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Protein science 28(12), 2112-2118 (2019). doi:10.1002/pro.3739, Flavin-dependent halogenases require reduced flavin adenine dinucleotide (FADH2 ), O2 , and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6-halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases., Published by Protein Society, Bethesda, Md.

The physiological relevance of contacts in crystal lattices often remains elusive. This was also the case for the complex between the invasion protein internalin B (InlB) from Listeria monocytogenes and its host cell receptor, the human receptor tyrosine kinase (RTK) MET. InlB is a MET agonist and induces bacterial host cell invasion. Activation of RTKs generally involves ligand-induced dimerization of the receptor ectodomain. The two currently available crystal structures of the InlB:MET complex show the same arrangement of InlB and MET in a 1:1 complex, but different dimeric 2:2 assemblies. Only one of these 2:2 assemblies is predicted to be stable by a computational procedure. This assembly is mainly stabilized by a contact between the Cap domain of InlB from one and the Sema domain of MET from another 1:1 complex. Here, we probe the physiological relevance of this interaction. We generated variants of the leucine-rich repeat (LRR) protein InlB by inserting an additional repeat between the first and the second LRR. This should allow formation of the 1:1 complex but disrupt the potential 2:2 complex involving the Cap-Sema contact due to steric distortions. A crystal structure of one of the engineered proteins showed that it folded properly. Binding affinity to MET was comparable to that of wild-type InlB. The InlB variant induced MET phosphorylation and cell scatter like wild-type InlB. These results suggest that the Cap-Sema interaction is not physiologically relevant and support the previously proposed assembly, in which a 2:2 InlB:MET complex is built around a ligand dimer.

Hyaluronic acid was obtained from filtrates of heat-killed cultures of Streptococcus pyogenes group A, strain K56, by simple ethanol precipitation and treatment with an adsorbent. The hyaluronic acid is pure as judged from chemical and sedimentation analyses. Particles of streptococcal bacteriophage 12/12 were isolated from phage-lysed group A streptococci by polyethylene glycol precipitation and isopyenic centrifugation. Electron micrographs of negatively stained preparations showed a typical Bradley group B virus with a long, flexible, cross-striated tail and a knob- or star-like structure at the distal tip of the tail. The hyaluronic acid is depolymerized upon incubation with the phage 12/12 virions. After extensive digestion, a mixture of at least four oligosaccharides is formed, the two smallest of which are a tetra- and octasaccharide terminating in reducing N-acetyl-D-glucosamine. The tetrasaccharide shows an absorption maximum at 231.5 nm with a molar extinction coefficient epsilon = 4820 litres X mole-1 X cm-1, and it is therefore concluded that the bacteriophage-borne hyaluronidase catalyses a beta-elimination. Accordingly it is classified as a hyaluronate lyase (EC 4.2.99.1).

PrP(C)-deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP(DeltaF)) suffer from neurodegeneration, which is rescued by full-length PrP(C). We now report that expression of PrP(DeltaCD), a PrP variant lacking 40 central residues (94-134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP(C) or PrP(C) lacking all octarepeats. Expression of a PrP(C) variant lacking eight residues (114-121) was innocuous in the presence or absence of full-length PrP(C), yet enhanced the toxicity of PrP(DeltaCD) and diminished that of PrP(DeltaF). Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP(C), whereas removal of residues 114-121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP(C), whose effector domain encompasses residues 94-134.

Pathogenic Yersinia species use a type III secretion (TTS) system to deliver a number of cytotoxic effector proteins directly into the mammalian host cell. To ensure effective translocation, several such effector proteins transiently bind to specific chaperones in the bacterial cytoplasm. Correspondingly, SycT is the chaperone of YopT, a cysteine protease that cleaves the membrane-anchor of Rho-GTPases in the host. We have analyzed the complex between YopT and SycT and determined the structure of SycT in three crystal forms. Biochemical studies indicate a stoichometric effector/chaperone ratio of 1:2 and the chaperone-binding site contains at least residues 52-103 of YopT. The crystal structures reveal a SycT homodimer with an overall fold similar to that of other TTS effector chaperones. In contrast to the canonical five-stranded anti-parallel beta-sheet flanked by three alpha-helices, SycT lacks the dimerization alpha-helix and has an additional beta-strand capable of undergoing a conformational change. The dimer interface consists of two beta-strands and the connecting loops. Two hydrophobic patches involved in effector binding in other TTS effector chaperones are also found in SycT. The structural similarity of SycT to other chaperones and the spatial conservation of effector-binding sites support the idea that TTS effector chaperones form a single functional and structural group.

