Dead or alive? Viability of chytrid zoospores shed from live amphibian hosts.

Pathogens vary in virulence and rates of transmission because of many differences in the host, the pathogen, and their environment. The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), affects amphibian hosts differently, causing extinction and population declines in some species but having limited effects on others. Phenotypic differences in zoospore production rates among Bd lineages likely contribute to some of the variation observed among host responses, although no studies have quantified the viability of zoospores shed from live animals. We compared host survivorship, infection intensity, shedding rates, and zoospore viability between 2 species of endangered tropical frogs, Hylomantis lemur and Atelopus zeteki, when exposed to a highly virulent lineage of Bd (JEL 423). We applied a dye to zoospores 30 to 60 min following animal soaks, to estimate shedding rate and proportion of live zoospores shed by different species. The average infection intensity for A. zeteki was nearly 17 times higher (31,455 ± 10,103 zoospore genomic equivalents [ZGEs]) than that of H. lemur (1832 ± 1086 ZGEs), and A. zeteki died earlier than H. lemur. The proportion of viable zoospores was ~80% in both species throughout the experiment, although A. zeteki produced many more zoospores, suggesting it may play a disproportionate role in spreading disease in communities where it occurs, because the large number of viable zoospores they produce might increase infection in other species where they are reintroduced.

Dynamics of an emerging disease drive large-scale amphibian population extinctions.

Epidemiological theory generally suggests that pathogens will not cause host extinctions because the pathogen should fade out when the host population is driven below some threshold density. An emerging infectious disease, chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is directly linked to the recent extinction or serious decline of hundreds of amphibian species. Despite continued spread of this pathogen into uninfected areas, the dynamics of the host-pathogen interaction remain unknown. We use fine-scale spatiotemporal data to describe (i) the invasion and spread of Bd through three lake basins, each containing multiple populations of the mountain yellow-legged frog, and (ii) the accompanying host-pathogen dynamics. Despite intensive sampling, Bd was not detected on frogs in study basins until just before epidemics began. Following Bd arrival in a basin, the disease spread to neighboring populations at approximately 700 m/yr in a wave-like pattern until all populations were infected. Within a population, infection prevalence rapidly reached 100% and infection intensity on individual frogs increased in parallel. Frog mass mortality began only when infection intensity reached a critical threshold and repeatedly led to extinction of populations. Our results indicate that the high growth rate and virulence of Bd allow the near-simultaneous infection and buildup of high infection intensities in all host individuals; subsequent host population crashes therefore occur before Bd is limited by density-dependent factors. Preventing infection intensities in host populations from reaching this threshold could provide an effective strategy to avoid the extinction of susceptible amphibian species in the wild.

Integration of a Cross-Ancestry Polygenic Model With Clinical Risk Factors Improves Breast Cancer Risk Stratification.

PURPOSE: To develop and validate a cross-ancestry integrated risk score (caIRS) that combines a cross-ancestry polygenic risk score (caPRS) with a clinical estimator for breast cancer (BC) risk. We hypothesized that the caIRS is a better predictor of BC risk than clinical risk factors across diverse ancestry groups. METHODS: We used diverse retrospective cohort data with longitudinal follow-up to develop a caPRS and integrate it with the Tyrer-Cuzick (T-C) clinical model. We tested the association between the caIRS and BC risk in two validation cohorts including > 130,000 women. We compared model discrimination for 5-year and remaining lifetime BC risk between the caIRS and T-C and assessed how the caIRS would affect screening in the clinic. RESULTS: The caIRS outperformed T-C alone for all populations tested in both validation cohorts and contributed significantly to risk prediction beyond T-C. The area under the receiver operating characteristic curve improved from 0.57 to 0.65, and the odds ratio per standard deviation increased from 1.35 (95% CI, 1.27 to 1.43) to 1.79 (95% CI, 1.70 to 1.88) in validation cohort 1 with similar improvements observed in validation cohort 2. We observed the largest gain in positive predictive value using the caIRS in Black/African American women across both validation cohorts, with an approximately two-fold increase and an equivalent negative predictive value as the T-C. In a multivariate, age-adjusted logistic regression model including both caIRS and T-C, caIRS remained significant, indicating that caIRS provides information over T-C alone. CONCLUSION: Adding a caPRS to the T-C model improves BC risk stratification for women of multiple ancestries, which could have implications for screening recommendations and prevention.

