L5 root compression caused by degenerative spinal stenosis of the L1-L2 and L2-L3 spaces.

STUDY DESIGN: A severe bilateral L5 root lesion associated with spinal stenosis at L1-L2 and L2-L3 is described. OBJECTIVE: To describe clinical findings and the difficulty in obtaining a correct diagnosis of L5 Root Compression. SUMMARY OF BACKGROUND DATA: The disorder reported in this study has not been reported previously. Only one similar case has been described in the literature: an L5 root compression at L1-L2 caused by disc herniation. METHODS: Diagnosis was obtained by using computed tomography scanning, magnetic resonance imaging, and computed tomography myelography. The findings at L5-S1 were minimal to justify the patient's clinical symptoms, but a detailed study of the upper levels revealed spinal stenosis at L1-L2 and L2-L3, which could have been causing L5 and S1 root compression. A decompressive laminectomy and partial facetectomy in both levels were performed. RESULTS: The patient's pain and claudication disappeared, and clinical symptoms associated with the right L5 root improved. The left L5 root deficit remained stable. CONCLUSION: An unusual case of L5 root compression caused by degenerative stenosis of L1-L2 and L2-L3 is described.

Intradural disc herniation associated with epidural gas.

STUDY DESIGN: A case report of a patient suffering from an intradural herniated disc associated with the presence of epidural gas. OBJECTIVE: To advise spine surgeons of the possible association of intradural disc herniation and epidural gas, to prevent overlooking intradural disc fragments during surgery. SUMMARY OF BACKGROUND DATA: Three cases of this rare association were published previously, something surprising given the relatively rare occurrence of intradural herniations and the presence of epidural gas. METHODS: A case is presented where such an association occurred, on the basis of preoperative examinations and intraoperative findings. The literature is reviewed for cases of herniated discs associated with epidural gas and for intradural herniations. RESULTS: During the open discectomy, after a negative epidural examination, an intradural examination was performed, revealing a disc herniation, which was removed. The patient's postoperative development was satisfactory. CONCLUSION: The possibility of an intradural herniated disc must always be considered when performing an open discectomy on a patient whose computed tomography scan reveals the presence of epidural gas. In the event that no clear disc herniation is found to justify the clinical symptoms or the previous radiologic findings, an intradural exploration may be indicated.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures.

To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.

Identification of a family of streptococcal surface proteins with extremely repetitive structure.

The group B Streptococcus (GBS) causes the majority of life-threatening bacterial infections in newborn children. Most GBS strains isolated from such infections express a surface protein, designated Rib, that confers protective immunity and therefore is of interest for analysis of pathogenetic mechanisms. Sequence analysis demonstrated that Rib has an exceptionally long signal peptide (55 amino acid residues) and 12 repeats (79 amino acid residues each) that account for >80% of the sequence of the mature protein. The repeats are identical even at the DNA level, indicating that an efficient mechanism operates to maintain a highly repetitive structure in Rib. The structure of Rib is similar to that of alpha, a previously characterized surface protein that is common among GBS strains lacking Rib. However, highly purified preparations of Rib and alpha did not cross-react immunologically, although the two proteins show extensive amino acid residue identity (47% in the repeat region). When analyzed in Western blots, Rib and alpha give rise to a regularly spaced ladder pattern, apparently due to hydrolysis of acid-labile Asp-Pro bonds in the repeats. We conclude that Rib and alpha are members of a novel family of streptococcal surface proteins with unusual repetitive structure.

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae.

OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport. DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae. METHODS: Thin-section immunoelectron microscopy studies were performed on S. cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins. RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane. p24 was localized in the nucleus or perinuclear membrane. However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane. CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane. Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

The large surface protein of hepatitis B virus is retained in the yeast endoplasmic reticulum and provokes its unique enlargement.