Summary Two Chinese domestic yak populations representing the Plateau type and the Huanhu Alpine type were analysed with 12 bovine microsatellite primers. All primer pairs functioned in the yak genome and polymorphism was found at all loci. The allele size ranges and frequencies of the two yak populations were similar and there was considerable overlap with the allele size ranges observed in cattle. Data for European cattle breeds was obtained from the Cattle Diversity Database (CaDBase) to interpret the heterozygosity and genetic distance estimates in yak populations. Heterozygosity estimated for the two yak populations was comparable with that of European cattle while Nei's Genetic Distance DA between the two yak populations was less than distances between the most closely related German cattle breeds. Bovine microsatellite primers proved to be a valuable tool for characterization of yak populations. Zusammenfassung Untersuchungen von zwei chinesischen Yak (Bos grunniens) Herden mittels Primersequenzen von Rindermikrosatelliten Primersequenzen von Rindermikrosatelliten wurden verwendet, um zwei chinesische Yak-Herden zu analysieren, eine vom Plateau-Typ und eine andere vom Huanhu-Bergtyp. Alle 12 Primerpaare fuhrten zu polymorphen Amplifikationsprodukten mit den DNA-Proben der Yaks. Der Allelgrosenbereich und die Allelfrequenzen aller 12 Marker waren in den beiden Yak-Herden sehr ahnlich. Zum Vergleich wurden Typisierungsergebnisse der 12 Mikrosatellitengenorte von 15 europaischen Rinderassen aus der Rinderdiversitatsdatenbank CaDBase entnommen. Ein groser Teil der Yak-Allele hatte die gleiche Grose wie beim Rind und auch der geschatzte Heterozygotiegrad der beiden Yak-Populationen war vergleichbar mit dem durchschnittlichen Heterozygotiegrad der 15 Rinderrassen. Die genetische Distanz (Nei's DA) zwischen beiden Yak-Herden wurde jedoch geringer geschatzt als zwischen zwei Paaren eng verwandter Rinderrassen. Die Ergebnisse zeigen, dass Sequenzinformationen von Rindermikrosatellitenprimern fur die Charakterisierung von Yak-Populationen auf der molekularen Ebene nutzlich sein konnen.

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self-assembles into rings and helices in a nucleotide-dependent manner, similar to human dynamin-1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three-dimensional map, which shows that the nucleotide-free complex consists of two layers with 11-fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide-dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission.

BACKGROUND Evaluation of effusion specimens for the presence of adenocarcinoma often is complicated by the presence of reactive mesothelial cells that can mimic adenocarcinoma. Ancillary studies, in particular immunohistochemistry, can be helpful in making this distinction. MOC-31 is an antibody that recently was reported to be useful in distinguishing adenocarcinoma from mesothelioma in tissue specimens. In this study we examined the utility of this antibody in pleural effusions. METHODS Eighty-nine archival, formalin fixed, paraffin embedded cell blocks representing 59 adenocarcinomas, 12 other neoplasms (including 6 mesotheliomas), and 18 reactive effusions were retrieved. After protease digestion, recut slides were immunostained with the MOC-31 antibody utilizing a modified avidin-biotin complex technique. Only membrane-based reactivity was considered as positive. RESULTS In two adenocarcinomas there was insufficient material remaining in the cell block. Among the 57 remaining cases, reactivity was observed in 54 cases. Reactivity also was observed in one of six mesotheliomas and one small cell carcinoma. The remaining cases, including all 18 reactive effusions, were nonreactive. In distinguishing adenocarcinoma from reactive mesothelial cells, the presence of MOC-31 reactivity was found to be 95% sensitive and 100% specific with a positive predictive value of 100% and a negative predictive value of 95%. CONCLUSIONS MOC-31 is useful in differentiating between adenocarcinoma and reactive mesothelial cells in pleural effusion specimens. Cancer (Cancer Cytopathol) 1999;87:390–4. © 1999 American Cancer Society.

