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The HrcQB protein from the plant pathogen Pseudomonas syringae is a core component of the bacterial type III secretion apparatus. The core consists of nine proteins widely conserved among animal and plant pathogens which also share sequence and structural similarities with proteins from the bacterial flagellum. Previous studies of the carboxy-terminal domain of HrcQB (HrcQB-C) and its flagellar homologue, FliN-C, have revealed extensive sequence and structural homologies, similar subcellular localization, and participation in analogous protein-protein interaction networks. It is not clear however whether the similarities between the two proteins extend to the level of quaternary association which is essential for the formation of higher-order structures within the TTSS. Even though the crystal structure of the FliN is a dimer, more detailed studies support a tetrameric donut-like association. However, both models, dimer and donut-like tetramer, are quite different from the crystallographic elongated dimer of dimers of the HrcQB-C. To resolve this discrepancy we performed a multidisciplinary investigation of the quaternary association of the HrcQB-C, including mass-spectrometry, electrophoresis in non-reductive conditions, gel filtration, glutaraldehyde cross-linking and small angle X-ray scattering. Our experiments indicate that stable tetramers of elongated shape are assembled in solution, in agreement with the results of crystallographic studies. Circular dichroism data are consistent with a dimer-dimer interface analogous to the one established in the crystal structure. Finally, molecular dynamics simulations reveal the relative orientation of the dimers forming the tetramers and the possible differences from that of the crystal structure.

Radical vaginal trachelectomy in patients with early-stage cervical cancer is an oncologically safe procedure in well-selected patients. Successful pregnancy in a patient with radical vaginal trachelectomy is possible, with two-thirds of pregnancies resulting in live birth. However, it presents a great challenge for assisted reproductive techniques and reproductive medicine in cases with subsequent severe cervical stenosis. This is a report of a 38-year-old patient who underwent radical vaginal trachelectomy at the age of 33 years for early stage (IA2) adenocarcinoma and subsequently presented with infertility due to cervical factors. The patient underwent ovarian stimulation using a novel SMART (Stimulation with Minimal Adverse effects, Retrieval and Transfer)-IVF protocol. As it was impossible to perform transcervical embryo transfer with an almost absent severely stenotic cervical opening, a transmyometrial embryo transfer under ultrasound guidance was performed. This resulted in a successful singleton full-term pregnancy delivered by Caesarean section at gestational age 37 weeks. As far as is known, this is the first reported case of successful pregnancy conceived by IVF with transmyometrial embryo transfer for a patient who had previously undergone radical vaginal trachelectomy.

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions. Reactions of premat-GO with Cu(II) were investigated using optical, EPR, and resonance Raman spectroscopy, SDS-PAGE, and X-ray crystallography. Premat-GO reacted anaerobically with excess Cu(II) to efficiently form the thioether bond but not the Y272 radical. A potential C228-copper coordinated intermediate (lambda max = 406 nm) in the processing reaction, which had not yet formed the C228-Y272 cross-link, was identified from the absorption spectrum. A copper-thiolate protein complex, with copper coordinated to C228, H496, and H581, was also observed in a 3 min anaerobic soak by X-ray crystallography, whereas a 24 h soak revealed the C228-Y272 thioether bond. In solution, addition of oxygenated buffer to premat-GO preincubated with excess Cu(II) generated the Y272 radical state. On the basis of these data, a mechanism for the formation of the C228-Y272 bond and tyrosyl radical generation is proposed. The 406 nm complex is demonstrated to be a catalytically competent processing intermediate under anaerobic conditions. We propose a potential mechanism which is in common with aerobic processing by Cu(II) until the step at which the second electron acceptor is required.

A total of 134 controlled natural IVF (nIVF) cycles were reviewed retrospectively and compared with 370 stimulated IVF (sIVF) cycles. The clinical pregnancy rate per embryo transfer following nIVF was 27% and 47% in sIVF cycles for patients aged less than 35. However, natural cycle patients could attempt consecutive cycles with much less impact on their lives, both medically and financially. In patients under 35 years of age, the choice of controlled nIVF reduces the cost and risk to the patient, permitting her to have multiple, consecutive attempts, and cumulatively offers a clinical pregnancy rate which approaches that of sIVF. The multiple pregnancy rate in nIVF is significantly reduced compared with sIVF treatment cycles. In patients over 35 years of age the benefits of nIVF were much less evident (clinical pregnancy rate: 8% per embryo transfer) and the opportunity to transfer multiple embryos in these patients seems to be advantageous.