The coding sequences for each of the three envelope proteins of hepatitis B virus (HBV), the major (S), middle (M), and large (L) surface proteins, were expressed in Saccharomyces cerevisiae. Analysis by immunoelectron microscopy of thin sections of yeast cells showed that production of L protein but not of M or S protein provoked morphological changes in the yeast endoplasmic reticulum. A large accumulation of membranous structures connected with the perinuclear cysternae and specifically labeled by a monoclonal antibody directed against the amino-terminal (preS1) sequence of the L protein, was observed. The L protein was post-translationally modified by N- and O-linked glycosylation, indicative of its entry into the yeast secretory pathway and by N-myristoylation of its amino-terminal glycine residue. Deletion of this glycine residue resulted in the synthesis of a nonmyristoylated L protein. Proliferation of the endoplasmic reticulum was comparable in cells producing either the myristoylated or nonmyristoylated L protein, indicating that myristoylation alone is not responsible for the induction of the abnormal membrane morphology.

Sequence analysis of the putative structural genes of hepatitis C virus from Japanese and European origin.

cDNA fragments encoding the putative structural genes of the hepatitis C genome were isolated from a plasma pool of Japanese non-A, non-B hepatitis patients and from sera of individual Spanish patients. From the Japanese plasma pool a series of E1 clones was obtained that showed 88-98% homology among each other, both at the nucleotide and amino acid level. Compared to the sequences published by the Chiron Corporation and Takeuchi et al., the amino acid homology was 75-79% and 91-94%, respectively. Analysis of the core and E2/NS1 genes showed a high conservation of the core sequence and a high sequence variation in the 5' end of the E2/NS1 gene. The E1 gene of one Spanish isolate showed greater homology to the Chiron than to the Japanese sequence. Another Spanish isolate was more homologous to the Japanese sequence indicating that both hepatitis C genotypes are present in Europe. Analysis of the E1 gene of an isolate derived from a single patient with a 5-year interval revealed nine nucleotide and five amino acid changes.

Cloning, partial sequence, expression, and antigenic analysis of the filamentous hemagglutinin gene of Bordetella pertussis.

The gene coding for the filamentous hemagglutinin (FHA), one of the main factors involved in mediating adherence of Bordetella pertussis to ciliated host cells, was cloned in Escherichia coli, and the 3,500-base-pair nucleotide sequence encoding the amino-terminal region was determined. Molecular cloning, together with the characterization of recombinant FHA-related proteins produced in E. coli, revealed that the primary translation product is a protein of about 370 kilodaltons (kDa). The mature 220-kDa FHA polypeptide secreted by B. pertussis is most probably generated by proteolytic processing that eliminates a carboxy-terminal portion of about 150 kDa. The 1,087 amino-terminal residues of the predicted FHA sequence showed a number of remarkable features. Extensive homology to the Serratia marcescens and Proteus mirabilis hemolysin proteins was found between amino acids 91 and 205 of the FHA sequence, suggesting involvement of this FHA domain in host cell binding or secretion of FHA from B. pertussis. In addition, two regions containing repetitive amino acid sequences were identified. One region, extending from residues 382 to 664, was formed by six repeats, and a second, extending from residues 701 to 912, contained three repeats. The reactivities of several recombinant FHA-derived proteins with a panel of monoclonal antibodies identified at least four epitopes composing an immunoreactive domain present in the carboxy-terminal moiety of the mature FHA.

Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae.

Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed. Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae. In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed. This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein. These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides. This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface. Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications.

Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen.

A host/vector system suitable for large-scale production of HBsAg has been constructed and optimized in terms of the expression plasmid and yeast host strain in order to permit fermentation to very high cell densities. The final expression plasmid contains the coding sequence of the major HBsAg protein (P24) flanked by the promoter sequences from a glycolytic gene and by the transcription-termination region of the ARG3 gene. The host/vector system was found to be genetically stable under large-scale fermentation conditions as demonstrated by nucleotide sequencing and restriction mapping experiments. The P24 protein is recovered from yeast as particles whose physiochemical properties are very similar to those of plasma-derived HBsAg.

Development of a hepatitis B vaccine from transformed yeast cells.