BACKGROUND Fine-needle aspiration biopsy (FNAB) is frequently used to diagnose mass lesions in the liver. Differentiating metastatic adenocarcinoma from primary hepatocellular carcinoma can be difficult. Despite a number of morphologic criteria, there remain occasional cases in which the cytologic features fail to resolve this differential reliably; in these cases ancillary studies may be useful. Recently, it has been reported that the antibody MOC-31 reliably separates metastatic adenocarcinoma from hepatocellular carcinoma. In this study we examine the utility of MOC-31 in liver FNAB material. METHODS Thirty-three archival, alcohol-fixed, paraffin-embedded cell blocks representing 17 cases of hepatocellular carcinomas and 16 cases of metastatic adenocarcinoma were retreived. After protease digestion, the sections were immunostained with the antibody MOC-31 (Dako, Carpinteria, CA) utilizing a modified avidin-biotin complex technique. Only membrane-based reactivity was considered positive. RESULTS In five cases there was insufficient diagnostic material remaining in the cell block for immunohistochemical staining. Among the remaining cases, MOC-31 reactivity was observed in 10 of 12 metastatic adenocarcinomas and 2 of 16 hepatocellular carcinomas. For metastatic adenocarcinoma the presence of MOC-31 reactivity yields a sensitivity of 83%, a specificity of 87%, a positive predictive value of 83%, and a negative predicitive value of 87%. CONCLUSIONS MOC-31 is useful in separating metastatic adenocarcinoma from hepatocellular carcinoma in FNAB cell block material. Cancer (Cancer Cytopathol) 1999;87:295–8. © 1999 American Cancer Society.

This report presents the fine-needle aspiration biopsy (FNAB) findings of a multicystic renal tumor in a 52-yr-old woman. The aspirate smears contained clusters of cells with large, irregular nuclei and cytoplasmic vacuoles. Subsequent nephrectomy revealed a multicystic nephroma (MCN). Although most common in childhood, MCN should always be considered in the FNAB differential diagnosis of a multicystic renal mass in adult patients. Even in cases where the diagnosis of MCN is considered, it may be difficult to distinguish from cystic renal cell carcinoma on the basis of radiographic and FNAB findings.

