En bloc resection for malignant colouterine fistula.

Colouterine fistula is a rare clinical entity. A literature search revealed only a few reports dealing with this complex problem, mostly resulting as a complication of diverticular disease of the colon. During a 4-month period, we diagnosed and successfully treated 2 women with a malignant colouterine fistula originating from a primary colorectal carcinoma invading the uterus. We herein report on our experience dealing with this kind of pathology, with special emphasis on the surgical technique used to resect the tumoral mass "en bloc".

A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.

A bicyclic and hsst2 selective somatostatin analogue: design, synthesis, conformational analysis and binding.

A backbone bridged and disulfide bridged bicyclic somatostatin analogue, compound 1 (PTR-3205), was designed and synthesized by solid-phase methodology. The binding of compound 1 to the five different somatostatin receptors, expressed in CHO or COS-7 cells, indicate a high degree of selectivity towards hsstr2. The three-dimensional structure of this compound has been determined in DMSO-d(6) and in water by 1H NMR and by molecular dynamics simulations. Similar backbone conformations were observed in both solvents. We have established direct evidence that the backbone of this bicyclic somatostatin analogue assumes a 'folded' conformation in solution, where the lactam ring extends roughly in the plane of the beta-turn. The pharmacophoric region Phe-(D)-Trp-Lys-Thr of compound 1 is in accord with that of both the Veber compound L-363,301 (Merck) and sandostatin. We believe that the enhanced selectivity towards the hsst2 receptor, in comparison with other analogues, is due to its large hydrophobic region, composed of the lactam ring and the Phe side chains at positions 1 and 8.

Novel long-acting somatostatin analog with endocrine selectivity: potent suppression of growth hormone but not of insulin.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a natural cyclic peptide inhibitor of pituitary, pancreatic, and gastrointestinal secretion. Its long-acting analogs are in clinical use for treatment of various endocrine syndromes and gastrointestinal anomalies. These analogs are more potent inhibitors of the endocrine release of GH, glucagon, and insulin than the native SRIF; hence, they do not display considerable physiological selectivity. Our goal was to design effective and physiologically selective SRIF analogs with potential therapeutic value. We employed an integrated approach consisting of screening of backbone cyclic peptide libraries constructed on the basis of molecular modeling of known SRIF agonists and of high throughput receptor binding assays with each of the five cloned human SRIF receptors (hsst1-5). By using this approach, we identified a novel, high affinity, enzymatically stable, and long-acting SRIF analog, PTR-3173, which binds with nanomolar affinity to human SRIF receptors hsst2, hsst4, and hsst5. The hsst5 and the rat sst5 (rsst5) forms have the same nanomolar affinity for this analog. In the human carcinoid-derived cell line BON-1, PTR-3173 inhibits forskolin-stimulated cAMP accumulation as efficiently as the drug octreotide, indicating its agonistic effect in this human cell system. In hormone secretion studies with rats, we found that PTR-3173 is 1000-fold and more than 10,000-fold more potent in inhibiting GH release than glucagon and insulin release, respectively. These results suggest that PTR-3173 is the first highly selective somatostatinergic analog for the in vivo inhibition of GH secretion, with minimal or no effect on glucagon and insulin release, respectively.

'Key paper' for covalent binding of proteins and its uses.

A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed 'Key paper' is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins.

Selective binding of lipid to glycoprotein was detected when [3H]palmitate-labeled Sindbis virus particles or viral-infected cells were disrupted by heating with sodium dodecyl sulfate, and glycoproteins were isolated by electrophoresis in sodium dodecyl sulfate/10% polyacrylamide gels. The smaller glycoprotein (E2) retained 2 to 3 times more labeled lipid than did the larger EI glycoprotein, and the cell-associated glycoprotein precursor (PE2) bound even less lipid. No lipid was associated with the nonglycosylated glycoproteins that accumulated in infected cells treated with tunicamycin. The labeled lipid remained bound to the glycoproteins after exhaustive extraction with chloroform/methanol of virus particles, infected-cell extracts, or isolated glycoproteins, but it could be extracted by chloroform/methanol after treating glycoproteins with mild alkali. Analysis by gas/liquid chromatography showed that 60% of the label was in palmitate and the balance of label was distributed between oleate and stearate. There were approximately 2 mol of fatty acid bound per mol of E1 glycoprotein. Proteolysis of the fatty acid-labeled glycoprotein with pepsin, thermolysin, and Pronase degraded the polypeptide to fragments that retained the fatty acids in an alkali-labile state. These data suggest that a covalent attachment of fatty acid may occur during maturation of the viral glycoproteins.

Altered E2 glycoprotein of Sindbis virus and its use in complementation studies.

