Accessory or additional renal arteries show no relevant effects on the width of the upper urinary tract: a 64-slice multidetector CT study in 1072 patients with 2132 kidneys.

OBJECTIVE: The aim of this study was to find out on an unselected patient group whether crossing vessels have an influence on the width of the renal pelvis and what independent predictors of these target variables exist. METHODS: In this cross-sectional study, 1072 patients with arterially contrasted CT scans were included. The 2132 kidneys were supplied by 2736 arteries. RESULTS: On the right side, there were 293 additional and accessory arteries in 286 patients, and on the left side there were 304 in 271 patients. 154 renal pelves were more than 15 mm wide. The greatest independent factor for hydronephrosis on one side was hydronephrosis on the contralateral side (p<0.0001 each). Independent predictors for the width of the renal pelvis on the right side were the width of the renal pelvis on the left, female gender, increasing age and height; for the left side, predictors were the width of the renal pelvis on the right, concrements, parapelvic cysts and great rotation of the upper pole of the kidney to dorsal. Crossing vessels had no influence on the development of hydronephrosis. Only anterior crossing vessels on the right side are associated with widening of the renal pelvis by 1 mm, without making it possible to identify the vessel as an independent factor in multivariate regression models. CONCLUSION: The width of the renal pelvis on the contralateral side is the strongest independent predictor for hydronephrosis and the width of the renal pelvis. There is no link between crossing vessels and the width of the renal pelvis.

Human papillomavirus type 11 (HPV-11) neutralizing antibodies in the serum and genital mucosal secretions of African green monkeys immunized with HPV-11 virus-like particles expressed in yeast.

It has been shown previously that immunization of animals with recombinant virus-like particles (VLPs) consisting of the viral capsid proteins L1 or L1 plus L2 protected animals against experimental viral challenge. However, none of these experimental models addresses the issue of whether systemic immunization with VLPs elicits a neutralizing antibody response in the genital mucosa. Such a response may be necessary to protect the uterine cervix against infection with genital human papillomavirus (HPV) types. African green monkeys systemically immunized with HPV-11 VLPs expressed in Saccharomyces cerevisiae and formulated on aluminum adjuvant elicited high-titered HPV-11 VLP-specific serum antibody responses. Sera from these immunized monkeys neutralized HPV-11 in the athymic mouse xenograft system. Significant levels of HPV-11-neutralizing antibodies also were observed in cervicovaginal secretions. These findings suggest that protection against HPV infection of the uterine cervix may be possible through systemic immunization with HPV VLPs.

Expression of the major capsid protein of human papillomavirus type 11 in Saccharomyces cerevisae.

The major capsid protein L1 of papillomaviruses expressed recombinantly or in infected cells has the intrinsic ability to form virus-like particles (VLPs) which display conformational epitopes necessary to elicit high-titered, virus-neutralizing antibodies. We have shown previously that the L1 gene of human papillomavirus type 6a (HPV6) can be expressed efficiently in Saccharomyces cerevisae (Sc) as VLPs. However, when we attempted to express the L1 gene cloned from the closely related HPV11 in yeast, few VLPs were observed in crude lysates. The lower expression level of HPV11 L1 protein was found to result from a truncation of the HPV11 L1 mRNA. Since sequence requirements for transcriptional termination in yeast are still unclear, the HPV6 L1 gene was used as the basis for the complete synthetic reconstruction of the entire 1506-bp HPV11 L1 gene. Expression of this HPV6/11 hybrid L1 gene in yeast resulted in predominantly full-length L1 mRNA and a > 7-fold increased level of production of HPV11 VLPs compared to that expressed by the wild-type HPV11 L1 gene. The VLPs were shown to display the conformational epitopes important to elicit virus-neutralizing antibodies.

Molecular cloning of an avian anti-Müllerian hormone homologue.

Anti-Müllerian hormone (AMH) is responsible for regression of the Müllerian ducts in males during embryonic development. This peptide hormone of the transforming growth factor-beta family is also believed to play a broader role in sex determination, affecting differentiation and morphogenesis of the testes. Accordingly, in mammals, AMH is produced at much higher levels in male fetuses than in female fetuses. In contrast, in birds, both male and female embryonic gonads produce AMH at high levels, although in males it is still responsible for regression of the Müllerian ducts. Its persistent expression by the embryonic ovaries and its role in female sex determination in birds is not understood. We have cloned an avian homologue to AMH. Avian AMH cDNA encodes a 644 amino acid protein that is 42% identical to human AMH overall with increased identity at the carboxyl terminus. Similarities to human AMH include motifs of sequence identity, a conserved putative plasmin cleavage site and cysteine alignments, and similar genomic intron/exon structure. Antibodies to recombinant avian AMH cross-react with recombinant human AMH and were used to show that avian AMH is glycosylated as has been shown for the human form. The avian AMH gene is transcribed in both male and female gonads but not in liver, heart, kidney or muscle.

Sequence conservation within the major capsid protein of human papillomavirus (HPV) type 18 and formation of HPV-18 virus-like particles in Saccharomyces cerevisiae.

