrhage). We excluded cysts that spontaneously resolved by 16 weeks' gestation., Results: Eight patients of 16,472 deliveries were identified with adnexal masses that satisfied the above criteria. Six patients underwent laparotomy in the first and/or the second trimester of pregnancy. In two of them emergency laparotomy were done due to torsion or haemorrhage as a complication of the adnexal masses. In all patients benign ovarian tumors were found. Two patients underwent transvaginal aspiration of simplex cysts due to subtorsion in the first trimester of gestation (negative results on cytological study). All of these eight patients had term deliveries. Two patients, due to obstetrical reasons, underwent caesarean section., Conclusion: The incidence of an adnexal mass during pregnancy in our population is consistent with what has been reported in the literature. We emphasize that transvaginal aspiration and drainage of symptomatic simplex cysts in the first trimester and percutaneous cysts in the second trimester can avert laparotomy. Our data support a randomised clinical study to determine optimal management of an adnexal mass in pregnancy.

Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N-terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo-protease inhibitor. The location of missense mutations, characterized in people suffering from Haim-Munk and Papillon-Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.

Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the catalytic unit of a primase enzyme, that of a approximately 320 residue fragment of Escherichia coli primase, determined at 2.9 A resolution. Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases. The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity., (Copyright 2000 Academic Press.)

A review of kinetic and structural data has enabled us to reconsider the definition of substrate binding sites in papain-like cysteine proteases. Only three substrate binding sites, S2, S1 and S1', involve main as well as side chain contacts between substrate and enzyme residues. Interactions between the enzymes and the substrate P3 and P2' residues are based on side chains (an exception is cathepsin B which is a carboxydipeptidase), so their interaction surface spreads over a relatively wide area. The location and definition of substrate binding sites beyond S3 and S2' is even more questionable.

Background: Cathepsin H is a lysosomal cysteine protease, involved in intracellular protein degradation. It is the only known mono-aminopeptidase in the papain-like family and is reported to be involved in tumor metastasis. The cathepsin H structure was determined in order to investigate the structural basis for its aminopeptidase activity and thus to provide the basis for structure-based design of synthetic inhibitors., Results: The crystal structure of native porcine cathepsin H was determined at 2.1 A resolution. The structure has the typical papain-family fold. The so-called mini-chain, the octapeptide EPQNCSAT, is attached via a disulfide bond to the body of the enzyme and bound in a narrowed active-site cleft, in the substrate-binding direction. The mini-chain fills the region that in related enzymes comprises the non-primed substrate-binding sites from S2 backwards., Conclusions: The crystal structure of cathepsin H reveals that the mini-chain has a definitive role in substrate recognition and that carbohydrate residues attached to the body of the enzyme are involved in positioning the mini-chain in the active-site cleft. Modeling of a substrate into the active-site cleft suggests that the negatively charged carboxyl group of the C terminus of the mini-chain acts as an anchor for the positively charged N-terminal amino group of a substrate. The observed displacements of the residues within the active-site cleft from their equivalent positions in the papain-like endopeptidases suggest that they form the structural basis for the positioning of both the mini-chain and the substrate, resulting in exopeptidase activity.

The structure of the wild-type human procathepsin B has been refined to a crystallographic R-value of 0.18 and R-free of 0.23 exploiting the data obtained from new crystals that diffract beyond 2.5 A resolution. The structure confirms two previously presented, lower-resolution structures. The structure of the propeptide chain folds on the surface of the enzyme domains and blocks access of substrate to the already formed active site. Abundant solvent molecules fill the cavities between the propeptide and the enzyme part of the molecule. The propeptide structure is compared with a substrate model in the S2, S1, S1' and S2' binding sites. In this crystal form the cathepsin B occluding loop residues adopt yet another conformation. The structures show that the occluding loop region between the residues Cys108 and Cys119 behaves quite independently from the rest of the structure and easily adapts to changes in environment. The variety of the observed conformations of the occluding loop is in agreement with other data showing that the loop is responsible for limiting cathepsin B activity to that of a carboxydipeptidase. The region before Cys108 is essentially the same as in the mature structure, whereas the region from Cys119 to Thr125 is raised compared to the mature form by the propeptide squeezed between it and the enzyme domains, surface. The structure strongly suggests that processing of procathepsin B during its autoactivation is not unimolecular., (Copyright 1997 Academic Press Limited.)

Late chorionic villus sampling (placental biopsy) under ultrasound guidance was carried out in 800 (80 per cent) cases in the second trimester and 200 (20 per cent) cases in the third trimester of pregnancy. Out of 1000 placental biopsies, 250 (25 per cent) were performed because of suspicious ultrasonographic findings. Colour Doppler was used to investigate the uteroplacental and fetal vessels in 300 (30 per cent) pregnancies before and after late chorionic villus sampling (CVS). In the same group, mean serum alpha-fetoprotein (AFP) levels increased after sampling in 20 (6.7 per cent) patients. In 20 patients (2 per cent), complications between sampling and delivery were found. A placental haematoma measuring 0.5-1 ml was seen at the sampling site in 4 (0.4 per cent) patients in the second trimester of pregnancy and in 3 (0.3 per cent) in the third trimester. Two (0.2 per cent) demonstrated fever, but there were no instances of chorioamnionitis. There were only three (0.3 per cent) spontaneous abortions 4-6 weeks after late CVS. However, there was no correlation between AFP elevation, placental haematoma, Doppler measurements, and spontaneous abortion. Cytogenetic findings were obtained in 990 (99 per cent) of 1000 placental samplings. We found 60 (6.0 per cent) chromosomal abnormalities. In the group with suspicious ultrasonic findings (250 cases), we found significant oligohydramnios in 125 (50 per cent) and significant polyhydramnios in 60 (24 per cent), and 45 (18 per cent) had chromosomal abnormalities. Among the 60 patients with chromosomal abnormalities, ultrasonographic findings in 10 (16.7 per cent) were detected after the 20th week of pregnancy. There were no significant differences in mean pulsatility index (PI) in the uteroplacental and fetal vessels before and after late CVS. Preliminary data from five trisomic fetuses (three trisomy 21 and two trisomy 18) showed abnormally increased umbilical PI and abnormally decreased middle cerebral artery PI.

