[Anatomy and vascularization on the male urethra and penis]. / Anatomia y vascularizacion de la uretra y el pene.

In this review we present an update on the anatomy and vascularization of the male urethra. The real objective of this review is to make the following chapters more understandable, both to know the physio-pathological mechanisms of urethral pathology and also to help us in their surgical management.

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame.

Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

Substrates and inhibitors of human T-cell leukemia virus type 1 (HTLV-1) proteinase.

Human T-cell leukemia virus type 1 (HTLV-1) proteinase, a 125 residue polypeptide, was chemically synthesized using the solid phase method. The crude product was purified, renaturated and proteolytic activity was tested using oligopeptide substrates derived from processing sites of various retroviral polyproteins. Cleavage of the oligopeptide substrates together with an initial study using a series of HIV-1 and MAV (myeloblastosis associated virus) proteinase inhibitors suggest that the substrate specificity of HTLV-1 proteinase is very close to that of BLV (bovine leukemia virus) proteinase and distinct from that of both HIV-1 and MAV proteinases.

Solid phase synthesis of the proteinase of bovine leukemia virus. Comparison of its specificity to that of HIV-2 proteinase.

The 126-residue proteinase (PR) of bovine leukemia virus (BLV) was synthesized by solid-phase peptide synthesis and its activity was shown using various oligopeptide substrates representing cleavage sites in BLV, human T-cell leukemia virus type 1 (HTLV-1), murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1). The specificity of the BLV PR was also compared to that of chemically synthesized human immunodeficiency virus type 2 (HIV-2) PR. Many of the peptides were cleaved at the expected site, however, 6 out of 15 were hydrolyzed only by one of the PRs. Furthermore, one BLV peptide was processed differently by the two enzymes. These results, together with the relative activities and the lack of inhibition of BLV PR by two HIV-1 PR inhibitors, suggest that the BLV PR specificity is substantially different from that of HIV PRs.

Studies on the role of the S4 substrate binding site of HIV proteinases.

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.

Subsite specificity of the proteinase from myeloblastosis associated virus.

The subsite requirements of the aspartic proteinase from the myeloblastosis-associated virus (MAV) for the cleavage of peptide substrates were studied with a series of synthetic peptides of general structure Ala-Thr-P4-P3-P2-P1*Nph-Val-Arg-Lys-Ala. The residues in positions P4, P3, P2 and P1 were varied and the kinetic parameters for the cleavage of substrates in 2.0 M NaCl were spectrophotometrically determined at pH 6.0 and 37 degrees C. The acceptance of amino acid residues in particular subsites is similar to that observed with the human immunodeficiency virus type 1 (HIV-1) proteinase in our earlier studies on the same substrate series: hydrophobic or aromatic residues are preferable in P1 position, a broad variety of residues are acceptable in P3 whereas the residues occupying P2 plays the decisive role in the substrate cleavage as evidenced by its dramatic influence on both kcat and Km values. The most remarkable difference between the two enzymes was found in P3 and P4 subsites. In P3, the introduction of negatively charged glutamate increases the substrate binding by the MAV proteinase 12-fold and decreases binding by the HIV-1 proteinase. In P4, Pro in this series is a favourable residue for the MAV proteinase and is strongly inacceptable for HIV-1 the proteinase. The pH profile of the cleavage was studied with a chromogenic substrate and differences between HIV-1 and MAV proteinases are discussed.

Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins.

The substrate specificity of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag-Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (Km and kcat) obtained for the different substrates showed several hundred-fold variation but were similar for the same substrate.

High-level expression of enzymatically active bovine leukemia virus proteinase in E. coli.

An E. coli plasmid expressing efficiently an artificial precursor of bovine leukemia virus (BLV) proteinase under transcriptional control of the phage T7 promoter was constructed. The expression product accumulates in the induced E. coli cells in the form of insoluble cytoplasmic inclusions. Solubilization of the inclusions and a refolding step yield almost pure and completely self-processed proteinase. Purification to homogeneity was achieved by ion-exchange chromatography and reverse-phase HPLC. On a preparative scale, a high yield of enzymatically active proteinase was obtained. An initial study using a series of synthetic peptide substrates shows a distinct substrate specificity of BLV proteinase.

Distribution of tyrosinated and detyrosinated alpha-tubulin revealed by mouse anti-peptide polyclonal antibodies.

Mouse anti-peptide antibodies that specifically react (in competitive ELISA and immunoblotting) with the corresponding C-terminal hepta- and octapeptides of alpha-tubulin differing in the terminal tyrosine and that hence recognize the post-translationally modified forms of alpha-tubulin are described. At the light microscopic level tyrosinated tubulin was demonstrated practically in all structures containing microtubules with the exception of the flagella of the spermatozoa of several species. The presence of detyrosinated Glu tubulin was very limited; occasional interphase microtubules, midbody and flagella of the spermatozoa only exhibited the positive reaction. The results compared to the recently published findings indicate that the different arrangement of microtubules assembled mostly of Glu tubulin can be distinguished by polyclonal antibodies against detyrosinated tubulin.