We have detected a Sindbis virus variant that contains a smaller-molecular-weight form of the viral glycoprotein E2. The molecular weight of the PE2 precursor and the glycosylation pattern of the smaller E2 are normal, thus indicating that this E2 is formed by an aberrant proteolytic cleavage. The altered E2 was detected in an RNA+ temperature-sensitive mutant that was defective in proteolytic cleavage, but the abnormal PE2-to-E2 reaction could be separated from the ts mutation and is not itself a temperature-sensitive defect. We used the variant E2 as a marker to monitor the complementation reaction between an RNA+ and an RNA- mutant and discovered that complementation was not reciprocal; the RNA defect was corrected by the RNA+ mutant gene products but the RNA+ defect was not complemented by any RNA- gene products. Other studies have shown that the smaller E2 is not preferentially selected during viral maturation and budding. No significant changes have been detected in the biological activity of virions with this altered E2 protein. Comparison of the electrophoretic migration of the E1 and E2 Sindbis viral glycoproteins in a two-dimensional polyacrylamide slab gel system that was first run in the absence of sulfhydryl-reducing reagent and then with beta-mercaptoethanol indicated that the mobility of E1, but not that of E2, was significantly altered by reduction.

Formation of a Sindbis virus nonstructural protein and its relation of 42S mRNA function.

Chicken embryo fibroblasts infected with an RNA- temperature-sensitive mutant (ts24) of Sindbis virus accumulated a large-molecular-weight protein (p200) when cells were shifted from the permissive to nonpermissive temperature. Appearance of p200 was accompanied by a decrease in the synthesis of viral structural proteins, but [35S]methionine tryptic peptides from p200 were different from those derived from a 140,000-molecular-weight polypeptide that contains the amino acid sequences of viral structural proteins. Among three other RNA- ts mutants that were tested for p200 formation, only one (ts21) produced this protein. The accumulation of p200 in ts24- and ts21-infected cells could be correlated with a shift in the formation of 42S and 26S viral RNA that led to an increase in the relative amounts of 42S RNA. These data indicate that p200 is translated from the nonstructural genes of the virion 42S RNA and further suggest that this RNA does not function effectively in vivo as an mRNA for the Sindbis virus structural proteins.

The regulatory nature of the phoB gene for alkaline phosphatase synthesis in Escherichia coli.

Quantitative measurements of alkaline phosphatase activity in various merozygotic combinations and electrophoretic analyses of periplasmic proteins derepressed by phosphate-starvation show that phoB is a positive regulatory gene.

Location of the genes controlling alkaline phosphatase on the F13 episome of Escherichia coli.

Interrupted mating experiments between F'13 and F(-) cells showed that the location on the F'13 episome of the genes controlling alkaline phosphatase is on the end proximal to the point of entry, in the order phoA proC phoB phoR tsx.

Pleiotropic effects of mutations involved in the regulation of Escherichia coli K-12 alkaline phosphatase.

Induction of alkaline phosphatase in wild-type Escherichia coli K-12 leads to the appearance of three new proteins in addition to alkaline phosphatase in the periplasmic space of the bacteria. These proteins are detected in autoradiograms of sodium dodecyl sulfate-acrylamide gel electropherograms of extracts from cells labeled with [(35)S]methionine. Studies with constitutive mutants defective in the three genes phoS, phoT, and phoR that have been shown to regulate alkaline phosphatase synthesis indicate that the three periplasmic proteins are coregulated with alkaline phosphatase. A mutant that has a deletion in the alkaline phosphatase structural gene phoA produces the three proteins, but a newly discovered mutant phoB that has a defect in the expression of alkaline phosphatase fails to produce the three proteins. phoB mutants are shown here to be unable to make detectable amounts of alkaline phosphatase polypeptides, as measured by immunoprecipitins or acrylamide gel electropherograms. On the basis of these results we suggest a new model for the regulation of alkaline phosphatase biosynthesis. In this model, a ternary complex composed of phoB(+) and phoR(+) gene products and an internal metabolite functions as a positive control element to regulate the transcription of several cistrons coding for periplasmic proteins.

Bacterial conjugation: an analysis of mixed recombinant clones.

A fraction of recombinant colonies resulting from conjugation is heterogenetic for unselected markers. Constitutivity for alkaline phosphatase synthesis (phoR) is studied as the unselected marker. The frequency of phoR heterogeneity depends on the genetic distance between phoR and the selected marker. Various models are considered which explain the formation of heterogenetic colonies (mixed clones), and experiments are described which test these models. It is concluded that the Hfr fragment can replicate and participate more than once in recombination thus yielding heterogenetic colonies.