The response regulator VicR from the Gram-positive bacterium Enterococcus faecalis forms part of the two-component signal transduction system of the YycFG subfamily. The structure of the DNA-binding domain of VicR, VicR(c), has been solved and belongs to the winged helix-turn-helix family. It is very similar to the DNA-binding domains of Escherichia coli PhoB and OmpR, despite low sequence similarity, but differs in two important loops. The alpha-loop, which links the two helices of the helix-turn-helix motif, is similar to that of PhoB, where it has been implicated in contacting the sigma subunit of RNA polymerase, but differs from that of OmpR. Conversely, the loop following the helix-turn-helix motif is similar to that of OmpR and differs from that of PhoB. YycF/VicR, PhoB and Bacillus subtilis PhoP regulators all recognize almost identical DNA sequences and although there is currently no experimental evidence linking this loop with the DNA, the structure is consistent with possible involvement in selective DNA recognition or binding.

Background: Adult orthotopic liver transplantation is associated with significant use of allogenic blood products, which places considerable demands on finite resources. This could be reduced by autologous red cell salvage use, and we evaluated its cost effectiveness in this prospective study., Methods: Intraoperative autotransfusion was used in 660 adult liver transplant patients between January 1997 and July 2002. These included 134 with acute liver failure, 62 retransplants, 90 alcohol-related, 183 viral, 98 cholestatic chronic liver diseases, and 93 with other etiologies., Results: The total volume of red blood cells transfused was 3641+/-315 ml, 2805+/-234 ml, 2603+/-443 ml, and 2785+/-337 ml for alcohol-related, viral, cholestatic, and others, respectively. Low preoperative hemoglobin was significantly associated with higher intraoperative transfusion requirements. Blood volumes transfused at retransplantation were significantly higher (7077+/-1110 ml vs. 2864+/-138 ml; P<0.001) than for acute liver failure and chronic liver disease. Autologous blood volumes transfused were similar in all diagnostic groups, but were significantly greater in retransplantation (2754+/-541 ml vs. 1524+/-77 ml; P<0.01). Venovenous bypass was significantly associated with higher transfusion requirements. Total savings per case were similar for all diagnostic groups but were greater in cases of retransplantation (864+/-222 pounds (1235+/-317 US dollars) vs. 238+/-24 pounds (340+/-34 US dollars; P<0.001). With the use of autologous transfusion over the study period, a cost saving of 131,901 pounds (188,618 US dollars) was achieved., Conclusions: Intraoperative red blood cell salvage and autologous transfusion is cost effective in adult liver transplantation. Currently, where optimum resource utilization and fiscal constraint are paramount in healthcare delivery, autologous transfusion is an important adjunct in liver transplantation.

Objective: We present the MRI features with pathologic correlation of aggressive fibromatosis, incorporating 203 cases over a 5-year period from the Royal Marsden Hospital Sarcoma Unit database., Materials and Methods: Sixty patients had imaging available for retrospective review of which 29 had preoperative MRI and final histopathologic diagnosis of aggressive fibromatosis., Results: The average age at diagnosis was 41.3 years with a female-to-male sex ratio of 1.2:1. Twenty lesions were extraabdominal; six, intraabdominal; and three, in the abdominal wall (classic desmoid). The average tumor size was 6.4 cm (range, 2.2-13.7 cm). Intraabdominal aggressive fibromatosis produced the largest tumors, averaging 9.5 cm. Most lesions were ovoid (52%) or infiltrative (34.5%) in outline with an irregular or lobulated contour (76%). The lesions crossed major fascial boundaries in 31% of cases overall and in 66% of patients referred for recurrent disease. On MRI, homogeneous isointensity or mild hyperintensity on T1-weighted images and heterogenous high signal on T2-weighted or STIR images were seen. All lesions enhanced after IV gadolinium, usually avidly. In contrast to previous reports, 38% of cases failed to show low signal on all pulse sequences and no abnormalities were seen in local bone structures. Histology showed sheets of bland spindle cells in dense collagen and did not vary with the MRI signal characteristics of the lesion. Patients referred for recurrent disease were most likely to have a recurrence after surgery. MRI and pathology findings did not predict recurrence., Conclusion: Accurate diagnosis and staging of aggressive fibromatosis by MRI have important treatment and prognostic implications.