Background. Resistance to chemotherapy is common in bulky hypoxic tumors such as epithelial ovarian cancer. Hyperbaric oxygen (HBO) oxygenates hypoxic tissues and promotes neovascularization. These unique properties of HBO may help overcome chemotherapy resistance by increasing both tumor perfusion and cellular sensitivity. This study was undertaken to determine if HBO increases the response of epithelial ovarian cancer to cisplatin chemotherapy. Methods. In Phase I,64 nu/nu mice were divided into four groups and subcutaneously inoculated with cells from the A2780 human epithelial ovarian cancer cell line. Group 1 served as controls. Group 2 received weekly intraperitoneal cisplatin (3.15 mg/kg). Group 3 was exposed to HBO (dives) at 2.4 atmospheres absolute pressure for 90 minutes, 7 days a week. Group 4 received both cisplatin and HBO. In Phase II, 72 mice were divided into two groups and similarly inoculated. Both groups received weekly intraperitoneal cisplatin (2.5 mg/kg). Group 1 was not exposed to HBO. Group 2 was exposed to HBO for 5 days a week. Results. Dramatic tumor neovascularization was found in tumors of mice exposed to HBO (P = 0.0001). There was significant (P = 0.014) tumor growth retardation in Phase I for mice receiving both cisplatin and HBO compared with those treated with cisplatin alone. This significance was noted after just two doses of cisplatin but subsequently lost due to reduced numbers of mice. In Phase II, neovascularization was detectable after 10 HBO treatments (2 weeks) and was maximal after 15 treatments (3 weeks). Conclusions. Hyperbaric oxygen increases vascularity in bulky tumors such as epithelial ovarian cancer. There appears to be a relationship between increased vascularity and enhanced response to chemotherapy that merits further investigation. Cancer 1995 ;75 :2313-22.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Contents: Biotechnological procedures are valuable tools for an efficient reproductive management in a commercial pig production. Artificial insemination, puberty induction, estrus synchronization, early pregnancy diagnosis and induction of parturition are routinely used since years already and have significant impact for the herd management of sows. Embryo transfer (ET) can also be efficiently applied. But surgical procedures are required for the recovery and transfer of embryos. ET related biotechniques such as cryopreservation of ova and embryos, in vitro production of embryos, cloning and gene transfer are still at an experimental stage but will most likely gain significance shortly. Inhalt: Anwendungsmoglichkeiten biotechnologischer Verfahren bei der Reproduktionssteuerung weiblicher Schweine Biotechnologische Verfahren sind wertvolle Hilfsmittel fur ein effizientes Fortpflan-zungsmanagement in der Schweineproduktion. Die kunstliche Besamung, die Auslosung der Geschlechtsreife, die Zyklussynchronisation, die fruhzeitige Trachtigkeits-diagnose sowie die Geburtsauslasung werden teilweise bereits seit vielen Jahren ge-nutzt und haben ganr erhebliche Bedeutung beim Herdenmanagement von Sauen. Auch die Embryotransfer-Technologie (ET) kann beim Schwein erfolgreich ange-wandt werden. Jedoch konnen Embryonen derzeit nur chirurgiich gewonnen und ubertragen werden. ET-assoziierte Biotechniken wie die Tiefgefrierkonservierung von Eizellen und Embryonen, die In-vitro-Produktion von Embryonen, das Klonen und der Gentransfer befinden sich derzeit noch im Experimentalstadium, werden aber in naher Zukunft wachsende Bedeutung erlangen.

Cytologic brushing is a safe and specific procedure for diagnosing carcinoma of the distal bile duct and proximal pancreatic duct. The vast majority of these lesions are pure adenocarcinomas. Occasionally, however, other morphologic subtypes may be encountered. We report our experience with two adenosquamous carcinomas of the bile duct diagnosed by cytologic brushing. Both patients presented clinically with jaundice and were found to have mass lesions obstructing the bile duct. The brush specimens were cellular and contained a mixture of glandular and squamous elements. The glandular component was characterized by cohesive aggregates of cells with hyperchromatic, overlapping nuclei. The squamous component contained clusters and individual cells with hyperchromatic oval-to-spindled nuclei and orangeophilic cytoplasm. Focally, the squamous elements appeared to gradually merge into the glandular component, and there were clusters of hybrid cells which were difficult to classify as squamous or glandular. Although uncommon, these 2 cases demonstrate that the cytologic features of adenosquamous carcinoma can be appreciated on cytologic brushing specimens.

— The specific activities of the enzymes responsible for remethylation of homocysteine to methionine. N5-methyltetrahydrofolate-homocysteine methyltransferase and betaine-homocysteine methyl-transferase, and of the enzyme responsible for transferring the β-carbon atom of serine to tetrahydrofolate. serine tetrahydrofolate 5,10-hydroxymethyltransferase, have been measured in brain, liver and kidney of the developing Rhesus monkey from mid-gestation, from birth and neonatal life to maturity. The specific activity of N5-methyltetrahydrofolate-homocysteine methyltransferase in all tissues is higher during late gestation and shortly after birth than it is in the adult, and in brain and liver it shows a positive correlation with increasing gestational age. Betaine-homocysteine methyltransferase activity is not measurable in brain. In liver it increases linearly during fetal and neonatal development until values found in the adult are reached. In kidney there is a sharp linear increase during the period of gestation studied. The drop at birth is followed by a sharp increase back to values noted at the end of gestation and thereafter a slow increase to values found in the adult. The specific activity of serine-tetrahydrofolate 5,10-hydroxymethyltransferase tends to be higher in fetal and early neonatal brain than it is in adult brain, whereas in liver and kidney it is low in the fetus and rises during development to reach values found in the adult some months after birth.

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