Individual and combined effects of multiple pathogens on Pacific treefrogs.

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens.

The application of a newly developed linear stapler preloaded with tissue reinforcement for distal pancreatectomy in the management of ovarian cancer.

Advanced ovarian cancer may extend into the spleen, and even the pancreatic tail, in which a splenectomy associated with distal pancreatectomy is crucial for optimal cytoreduction. A new linear stapler preloaded with tissue reinforcement is currently introduced. We herein report the first three cases of successful application of this device for distal pancreatectomy performed during cytoreductive surgery for ovarian cancer.

The risk of post-molar gestational trophoblastic neoplasia is higher in heterozygous than in homozygous complete hydatidiform moles.

BACKGROUND: Complete hydatidiform mole (CHM) is a high-risk pregnancy for gestational trophoblastic neoplasia (GTN). Patients with CHM have a 10-30% chance of trophoblastic sequelae. CHM includes androgenic homozygous (monospermic) and androgenic heterozygous (dispermic) moles. It is controversial whether the risk of GTN is higher with heterozygous than with homozygous CHM. A prospective cohort study was conducted to assess risk of GTN in homozygous and heterozygous CHM using short tandem repeat (STR) polymorphisms, and a meta-analysis of previous reports. METHODS: Twenty-eight consecutive molar pregnancies were evacuated and followed by regular hCG measurements to detect GTN. Persistent GTN was diagnosed according to the International Federation of Gynecology and Obstetrics 2000 system. Cytogenesis of the mole was determined by STR polymorphisms of molar tissue and parental blood. A meta-analysis of the GTN rate from previous reports was conducted using Mantel-Haenszel methods. RESULTS: Of 28 molar pregnancies, 24 were homozygous and three were heterozygous CHM. The remaining mole was diandric triploidy (a partial hydatidiform mole). Of the 24 homozygous CHMs, six (25%) cases developed GTN and received chemotherapy. Meanwhile, all three cases (100%) of heterozygous mole developed GTN and needed chemotherapy. The GTN risk was higher in heterozygous (P = 0.029, Fisher's exact test) than homozygous moles. A systematic review revealed only five previous reports (with more than 15 cytogenetically diagnosed cases), and the pooled relative risk of persistent GTN for heterozygous mole was not significant (odds ratio, 2.0; 95% confidence interval, 0.98-4.07). CONCLUSIONS: Heterozygous CHM had a higher risk for GTN than homozygous CHM.

Pressurized intraperitoneal aerosol chemotherapy: a review of the introduction of a new surgical technology using the IDEAL framework.

BACKGROUND: The IDEAL (Idea, Development, Evaluation, Assessment, Long-term study) framework is a scheme of investigation for innovative surgical therapeutic interventions. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a procedure based on laparoscopy to deliver intraperitoneal chemotherapy for peritoneal metastases, introduced in 2011. The aim of this article was to review literature on PIPAC and assess whether development of the technique has followed the IDEAL framework. METHODS: A search of MEDLINE and Embase was carried out to identify scientific reports on PIPAC published between January 2000 and February 2019. The studies were categorized according to the IDEAL stages. RESULTS: Eighty-six original research papers on PIPAC were identified. There were 23 stage 0, 18 stage 1, 25 stage 2a and six stage 2b studies. Protocol papers for stage 1, 2b and 3 studies, and trial registrations for stage 2a studies, were also identified. The number of centres publishing reports and the number of publications has increased each year. Overall, there has been progression through the IDEAL stages; however, about 60 per cent of clinical reports published in 2018 were stage 1 Idea-type studies. CONCLUSION: Since its introduction, studies investigating PIPAC have progressed in line with the IDEAL framework. However, the majority of studies reported recently were stage 0 and 1 studies.