A wild-type human procathepsin B was expressed, crystallized in two crystal forms and its crystal structure determined at 3.2 and 3.3 Angstroms resolution. The structure reveals that the propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. The structure of the enzymatically active domains is virtually identical to that of the native enzyme [Musil et al. (1991) EMBO J. 10, 2321-2330]: the main difference is that the occluding loop residues are lifted above the body of the mature enzyme, supporting the propeptide structure.

We studied the outcome of fetuses in whom cystic hygroma was diagnosed in the first and early second-trimester of pregnancy using transvaginal ultrasonography. The purpose of this study was to evaluate the association between fetal cystic hygroma and fetal cytogenetic abnormalities, and the long-term prognosis. Thirty-five consecutive fetuses between 9.1 and 13.4 weeks of gestation diagnosed as having a nuchal hygroma were evaluated ultrasonographically and karyotyped. Those with a normal chromosome complement were ultrasonographically monitored throughout the remainder of the pregnancy to document the resolution of the hygroma. Eighteen of thirty-five fetuses were found to have a normal karyotype and five of these were aborted electively. The hygromas resolved in ten of these karyotypically normal fetuses within four weeks of initial diagnosis and they were phenotypically normal at birth. Seventeen fetuses were karyotypically abnormal with trisomy twenty-one being the most common abnormality. Prenatal cytogenetic analysis should be offered to women with fetal cystic hygroma diagnosed in the first trimester. A normal outcome is likely in those without chromosome abnormalities.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.

Transabdominal placental biopsy under ultrasound guidance was carried out in 260 cases in the second trimester and 50 cases in the third trimester of pregnancy. Placental tissue was aspirated using an 18 or 20 gauge needle. In a total of 310 placental biopsies in the second and third trimester, 100 were performed because of suspicious ultrasonographic findings. Placental biopsy is simple in the presence of severe oligohydramnios where fetal blood sampling is usually more difficult. Oligohydramnios and polyhydramnios were the ultrasonographic findings in 50% of cases and were found to be associated with 30% of abnormal chromosomal findings. There was one (0.3%) abortion within two weeks following placental biopsy. Placental biopsy did not affect the outcome of the pregnancy.

In 126 pregnant women of the gestational age from the 28th to the 44th gestational week, the biparietal diameter, the abdomen diameter, and the placental maturity after Granum et al. were determined. Amniocentesis was performed for obstetric indication. Also the L/S ratio and the cytological estimation of the gestational age were recorded. The first degree of the placental maturity was found in 37, the second degree in 3, and the third degree in 36 pregnant women. In the first group, BPP was 8.38 +/- 0.75, the abdomen diameter 8.30 +/- 0.63, and pulmonary maturity in 73% of women according to the L/S ratio and in 91.9% according to cytological estimation. In the second group, BPP was 9.15 +/- 0.65, the abdomen diameter 9.03 +/- 0.83, and in 77.9% of women the L/S ratio was higher than 2 but in 56.6% of women, according to cytological estimation, there was a good pulmonary maturity. At the third degree of placental maturity, BPP was 9.49 +/- 0.60, the abdomen diameter 9.63 +/- 0.92, and a good pulmonary maturity in 94.4% of women. The RDS was not observed in 5.6% of newborns with the L/S ratio below 2. According to cytological estimation, BPP below 9.15 was found in pre-term labour and higher than 9.15 in at-term births when in 94% of women the L/S ratio was higher than 2.

In six patients estrogens and gonadotropins were low and the prolactin concentration was increased (36-128 micrograms/l). Amenorrhea in all patients lasted 8-60 months, while galactorrhea was observed in five patients. By the X-ray of the sella turcica and neurologic and ophthalmologic examinations no signs of an intrasellar expansive process were revealed. The progesterone test was negative in four patients and the clomiphene test in all six patients. The LH-RH test (100 micrograms i. m.) showed the increased LH and FSH concentrations in all six patients. They all became pregnant in the course of the bromocryptine treatment (5-7.5 mg daily). Pregnanediol and prolactin concentrations were recorded every 14 days in the first 16 weeks of pregnancy, both in six patients and in eleven controls with spontaneous pregnancy. The pregnanediol concentrations proved somewhat higher in the control group than in patients having become pregnant under the bromocryptine therapy, but the differences are not statistically significant. However, the prolactin concentrations were significantly lower in controls than in pregnant women with the hyperprolactinemic anovulatory syndrome (p less than 0.01) in the sixth to the twelfth week of pregnancy.

In addition to being the best method for the diagnosis of the hydatidiform mole, ultrasound is also used in the surgical treatment of the mole. The procedure of the evacuation of the complete hydatidiform mole by vacuum suction and continuous ultrasonic monitoring is described. The method is illustrated by three cases.

From 1971 to 1975, when beta sympathicomimetics were not used, from total 13 561 deliveries 6.06% were preterm deliveries. In years 1976-1980, when these tocolytics are routinely used from total 15 242 deliveries 5.24% were preterm deliveries. Significance is statistically characteristic, p = = 0,01. In the anamnesis of all parturients in both periods, frequency of factors which effect on the rate of preterm deliveries is equal.