Ectopic thymus in the neck; a case report and review of the literature.

A soft, poorly defined mass in the right upper neck of a 7-week-old boy was shown on histology to be ectopic thymus. As aberrant thymic tissue often does change into cysts or neoplasms removal is the treatment of choice. Its persistence in the upper neck seems to be very rare. Embryology, incidence, differential diagnosis and treatment are discussed with a review of the literature.

Synthesis of homologous peptides using fragment condensation: analogs of an HIV proteinase substrate.

Two protected peptides Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)OH and Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)-ProOH were synthesized on a resin substituted by 9-(hydroxymethyl)-2-fluoreneacetic acid. After cleavage with piperidine/DMF, desalting, and activation, these peptides were used for the synthesis of 11 analogs of an HIV proteinase nonapeptide substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 using fragment condensation in solid phase. The fragment condensation was made in an ultrasonic bath. Using only 2 equivalents of the activated peptide in a DMF solution, this reaction was complete in 2 h. All nonapeptides were assayed as substrates for HIV-1 and HIV-2 proteinases.

Immunohistochemical detection of bcl-2 protein in liver lesions: bcl-2 protein is expressed in hepatocellular carcinomas but not in liver cell dysplasia.

A proto-oncogene, bcl-2, encodes a protein that inhibits programmed cell death (apoptosis) and may play a role in cell and tissue differentiation. As bcl-2 appears to be involved in the turn-over of stem or precursor cells, it is thought to be operational in carcinogenesis pathways. However, apart from certain lymphomas, only limited data are available on the frequency of its expression in solid tumors. Immunohistochemical analysis with an antibody specific for bcl-2 protein was used to detect the protein in hepatocellular carcinomas and in one of the putative precursor lesions, liver cell dysplasia. We detected bcl-2 protein in 5 of 37 hepatocellular carcinomas. Immunoreactivity was not related to type, grade, or extent of PCNA staining of the tumours. No bcl-2 protein staining was observed in three types of liver cell dysplasia. Thus, bcl-2 is abnormally expressed in some hepatocellular carcinomas but not in potential tumour precursor cells.

Cloning, bacterial expression, and characterization of the Mason-Pfizer monkey virus proteinase.

We have cloned and expressed the 3' region of the Mason-Pfizer monkey virus pro gene in Escherichia coli. The recombinant 26-kDa precursor undergoes rapid self-processing both in E. coli and in vitro at the NH2 terminus, yielding a proteolytically active 17-kDa protein, p17. This initial cleavage is followed in vitro by a much slower self-processing that leads to emergence of proteolytically active p12 and a COOH-terminal cleavage product p5. We have found the NH2-terminal processing site of both the p17 and p12 to be identical and similar to the amino terminus of the mouse mammary tumor virus proteinase. We have also identified the COOH-terminal processing site of the p12 form. Using purified recombinant proteins and synthetic oligopeptide substrates based on naturally occurring retroviral processing sites, we have determined the enzymatic activity and specificity of the Mason-Pfizer monkey virus proteinase to be more closely related to that of myeloblastosis-associated virus proteinase rather than that of the Human immunodeficiency virus type 1 proteinase. Inhibition studies using peptide inhibitors support these results.

Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates.

The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.

[A highly sialylated, embryonic form of the neural cell adhesion molecule in Wilms tumor: identification of a cell adhesion molecule as a onco-developmental antigen]. / Nachweis der hochsialylierten, embryonalen Form des Neuralen Zelladhäsionsmoleküles im Wilms Tumor: Identifizierung eines Zelladhäsionsmoleküles als onko-developmentales Antigen.

The neural cell adhesion molecule NCAM is a general Ca2+ -independent cell adhesion molecule which exerts important functions during the development of the nervous system. NCAM polypeptides exist in various isoforms all of which have a similar extracellular domain structure containing a homophilic binding site for establishment of cell-cell contacts. A common structural feature of NCAM is the presence of homopolymers of alpha 2,8-linked sialic acid residues, the so-called polysialic acid, which is developmentally regulated. It undergoes a change from a highly less sialylated short chain form from embryonic to adult life. The polysialic acid regulates the homophilic adhesive properties of NCAM. The highly sialylated form of NCAM typically found in developing tissues decreases homophilic NCAM-NCAM interactions and, therefore, cell-cell contacts due to its large excluded volume and electric repulsive forces. We have used a monoclonal antibody against polysialic acid, polyclonal antibodies against NCAM, polysialic acid specific bacteriophage-encoded endoneuraminidases and a NCAM cDNA to investigate this molecule by light and electron microscopic immunolabeling, in situ hybridization and immunochemistry. The highly sialylated form of NCAM was found to be expressed in a developmentally-regulated fashion in embryonic kidney, undetectable in the normal adult kidney and re-expressed in Wilms tumor. In Wilms tumor the blastemal cell masses as well as epithelial differentiations such as tubules and glomeruloid bodies were immunolabeled whereas the stroma did not label for polysialic acid. Combination of immunoprecipitation and immunoblotting using antibodies against polysialic acid and NCAM, respectively, directly demonstrated structural relationship of renal polysialic acid with NCAM polypeptide. By immunoblot analysis two NCAM isoforms of approximately 120 and 140 kD were found in Wilms tumor. Immuno-electron microscopy provided direct morphological evidence for prevention of cell-cell contacts due to the presence of a thick cell surface coat composed of polysialic acid. In nephroblastomatosis complexes only blastemal cells exhibited immunocytochemically detectable polysialic acid. All other investigated kidney tumors, i.e. clear cell sarcoma, malignant rhabdoid tumor, cystic nephroma and renal cell carcinoma, were negative. These data allow the conclusion that the highly sialyated, embryonic form of NCAM is an onco-developmental antigen in human kidney. At the same time this is the first observation that a molecule with a well defined role for controlled cell migration and differentiation during embryonic organ development represents an onco-developmental antigen.