ANTECEDENTES: El marco conceptual IDEAL (Idea, Desarrollo, Exploración, Evaluación y Estudio a largo plazo) es un esquema de investigación para intervenciones quirúrgicas innovadoras. La quimioterapia intraperitoneal presurizada con aerosol (Pressurised Intraperitoneal Aerosol Chemotherapy, PIPAC) es un procedimiento introducido en 2011 y basado en la laparoscopia para administrar quimioterapia intraperitoneal en las metástasis peritoneales. El objetivo de este manuscrito era revisar la literatura sobre PIPAC y evaluar si el desarrollo de la técnica se ha hecho siguiendo el marco IDEAL. MÉTODOS: Se realizó una búsqueda en Medline y Embase para identificar publicaciones científicas sobre PIPAC aparecidas entre enero de 2000 y febrero de 2019. Los estudios se clasificaron según las etapas IDEAL. RESULTADOS: Se identificaron 86 trabajos de investigación originales sobre PIPAC. Hubo 23 estudios de la etapa 0, 18 de la etapa 1, 25 de la etapa 2a y 6 de la etapa 2b. También se identificaron protocolos para estudios de las etapas 1, 2b y 3, así como registros de ensayos para estudios de la etapa 2a. El número de centros que publican trabajos y el número de publicaciones ha aumentado cada año. En general, ha habido una progresión a través de las etapas IDEAL; sin embargo, aproximadamente el 60% de los informes clínicos publicados en 2018 fueron estudios tipo "Idea" de etapa 1. CONCLUSIÓN: Desde su introducción, los estudios que investigan PIPAC han progresado en la línea del marco IDEAL. Sin embargo, la mayoría de los estudios publicados recientemente fueron estudios de las etapas 0 y 1.

Serum YKL-40 as a marker for cervical adenocarcinoma.

BACKGROUND: The current study examined the clinical usefulness of YKL-40 in detection and prognosis of uterine cervical cancer. PATIENTS AND METHODS: Serum levels of YKL-40, cancer antigen 125 (CA 125), carbohydrate antigen 19-9 (CA19-9), and squamous cell carcinoma (SCC) antigen were determined by enzyme-linked immunosorbent assay in women with benign gynecologic disease (n=24), cervical malignancy (SCC, n=104; adenocarcinoma, n=37), and age-matched healthy controls (n=45). Immunohistochemical analysis for local YKL-40 expression was carried out on 28 adenocarcinomas. RESULTS: Receiver operating characteristic curve analysis showed that YKL-40 [area under the curve (AUC)=0.882] was significantly better at discriminating adenocarcinoma from healthy control than SCC antigen, CA 125, and CA19-9. For SCC, YKL-40 (AUC=0.898) carried out similarly to SCC antigen and was better than CA 125 and CA19-9. Using a cut-off YKL-40 value of 92.2 ng/ml, sensitivity of YKL-40 in stage I adenocarcinoma (68%) was higher than that of the other three markers (11%-21%). Tumor-associated macrophages showed immunoreactivity for YKL-40 in 2 of 28 adenocarcinoma tissue samples, but adenocarcinoma cells themselves were nonimmunoreactive in all samples. Multivariate Cox regression analysis revealed that elevated pretreatment YKL-40 levels predicted unfavorable prognosis, independent of International Federation of Gynecology and Obstetrics stage and age at diagnosis. CONCLUSIONS: Pretreatment serum YKL-40 level is a possible prognosticator of cervical adenocarcinoma.

Concurrent daily cisplatin and extended-field radiation therapy for carcinoma of the cervix.