The production in yeast cells of the recombinant DNA hepatitis B vaccine of SmithKline Biologicals involves an optimized fermentation process followed by cell disruption and extraction, together with other soluble yeast components of the surface antigen of the hepatitis B virus. The subsequent purification process includes precipitation steps, ion exchange and gel permeation chromatography, and caesium chloride ultracentrifugation. The yeast-derived antigen occurs as spherical particles containing the non-glycosylated HBsAg polypeptide, lipid, and Tween 20. The purity of the polypeptide is above 95% and confirmed by the absence of an immune response to yeast-derived contaminants in vaccinees. Yeast DNA levels were less than 10 pg/vaccine dose. Various biochemical analyses showed that the recombinant polypeptide was faithfully expressed and did not undergo unwanted processing or degradation during fermentation or purification. These results indicate that the recombinant HBsAg can be effectively produced in yeast and processed to a high degree of purity to yield HBsAg particles displaying most of the characteristic properties of plasma-derived HBsAg.

High-level production and isolation of human recombinant alpha 1-proteinase inhibitor in yeast.

The cDNA coding for mature human alpha 1-proteinase inhibitor (alpha 1-PI) has been inserted into a variety of yeast expression vectors. Yeast cells transformed with these plasmids were then assayed for the production of mature, unglycosylated alpha 1-PI. The production level is optimal when the recombinant plasmid carries the TDH promoter, the complete 2mu and the leu2D selection marker. Biologically active recombinant alpha 1-PI can be purified either analytically, by affinity chromatography using a monoclonal antibody, or on a large scale, by a procedure involving precipitation of high-Mr yeast material with polyethylene glycol 3300 followed by successive chromatography on DEAE-agarose, Zn-chelate agarose, kappa-chain agarose, heparin-agarose and aminohexyl-agarose.

Expression of cloned human haptoglobin and alpha 1-antitrypsin complementary DNAs in Saccharomyces cerevisiae.

Nucleotide sequences coding either for human preprohaptoglobin or for prohaptoglobin have been placed under the control of yeast ARG3 expression signals. Recombinant plasmids pRIT12598 and pRIT12597 express the prepro- and the pro-form of alpha 2 beta haptoglobin respectively, but at very low levels. Comparison with the expression of human pre- and mature alpha 1-antitrypsin cDNAs, cloned in the same expression vector, reveals large differences in the levels of specific proteins produced in yeast, although specific mRNA levels are similar. It is shown that presence or absence of the signal sequence in the cDNA construction results in a 20- to 30-fold difference in the yields of heterologous products. However, since haptoglobin and alpha 1-antitrypsin behave differently, the difference in expression for prohaptoglobin compared with the expression of mature alpha 1-antitrypsin is about three orders of magnitude. In addition, we provide evidence that glycosylation of both proteins can occur in yeast only when the signal sequence is present in the DNA constructions.

Production in yeast of hepatitis B surface antigen by R-DNA technology.

Yeast synthesizes the surface antigen protein of Hepatitis B virus when the structural gene is fused to the promoter from the ARG3 gene. Analysis of extracts and total cells shows that the primary translation product can be identified as a poorly antigenic monomer with an estimated molecular weight of 22K. In cell extracts Y-HBsAg is in the form of 20 nm particles which, like serum derived particles, are highly immunogenic in mice and monkeys. Yeast derived surface antigen is thus a viable alternative to the present serum derived HBV vaccines.

Expression of human alpha 1-antitrypsin cDNA in the yeast Saccharomyces cerevisiae.

Nucleotide sequences coding either for the precursor or the mature form of human alpha 1-antitrypsin have been placed under the control of the yeast ARG3 expression signals. Recombinant plasmids pRIT10782 and pRIT10787 express the precursor or the mature alpha 1-antitrypsin species, respectively, in two different yeast strains, with yields ranging between 0.3 and 1% of total soluble proteins. The alpha 1-antitrypsin synthesized in yeasts was specifically recognized by polyclonal and monoclonal antibodies raised against human alpha 1-antitrypsin. In addition, it was shown to be biologically active in its mature form only, with optimal activity in a peptidase-deficient yeast strain.