The major capsid protein L1 of human papillomaviruses (HPVs) has been identified as a promising candidate antigen for a prophylactic HPV vaccine. Since amino acid sequence heterogeneity has been demonstrated for the L1 genes within individual HPV types, nucleotide sequences of L1 were determined from six HPV-18 clinical isolates and the cervical carcinoma cell line SW756 and compared to the published HPV-18 prototype sequence. The sequences were almost identical between the clinical isolates and SW756 but differed markedly from the published prototype sequence. Resequencing the prototype HPV-18 revealed that these differences were due to sequencing artifacts of the prototype HPV-18 sequence archived in GenBank. Thus, the HPV-18 L1 genes seem to display a very high level of sequence conservation. The HPV-18 L1 gene derived from SW756 was expressed in Saccharomyces cerevisiae and self-assembly of the L1 protein into virus-like particles was demonstrated.

Resistance to fever induction and impaired acute-phase response in interleukin-1 beta-deficient mice.

We used gene targeting in embryonic stem cells to introduce an IL-1 beta null allele in mice. The IL-1 beta-deficient mice develop normally and are apparently healthy and fertile. The IL-1 beta null mice responded normally in models of contact and delayed-type hypersensitivity or following bacterial endotoxin LPS-induced inflammation. The IL-1 beta-deficient mice showed equivalent resistance to Listeria monocytogenes compared with wild-type controls. In contrast, when challenged with turpentine, which causes localized inflammation and tissue injury, the IL-1 beta mutant mice exhibited an impaired acute-phase inflammatory response and were completely resistant to fever development and anorexia. These results highlight a central role for IL-1 beta as a pyrogen and a mediator of the acute-phase response in a subset of inflammatory disease models, and support the notion that blocking the action of a single key cytokine can alter the course of specific immune and inflammatory responses.

Sequence determination of human papillomavirus type 6a and assembly of virus-like particles in Saccharomyces cerevisiae.

Human papillomavirus 6a (HPV6a), the most abundant HPV6 subtype, was detected in a vulvar condyloma acuminatum. The complete genome of HPV6a was cloned, and its DNA sequence was shown to be over 97% identical to the HPV6b sequence. Of the eight open reading frames (ORFs) of HPV6a, only the imputed amino acid sequence of the major capsid protein L1 was identical to the corresponding HPV6b sequence; all other HPV6a ORFs showed amino acid changes compared to the HPV6b ORFs. The HPV6a L1 or the L1 + L2 ORFs were expressed in the yeast Saccharomyces cerevisiae. Self-assembly of the L1 capsid protein into virus-like particles (VLPs) was demonstrated both in the L1 as well as L1 + L2 coexpressing yeast strains. Copurification of the L1 and L2 proteins showed complex formation of the L1 and L2 proteins in the yeast-derived VLPs of coexpressing strains.

Site-directed analysis of the functional domains in the factor Xa inhibitor tick anticoagulant peptide: identification of two distinct regions that constitute the enzyme recognition sites.

Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.

Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site.

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae.

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae.

The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides. To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter. The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera. Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process. Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution. The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with [3H]myristic acid; in addition the amino terminus of the final purified protein was blocked. Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis. In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease. The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease.

Galactosylceramide-beta-galactosidase deficiency in association with cherry red spot.

A 13-month-old white girl was the product of a normal pregnancy and delivered by caesarean section for breech presentation. Regression of motor milestones started by 11 months, when delayed language development was also noted. She was normocephalic without major dysmorphic features or organomegaly. Fundus examination disclosed a subtle cherry red spot bilaterally. No startle response was elicited. By 17 months she was extremely irritable and unable to tolerate liquids; there was symmetrical spasticity and florid cherry red spots. She died at 18 months of age. A systematic search for conditions associated with a cherry red spot was unrevealing. The absence of galactosylceramide galactosidase activity was unexpected and was confirmed on three occasions in two laboratories. Lactosylceramide I content, an enzyme thought to be identical to galactosylceramide-beta-galactosidase, was significantly decreased. The presence of a cherry red spot in Krabbe's disease, indicative of neuronal storage, has not been previously recognized. The existence of this variant has implications for genetic and biochemical studies.

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.

High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus.

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.

Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae.

As a model system for the study of factors affecting gene expression, hepatitis B virus core antigen (HBcAg) has been expressed in the yeast Saccharomyces cerevisiae. The singularly high levels of expression achieved are approx. 40% of the soluble yeast protein. The HBcAg polypeptides are present as 28-nm particles which are morphologically indistinguishable from HBcAg particles in human plasma and are highly immunogenic in mice. The plasmid construction employed to achieve these very high levels of expression utilizes the constitutively active yeast promoter from the GAP491 gene which is fused in a way that all non-translated sequences flanking the HBcAg coding region are yeast-derived. Hybrid constructions containing 3'-nontranslated viral DNA (yeast 5') or 5'-nontranslated viral DNA (yeast 3') as well as a construction with both 5'- and 3'-nontranslated viral DNA also have been made. A comparison of these constructions for levels of HBcAg expression indicates that the strongest contributor to the high levels of protein is the presence of 5'-flanking sequences which are yeast-derived; secondarily, a significant improvement can be achieved if the 3'-flanking sequences also are yeast-derived. The high abundance of HBcAg in the highest producer is explicable in part on the basis of the very high stability in yeast cells of HBcAg polypeptides. Analysis of the HBcAg coding sequence reveals a very low index of codon bias for S. cerevisiae, largely discounting codon usage as a contributor to the high level of protein obtained.