The aim of this study was to assess acute toxicities of concurrent low-dose daily cisplatin and extended-field radiation therapy (EFRT) for carcinoma of the uterine cervix. Fifteen women with cervical cancer who were treated with concurrent daily low-dose cisplatin and EFRT were analyzed. Daily cisplatin dose was fixed to 8 mg/m(2), which was determined in the preceding phase I study using pelvic radiotherapy. Twelve patients underwent either combined external beam radiation therapy and intracavitary brachytherapy or external beam radiation therapy alone. Three other patients were treated with adjuvant chemoradiotherapy after surgery. A total dose of EFRT ranged from 40 to 45 Gy, with an additional boost to the gross tumor volume up to 50.4-55 Gy. A median total dose of cisplatin during entire radiation therapy course was 224 mg/m(2) (range, 200-240 mg/m(2)). In 14 of 15 patients (93%), daily cisplatin could be delivered continuously as planned without any modification. Administration of cisplatin had to be interrupted in only one patient for only 3 days. Fourteen patients developed grade 2 or worse leukopenia including five after treatment, grade 2 in four, grade 3 in eight, and grade 4 in two. Grade 3 thrombocytopenia was observed in three patients. Grade 2 or worse anemia was observed in 12. Three patients had grade 3 nonhematologic toxicities, diarrhea in two, and nausea/vomiting in one. Although moderate to severe hematologic toxicities are common, this study suggests that concurrent low-dose daily cisplatin and EFRT are feasible. A cumulative cisplatin dose of greater than 200 mg/m(2) during radiation therapy could be achieved by using daily cisplatin dose of 8 mg/m(2).

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth.

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host's skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.

A peptide aptamer to antagonize BCL-6 function.

BCL-6 is a transcription factor essential for germinal centre B-cell development. The BCL-6 gene is involved in diffuse large-cell lymphoma and overexpressed in other types of non-Hodgkin's lymphoma and in high-grade breast cancer. BCL-6 is a transcriptional repressor whose N-terminal POZ domain mediates protein-protein interactions to exert its effects. Reasoning that disruption of POZ domain-mediated interactions may be an effective route to antagonizing the effects of BCL-6 in lymphoma, we screened a library for peptide aptamers that specifically bind to BCL-6 POZ and not the POZ domains of related proteins and describe here the first of these reagents, Apt48. Apt48 binds BCL-6 POZ in a manner distinct from the transcriptional corepressor SMRT, yet was found to prevent BCL-6-mediated repression of a luciferase reporter gene. Apt48 also reproduced several previously validated effects of BCL-6 inhibition. Notably, expression of the differentiation markers CD69, Blimp-1 and cyclin D2 was increased in B-cell lines when Apt48 was expressed. We also show that expression of Apt48 restores cytokine-mediated growth arrest to BCL-6 overexpressing cells. Thus, we have identified a peptide aptamer that affects a function of BCL-6 that is required to prevent differentiation of proliferating B cells.

Cis-diamminedichloroplatinum(II) nephrotoxicity and its relationship to renal gamma-glutamyl transpeptidase and glutathione.

To investigate the mechanism of cis-diamminedichloroplatinum(II) (cisplatin) nephrotoxicity, male Sprague-Dawley rats were given one injection of cisplatin (6 mg/kg i.v.). Urinary levels of amino acids and gamma-glutamyl transpeptidase were monitored for 8 days; kidney homogenate content of gamma-glutamyl transpeptidase was followed for 50 hr, and that of selenium-dependent glutathione peroxidase and total glutathione was followed for 4 hr. Peak urinary levels of amino acids and gamma-glutamyl transpeptidase occurred 4.5 hr after the i.v. dose. Glutamine, glycine, and ethanolamine were all elevated greater than 20 times that of the control at 4.5 hr and were still significantly elevated at 50 hr. Total renal glutathione content increased 51 +/- 17% (S.D.) of control values 20 min after cisplatin was given, before returning to base-line levels. No depletion of either renal glutathione or glutathione peroxidase was detected over the time interval studied. These results demonstrate an earlier physiological impairment than has hitherto been shown. Furthermore, depletion of glutathione and glutathione peroxidase does not occur in the rat kidney following therapeutic doses of cisplatin, in contrast to the changes observed in cardiac tissue following doxorubicin treatment.