Purpose: Colorectal cancers that display high-degree mi-crosatellite instability are associated with an improved prognosis and evidence of an activated host immune response. Molecular analyses have suggested that heat shock proteins, a family of proteins that have key immunologic functions, are upregulated in these cancers. We aimed to explore the expression of heat shock proteins 70 and 110 and their relationship to microsatellite instability, survival, and other clinicopathologic parameters., Methods: Twenty-six colorectal cancers that displayed microsatellite instability were matched by age, stage, and site in the colorectum to 26 microsatellite-stable cancers. Immunohistochemistry was used to detect expression of both markers., Results: The microsatellite-unstable group showed significantly higher expression of heat shock protein 70 than the microsatellite-stable group (P = 0.006), and patients undergoing curative resections for unstable cancers had improved prognosis compared with their stable counterparts (P = 0.026). Significantly, in a multivariate survival analysis, low or absent heat shock protein 70 expression was independently associated with a poor outcome (P = 0.001)., Conclusions: Heat shock protein 70 has known functions that promote antitumor immune responses. Its overexpression in colorectal cancers with microsatellite instability may be pivotal to explaining these tumors' enhanced immunogenicity and improved prognosis.

The recovery of a mature oocyte from a natural cycle followed by IVF (nIVF) is an attractive alternative to conventional IVF, involving ovarian stimulation, in the treatment of female infertility. Similarly, surgical recovery of spermatozoa from the epididymis by percutaneous sperm aspiration (PESA) has simplified the treatment of men with obstructive azoospermia. A couple sought treatment for diminished ovarian reserve and male factor infertility using IVF. A mature oocyte was retrieved and was inseminated by intracytoplasmic sperm injection (ICSI), following recovery of spermatozoa by PESA. A good quality embryo was transferred. A viable pregnancy was confirmed by ultrasound scan. A healthy baby boy was delivered naturally at 37 weeks gestation. This study reports the first ongoing clinical pregnancy and subsequent birth resulting from ICSI of spermatozoa retrieved by PESA into an oocyte recovered during a natural cycle. The use of a combination of less invasive assisted reproductive techniques (PESA and nIVF) can overcome barriers to fertility.

We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.

Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.

Purpose: To examine the reproductive performance of hepatitis B (HBV) and C (HCV) discordant couples following IVF-ET., Methods: A retrospective cohort study of 25 IVF-ET cycles in HBV and HCV discordant couples was performed. Thirteen patients in the study cohort were discordant for HBV (10 males and 3 females), and 12 (9 males and 3 females) for HCV. Twenty-seven consecutive age matched patients comprised the control group. All patients underwent controlled ovarian hyperstimulation using the long downregulation protocol followed by IVF or ICSI., Results: Patients in the three groups (HBV, HCV, and controls) had similar ages, and day 3 FSH concentrations. Despite comparable response to COH, and similar fertilization, and cleavage rates in the three groups, couples discordant for HBV or HCV had significantly poorer implantation and pregnancy rates (7.7%, 0% respectively) compared with controls (41%)., Conclusions: Despite comparable response to COH, HBV and HCV positive discordant couples, have significantly lower implantation and pregnancy rates compared with age-matched controls.

Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants. In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum. The HrcQ(B) protein, a component of the secretion apparatus of Pseudomonas syringae with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ(B)-C). Here, we report the crystal structure of HrcQ(B)-C and show that this domain retains the ability of the full-length protein to interact with other type III components. A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein-protein interactions. Based on the analogies between HrcQ(B) and its flagellum homologues, we propose that HrcQ(B)-C participates in the formation of a C-ring-like assembly.

GO (galactose oxidase; E.C. 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper ion and an amino acid-derived cofactor. The enzyme is produced by the filamentous fungus Fusarium graminearum as an extracellular enzyme. The enzyme has been extensively studied by structural, spectroscopic, kinetic and mutational approaches that have provided insight into the catalytic mechanism of this radical enzyme. One of the most intriguing features of the enzyme is the post-translational generation of an organic cofactor from active-site amino acid residues. Biogenesis of this cofactor involves the autocatalytic formation of a thioether bond between Cys-228 and Tyr-272, the latter being one of the copper ligands. Formation of this active-site feature is closely linked to the loss of an N-terminal 17 amino acid prosequence. When copper and oxygen are added to this pro-form of GO (pro GO), purified in copper-free conditions from the heterologous host Aspergillus nidulans, mature GO is formed by an autocatalytic process. Structural comparison of pro GO with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main-chain position. Some side chains of the active-site residues differ significantly from their positions in the mature enzyme. These structural effects of the prosequence suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. The prosequence is not mandatory for processing, as a recombinant form of GO lacking this region and purified under copper-free conditions can also be processed in an autocatalytic copper- and oxygen-dependent manner.