Detection of hepatitis B virus DNA in the liver of children with chronic hepatitis B by in situ hybridization and its relation to other viral markers.

The aim of the study was to detect hepatitis B virus (HBV) DNA by in situ hybridization (ISH) with a 35S-labeled radioactive probe in frozen liver biopsy tissue sections of 63 hepatitis B virus surface antigen (HBsAg)-positive children. The results were compared to other markers of viral replication. HBV DNA was detected in 48 children. Of the 15 negative cases, four had hepatitis B envelope antigen (HBeAg), 10 anti-HBe, and one neither HBeAg nor anti-HBe. Free HBV DNA in serum and liver was positive in one patient. Forty of the positive children were HBeAg- and six anti-HBe-positive; two were negative for both. Of 45 36 had HBV DNA in serum. In 38 of 47 HBV DNA and in 31 of 42 HBcAg could be detected in the liver. The HBV DNA signals were located mainly over the cytoplasm of hepatocytes. The distribution of HBV DNA in the tissue was classified as homogeneous, inhomogeneous with focal patches, and focal. It is concluded that in situ hybridization is a reliable method for detection of HBV DNA in liver tissue of children with chronic hepatitis B. The technique, which can be applied to small amounts of liver tissue, provides informations about the distribution of replicative viral sequences, complementing laboratory data, liver histochemistry, and histology.

Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.

The long chain form of polysialic acid characteristic of the low adhesive embryonic form of the neural cell adhesion molecule NCAM is temporally and spatially expressed in developing kidney but undetectable in normal adult kidney. Therefore, this molecule represents a developmentally regulated antigen in kidney contrasted with neural tissue, where it is also detectable in the adult brain. This investigation of 25 Wilms' tumors comprising all different histologic types demonstrates expression of this molecule under conditions of malignant growth. Immunostaining was observed in Wilms' tumors with both a monoclonal anti-polysialic acid antibody and a polyclonal anti-NCAM polypeptide antiserum. Intense cell surface staining sensitive to endosialidases specifically hydrolyzing alpha 2,8 linked (poly)sialic acid was detectable in blastemal regions, and weaker, variable labeling was seen over tubules and glomeruloid bodies. The stroma was not stained. This is evidence indicating that Wilms' tumor originates from the embryonic equivalent of induced metanephrogenic mesenchyme. It seems unlikely however, that the stroma is derived from the blastema. The same high molecular mass broad band typical of the embryonic form of NCAM was revealed by immunoblot analysis of homogenates from Wilms' tumor as well as from embryonic kidney and brain. In situ hybridization demonstrated the presence of mRNA for NCAM in all but stromal elements of Wilm's tumors. Thus, polysialic acid is present on NCAM and represents a new oncodevelopmental antigen in human kidney. Polysialic acid was greatly reduced or absent by immunohistochemistry and immunoblotting in necrotic tumor areas.

Blastemal cells of nephroblastomatosis complex share an onco-developmental antigen with embryonic kidney and Wilms' tumor. An immunohistochemical study on polysialic acid distribution.

Previous investigations on polysialic acid of the neural cell adhesion molecule NCAM in human kidney have demonstrated its presence during nephrogenesis in embryonic kidney, absence in normal adult kidney, and reexpression in Wilms' tumor. These data showed that polysialic acid of NCAM is an onco-developmental antigen in human kidney and provided more direct evidence for the metanephric origin of Wilms' tumor. In the present study, five cases of Wilms' tumor associated with nephroblastomatosis complexes were immunohistochemically investigated with a monoclonal antibody for the presence of polysialic acid. Regardless of the type of nephroblastomatosis complex, ie, renal nodular blastema, simple tubular metanephric hamartoma, sclerosing metanephric hamartoma with adenoma, or incipient Wilms' tumor, immunoreactivity for polysialic acid was found in the blastemal cells, but was undetectable in all other structural elements. Because only blastemal cells exhibited a characteristic feature of embryonal differentiating metanephric derivatives, it appears that Wilms' tumor has its origin not exclusively in nodular renal blastema but rather in blastemal cells present in the various forms of nephroblastomatosis complex. The presence of polysialic acid of NCAM in blastemal cells in such lesions indicates that further events in addition to the expression of the embryonic form of this cell adhesion molecule may be involved in the pathogenesis of Wilms' tumor.