Modular emergency general surgery training: A pilot study of a novel programme.

Introduction Pan-speciality consensus guidance advocates mandatory emergency general surgery (EGS) training modules for specialist registrars (StRs). This pilot study evaluated the impact of EGS modules aimed at StRs over 1 year. Methods Eleven StRs were allocated a focused 4-week EGS module, in addition to the standard 1:12 on-call duty rota, in a tertiary surgical centre. Primary outcome measures included the number of indicative emergency operations and validated Procedure Based Assessments (PBAs) performed, both during the EGS module and over the training year. Results StRs performed a median of 11 (range 5-15) laparotomies during the EGS module versus 31 (range 9-49) over the whole training year. StRs attended 43.7% of available laparotomies during the module (range 24.1-63.7%). EGS modules provided more than one-third of the total emergency laparotomy experience, and a quarter of the emergency colectomy, appendicectomy and Hartmann's procedure experience. There were no differences in EGS module-related outcomes between junior and senior StRs. Significantly more PBAs related to laparotomy and segmental colectomy were completed during EGS modules than the on-call duty rota, at 32% versus 14% (p<0.001) and 48% versus 22% (p=0.019), respectively. Performance levels were maintained following module completion. Conclusions These findings provide an important baseline when considering future modular EGS training.

Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons.

The development of neuronal excitability involves the coordinated expression of different voltage-gated ion channels. We have characterized the expression of two sensory neuron-specific tetrodotoxin-resistant sodium channel alpha subunits, Na(v)1. (SNS/PN3) and Na(v)1.9 (SNS2/NaN), in developing rat lumbar dorsal root ganglia (DRGs). Expression of both Na(v)1.8 and Na(v)1.9 increases with age, beginning at embryonic day (E) 15 and E17, respectively, and reaching adult levels by postnatal day 7. Their distribution is restricted mainly to those subpopulations of primary sensory neurons in developing and adult DRGs that give rise to unmyelinated C-fibers (neurofilament 200 negative). Na(v)1.8 is expressed in a higher proportion of neuronal profiles than Na(v)1.9 at all stages during development, as in the adult. At E17, almost all Na(v)1.8-expressing neurons also express the high-affinity NGF receptor TrkA, and only a small proportion bind to IB4, a marker for c-ret-expressing (glial-derived neurotrophic factor-responsive) neurons. Because IB4 binding neurons differentiate from TrkA neurons in the postnatal period, the proportion of Na(v)1.8 cells that bind to IB4 increases, in parallel with a decrease in the proportion of Na(v)1.8-TrkA co-expressing cells. In contrast, an equal number of Na(v)1.9 cells bind IB4 and TrkA in embryonic life. The differential expression of Na(v)1.8 and Na(v)1.9 in late embryonic development, with their distinctive kinetic properties, may contribute to the development of spontaneous and stimulus-evoked excitability in small diameter primary sensory neurons in the perinatal period and the activity-dependent changes in differentiation they produce.

Analysis of the relationship between enzyme activity and its internal motion using nuclear magnetic resonance: 15N relaxation studies of wild-type and mutant lysozyme.

A mutant lysozyme where R14 and H15 are deleted together has higher activity and a similar binding ability to an inhibitor, trimer of N-acetylglucosamine ((NAG)3), compared with wild-type lysozyme. Since this has been attributed to intrinsic protein dynamic properties, we investigated the relationship between the activity and the internal motions of proteins. Backbone dynamics of the free and the complex forms with the (NAG)3 have been studied by measurement of the 15N T1 and T2 relaxation rates and NOE determinations at 600 MHz. Analysis of the data using the model-free formalism showed that the generalized order parameters (S2) were almost the same in wild-type and mutant lysozyme in unbound state, indicating that the mutation had little effect on the global internal motions. On the other hand, in the presence of (NAG)3, although some signals located around the active site were broadened or decreased in intensity because of strong perturbation by (NAG)3, there were several residues that showed increased or decreased backbone S2 in the complexed lysozymes. A comparison of the internal motions of the wild-type and mutant complexes showed a number of distinct dynamic differences between them. In particular, many residues located at or near active-site regions (turn 1, strand 2, turn 2 and long loop), displayed greater backbone dynamics reflecting the order parameter in mutant complex relative to mutant free. Furthermore, the Rex values at the loop C-D region, which was considered to be important for enzymatic activity, significantly increased. From these results, it was suggested that variations in the dynamics of these regions may play an important role in the enzyme activity.