Purpose: To investigate whether the consecutive embryo transfer of day 3 embryos and of blastocyst protects against failure to reach embryo transfer and provides additional pregnancies., Methods: An embryo transfer was performed on day 3 following which all remaining embryos were cultured to the blastocyst stage for a possible second transfer., Results: One hundred and forty-two patients were selected for extended culture. Thirty-two of these patients did not develop blastocysts in culture, however, there were 12 pregnancies achieved in this group., Conclusions: The consecutive transfer of day 3 embryos and blastocysts can prevent the total loss of a cycle when embryos fail to develop to the blastocyst stage in culture and thereby provide additional pregnancies.

Objective: To establish the relative success of treatment by unstimulated in vitro maturation (IVM) of oocytes or stimulated in vitro fertilization (IVF) in women with polycystic ovaries undergoing assisted conception treatment., Methods: The case-control study included 107 IVM and 107 IVF cycles matched for age and cause of infertility. In vitro maturation patients underwent transvaginal recovery of immature oocytes during an unstimulated cycle, in vitro oocyte maturation, and fertilization. Those in the IVF group underwent ovarian stimulation after pituitary suppression. Embryos were transferred in the same cycle in both groups. Main outcome measures included numbers of mature oocytes and embryos produced, and rates of implantation, pregnancy, live birth, and complications., Results: In the IVM group after in vitro culture, 7.8 mature oocytes and 6.1 embryos were obtained per retrieval. With IVF, 12.0 mature oocytes (P <.01) and 9.3 embryos (P <.01) were obtained. The IVM pregnancy and live birth rates per retrieval were 26.2% and 15.9% compared with 38.3% and 26.2% for IVF (nonsignificant). The implantation rate of IVF-derived embryos was higher (17.1% versus 9.5%) than that for IVM (P <.01). There were 12 cases (11.2%) of moderate or severe ovarian hyperstimulation syndrome in IVF patients, compared with none in the IVM group (P <.01)., Conclusion: Our results suggest that for women with polycystic ovaries who require assisted conception, IVM is a promising alternative to conventional IVF treatment.

Copper amine oxidases are homodimeric enzymes containing one Cu(2+) ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5-9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (K(b)) against pH is bell-shaped, indicating two pK(a)s of 5.8 and approximately 8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the K(b) versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.

Insulin-like growth factor-I (IGF-I) has potent mitogenic and anti-apoptotic effects that give it a critical role in the regulation of rapidly renewing epithelial cell populations such as those found in the colon. Recent evidence has implicated circulating IGF-I levels as an important determinant of colorectal cancer risk, but the role, if any, of its autocrine/paracrine expression remains unexplored. Therefore, we investigated the local expression of IGF-I and IGF-I type I receptor (IGF-IR) in 50 paired normal colon and carcinoma samples. IGF-IR mRNA was present in all samples, whereas IGF-I mRNA was detected in only 30 normal (60%) and 27 tumour (54%) biopsies. Samples that did not express IGF-I mRNA had no IGF-I peptide detectable by immunocytochemistry. The absence of local IGF-1 expression was associated with significantly reduced mRNA levels specifying the proliferating cell nuclear antigen and c-myc, as well as Cox-2, and vascular endothelial growth factor - gene products that regulate angiogenesis. The biological relevance of this finding is suggested by the significant association between local IGF-I mRNA levels and microvessel density in the colorectal cancers., (Copyright 2002 S. Karger AG, Basel)

In the Gram-positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled by the transcriptional regulator AhrC. The hexameric AhrC protein binds in an L-arginine-dependent manner to pseudo-palindromic operators within the promoter regions of arginine biosynthetic and catabolic gene clusters. AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription of catabolic genes. The crystal structure of AhrC has been determined at 2.7 A resolution. Each subunit of the protein has two domains. The C-terminal domains are arranged with 32 point-group symmetry and mediate the major intersubunit interactions. The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not obey strict symmetry. A structural comparison of AhrC with the arginine repressor from the thermophile B. stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition, but also reveals some striking sequence differences, especially within the C-terminal oligomerization domain, which may contribute to the different thermostabilities of the proteins. Comparison of the crystal structure of AhrC with a 30 A resolution model obtained by combining X-ray structure-factor amplitudes with phases derived from electron-microscopic analyses of AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more widely useful for obtaining low-resolution phase information.