Pielolitotomía laparoscópica en riñón ectópico pélvico: informe de un caso y revisión de la literatura / Laparoscopic pyelolithotomy in pelvic ectopic kidney: Case report and literature review

Introducción: Los riñones pélvicos son una malformación congénita poco frecuente, pero con una tasa de complicaciones no despreciable, entre ellas el desarrollo de litiasis renales. Su manejo quirúrgico puede resultar complejo. El objetivo de este trabajo fue realizar una revisión de la literatura disponible sobre el tratamiento de la litiasis en riñones ectópicos.Material y métodosDescripción de un caso de pielolitotomía laparoscópica transperitoneal para el tratamiento de litiasis calicial inferior en riñón pélvico derecho. Se realizó una revisión de la literatura mediante PubMed. Se buscaron los siguientes términos: «pelvic ectopic kidney», « ureterorenoscopy», «extracorporeal lithotripsy», «NLPC», «pyelolithotomy». Se incluyeron artículos originales, metaanálisis, revisiones e informes de casos.ResultadosSe excluyeron 130 artículos por título o duplicación. Se evaluaron 62 resúmenes y 50 artículos de texto completo. La tasa libre de cálculos fue del 75% (LEOCH), 85% (URS-f), 85-90% (NLPC) y 100% (pielolitotomía laparoscópica).ConclusiónFactores como el tamaño de la litiasis, densidad y localización de la misma, así como las alteraciones anatómicas del tracto urinario superior, influyen en la elección de la vía de abordaje terapéutica (retrógrada, percutánea y/ o laparoscópica/robótica). (AU)

Introduction: Pelvic kidney is a rare congenital anomaly. The ectopic kidney is more susceptible to developing lithiasis. The management of this type of lithiasis is a challenge. The objective of this paper was to conduct a review of available literature on the treatment of stone in ectopic kidney.Material and methodsDescription of a case of transperitoneal laparoscopic pyelolithotomy for the treatment of inferior calyceal lithiasis in a right pelvic kidney. A literature review was performed by using Pubmed. The following terms and combination terms were searched: «pelvic ectopic kidney», «ureterorenoscopy», «extracorporeal lithotripsy», «PCNL», «pyelolithotomy». We included original articles, meta-analysis, review and case reports.Results130 articles were excluded by title or duplication. 62 abstracts articles and them 50 full text articles were evaluated. Stone free rate were 75% (SLW), 85% (URSf), 85-90% (PCNL) and 100% (laparoscopic pyelolithotomy). The literature on treatment on pelvic kidney is poor.ConclusionFactors such stone size, density and location, and upper urinary tract abnormalities, influence the choice of therapeutic approach (retrograde, percutaneous and/or laparoscopic/robotic). Laparoscopic pyelolithotomy is a safe and minimally invasive treatment option for large kidney stones with unfavorable anatomy for the endoscopic approach. (AU)

[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.

Anatomía y vascularización de la uretra masculina / Anatomy and vascularization of the male urethra and penis

En la presente revisión presentamos una actualización de la anatomía y vascularización de la uretra masculina. En realidad con esta revisión intentamos que los próximos capítulos del monográfico sean más accesibles a la compresión, tanto para conocer los mecanismos fisiopatológicos de la patología uretral como para ayudarnos al manejo quirúrgico de la misma

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

Non-sequential vasopressin peptides. Stereochemistry and biological activity.

Two cyclic disulfides of structure Cys-Tyr-Arg-Arg-Tyr-Cys-NH2 (1) and Cys-Tyr(Me)-Arg-Arg-Tyr(Me)-Cys-NH2 (2), two nonapeptide derivatives of 1 extended at the C-terminal with Pro-Arg-Gly-NH2 (3) or Pro-D-Arg-Gly-NH2 (4) and derivatives of 3 and 4 having Mpr in position 1, i.e. analogs (5) and (6), respectively, were synthesized, and their stereochemistry and biological activity were studied. All the peptides displayed low dose-dependent uterotonic activity in vitro and antidiuretic activity in vivo. None of the peptides increased the blood pressure of the experimental animals. Compounds 2, 4 and 6 showed a low inhibitory effect on AVP pressor activity; compound 6, in addition, displays a significant and long-lasting vasodepressor effect. NMR measurements indicated the existence of hydrogen bond between the amino acid residues in positions 2,5 and 3,4 of peptides 1 and 2, and side-chain interactions between amino acid residues in positions 2,3 and 4,5 of peptide 1. No such side-chain interactions were detected in peptide 2.

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.

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.

Synthesis and evaluation of a non-radioactive gene probe for the detection of C.perfringens alpha toxin.

The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay. A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124. In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc. None of the strains failed in hybridization. Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity. Specificity of the probe was shown with various strains of other bacterial species. None different Clostridia sp. tested, e.g. C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C. septicum and C.histolyticum, hybridized with the plc specific probe. Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results. Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

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.

Semisynthetic insulin analogues modified in positions B24, B25 and B29.

New semisynthetic analogues of human insulin, modified in the C-terminal region of the B-chain, were prepared to refine our understanding of the importance of particular amino acid residues in the expression of hormone biological properties. The following insulin analogues were synthesized by trypsin-catalyzed peptide-bond formation between the C-terminal arginineB22 of des-octapeptide(B23-B30)-insulin and synthetic octapeptides with the epsilon-amino group of lysineB29 protected by a phenylacetyl group: [L-Lys(Pac)B29]insulin, [D-PheB24,B25,L-Lys(Pac)B29]insulin and [D-Phe(p-Et)B24, L-Lys(Pac)B29]insulin. Enzymatic deprotection using immobilized penicillin amidohydrolase yielded: human insulin, [D-PheB24,B25]insulin and [DPhe(p-Et)B24]-insulin. Biological in vitro potencies (specific binding to cultured human lymphocytes IM-9 and lipogenic potency in isolated rat adipocytes) of the semisynthetic analogues were estimated, ranging from 0.2 to 100% relative to porcine insulin.

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.

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.

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.

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

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.

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.

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.

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.

Specificity studies on retroviral proteinase from myeloblastosis-associated virus.

The specificity of the p15 proteinase of myeloblastosis-associated virus (MAV) was tested with nonviral high molecular weight substrates and with synthetic peptides. Peptides with sequences spanning known cleavage sites in viral polyproteins of Rous sarcoma virus (RSV) and avian leukemia viruses, as well as in BSA and HSA, were synthesized, and the rate of their cleavage by the MAV proteinase was compared. Synthetic peptides require for successful cleavage at least 4 residues at the N-terminal side and 3 residues at the C-terminal side. The proteinase shows a preference for hydrophobic residues with bulky side chains (Met, Tyr, Phe) in P3, although Arg and Gln can also be accepted. Small hydrophobic residues are required in P2 and P2', and large hydrophobic residues (Tyr, Met, Phe/p-nitro-Phe) are preferred in both P1 and P1'. The difference between the specificity of the p15 proteinase and that of the HIV-1 proteinase mostly pertains to position P2' of the substrate, where bulkier side chains are accepted by the HIV-1 proteinase (Richards et al., 1990). A good chromogenic substrate for the MAV and RSV proteinases was developed and used to further characterize the MAV proteinase activity with respect to ionic strength and pH. The activity of the proteinase is strongly dependent on ionic strength and pH. Both the kcat and Km values contribute to a higher cleavage efficiency at higher salt concentrations and show a bell-shaped pH dependence curve with a sharp maximum at pH 5.5 (kcat) and 6.5 (Km).