Tertiary structure of erabutoxin b in aqueous solution as elucidated by two-dimensional nuclear magnetic resonance.

The three-dimensional structure of erabutoxin b, a short-chain neurotoxic peptide purified from the venom of the sea snake Laticauda semifasciata, was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing-based calculations. On the basis of 883 assigned nuclear Overhauser effect (NOE) connectivities, 676 final distance constraints were derived and used together with 38 torsion angle (phi, chi 1) constraints, four distance constraints derived from disulfide bridges and 30 distance constraints derived from hydrogen bonds. A total of 14 converged structures were obtained from 50 runs of calculations. The atomic root-mean-square difference about the mean coordinate positions (excluding the residues 18 to 22) is 0.60 A for backbone atoms (N, C alpha and C'). The protein consists of a core region from which three finger-like loops emerge outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 2 to 5 and 13 to 16, a three-stranded antiparallel beta-sheet involving residues 23 to 30, 35 to 41 and 50 to 56, and four disulfide bridges in the core region. Comparison with two crystal structures of erabutoxin b at 1.4 A and 1.7 A resolution indicated that the solution and the crystal structures were very similar, but less defined regions were observed at the localized region of the tip of the central loop and the outside of the third loop in solution. Other short-chain alpha-neurotoxins showed structural characteristics similar to those of erabutoxin b.

Crystallization and X-ray studies of the DNA-binding domain of OmpR protein, a positive regulator involved in activation of osmoregulatory genes in Escherichia coli.

The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively. The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method. X-ray studies show that the crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with a = b = 60.4 A, c = 58.8 A and gamma = 120 degrees. The asymmetric unit contains one molecule. The crystals diffract to at least 3 A resolution and are suitable for X-ray structure analysis.

NMR structure of the Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23: another example of an ancestral beta gamma-crystallin precursor structure.

The Streptomyces metalloproteinase inhibitor, SMPI, isolated from Streptomyces nigrescens TK-23, is a proteinaceous metalloproteinase inhibitor, and consists of 102 amino acid residues with two disulfide bridges. SMPI specifically inhibits metalloproteinases such as thermolysin. In the present work, the solution structure of SMPI was determined on the basis of 1536 nuclear Overhauser enhancement derived distance restraints and 52 dihedral angle restraints obtained from three-bond spin coupling constants. The final ensemble of 20 NMR structures overlaid onto their mean coordinate with backbone (N, Calpha, C') r.m.s.d. values of 0. 45(+/-0.11) A and 0.57(+/-0.18) A for residues 6 to 99 and the entire 102 residues, respectively. SMPI is essentially composed of two beta-sheets, each consisting of four antiparallel beta-strands. The structure can be considered as two Greek key motifs with 2-fold internal symmetry, a Greek key beta-barrel. One unique structural feature found in SMPI is in its extension between the first and second strands of the second Greek key motif. Interestingly, this extended segment is known to be involved in the inhibitory activity of SMPI. In the absence of sequence similarity, the SMPI structure shows clear similarity to both domains of the eye lens crystallins, both domains of the calcium sensor protein-S, as well as the single-domain yeast killer toxin. The yeast killer toxin structure was thought to be a precursor of the two-domain beta gamma-crystallin proteins, because of its structural similarity to each domain of the beta gamma-crystallins. SMPI thus provides another example of a single-domain protein structure that corresponds to the ancestral fold from which the two-domain proteins in the beta gamma-crystallin superfamily are believed to have evolved.