Production of a Ric c3 hypo-allergen with no IgE binding or anaphylactogenic activity.

Several studies have been carried out to expand the use of Ricinus communis L. castor bean (Ricinus communis L castor bean.). This oilseed finds appropriate conditions for its development in Brazil, with more than 700 applications. The main allergens of this plant are Ric c1 and Ric c3, that cross-react with various aeroallergens and food allergens such as peanuts, soybeans, corn, and wheat. This study aimed to determine the effect of mutations in Ric c3 amino acid residues known to affect IgE binding and allergy challenges. Based on the Ric c3 structure, B-cell epitopes, and amino acid involved in IgE binding, we produce recombinant mutant protein, mrRic c3, secreted from E. coli. Strategic glutamic acid residues in IgE-biding regions were changed by Leucine. The allergenicity of mrRic c3 was evaluated by determination of IgE, IgG1, and total IgG in immunized Balb/c mice and by degranulation assays of mast cells isolated from Wistar rats. The mrRic c3 presented a percentage of mast cell degranulation close to that seen in the negative control, and the immunization of mice with mrRic c3 presented lower levels of IgE and IgG1 than the group treated with the protein without mutations. The mutant mrRic c3 had an altered structure and reduced ability to stimulate pro-inflammatory responses and bind IgE but retained its ability to induce blocking antibodies. Thus, producing a hypoallergenic mutant allergen (mrRic c3) may be essential in developing new AIT strategies.

Femoral hernia repair. Kugel retroparietal approach versus plug alloplasty: a prospective study.

Plug insertion for primary femoral hernia repair may cause p.o. discomfort. The Kugel technique may avoid this problem. Patients' satisfaction to the Kugel and the plug techniques is compared in the present study. Demographics, surgical, outcome and analgesic consumption data of 26 patients treated for with the plug technique (P group) are compared with 24 operated with the Kugel patch (K group). Patients' p.o. discomfort to the two procedures was measured with quantitative (VAS score) and a qualitative (the short form of McGill pain questionnaire, SF-MPQ) methods, and compared. P group presented higher early p.o. pain (P<0.001), higher analgesic consumption and a significative delay in the return to physical activity (P<0.001). SF-MPQ scores at p.o. day 8, day 30 and month 6 were significantly lower for K group (P<0.001, P<0.001, P<0.005). The Kugel technique for femoral hernia treatment seems to cause less p.o. discomfort to patients than the plug technique.

Kugel hernia repair: open "mini-invasive" technique. Personal experience on 620 patients.

A large monoinstitutional series adopting the Kugel retroparietal technique for inguinal hernia surgery is analysed. Our aim is to assess the "mini-invasiveness" of this technique. Six hundred and twenty patients (pts) affected by monolateral inguinal hernia were treated with a preperitoneal alloplasty with a posterior approach (Kugel hernia repair, KHR) between January 2002 and September 2004. The surgical incision extension was 3.5 cm on average (range 2-4.5). The mean operation time was 33 min (range 20-45). Spinal anaesthesia and ambulatory procedure were applied in 595 cases (96%). Postoperative complications affected 20 pts (3%). The postoperative pain was well controlled. No chronic neuropathic pain was registered at follow-up. Patients resumed work after an average of 9 days (range 7-12) from operation. Recurrence rate was 0.8%. Conclusions. The Kugel hernia repair satisfies the standards to be awarded as a "mini-invasive" technique.

[D2 gastrectomy in the surgical treatment of gastric cancer]. / Gastrectomia D2 nel trattamento chirurgico del cancro gastrico.

The study is conducted on a series of 57 patients treated with D2 gastrectomy with curative intent for gastric cancer between January 2000 and December 2004, at Policlinico Multimedica (Milan). Postoperative mortality was 2%. Recurrence rate was 10%. The overall survival of the series is 36% at 4 years follow-up. Negative prognostic factors were: high grade tumor, locally advanced primary disease, presence of lymph node metastases, advanced stage of disease and recurrent disease at follow-up. The data of the study are comparable to those in the literature.

[Intraperitoneal hyperthermic chemotherapy. Preliminary experience with 22 consecutive patients]. / Chemioterapia ipertermica intraperitoneale. Esperienza preliminare in 22 pazienti consecutivi.

The study analyses the results of surgical treatment and intraperitoneal hyperthermic chemotherapy on a group of 22 patients, affected by peritoneal carcinomatosis of different origins, and treated at Policlinico Multimedica (Milan) between June 2001 and December 2004. Surgical major complications were present in the 23% of the patients, and post-operative mortality rate was 13%. None of the patients presented chemotherapy related toxicity. Six patients died within 2 and 40 months after surgery, while 13 are alive within 4 and 40 months after operation.

Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1.

In the yeast Saccharomyces cerevisiae, the first step in lactate metabolism is its transport across the plasma membrane, a proton symport process mediated by the product of the gene JEN1. Under aerobic conditions, the expression of JEN1 is regulated by the carbon source: the gene is repressed by glucose and induced by non-fermentable substrates. JEN1 expression is also controlled by oxygen availability, but is unaffected by the absence of haem biosynthesis. JEN1 is negatively regulated by the repressors Mig1p and Mig2p, and requires Cat8p for full derepression. In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression. We also provide evidence for transcriptional stimulation of JEN1 by the protein kinase Snf1p. Data are presented which provide a better understanding of the molecular mechanisms implicated in the co-regulation of genes involved in the metabolism of lactate.

Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1.

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193-203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.

Cloning and characterization of the lactate-specific inducible gene KlCYB2, encoding the cytochrome b(2) of Kluyveromyces lactis.

In yeast the utilization of lactate requires two enzymes, the D and L-lactate ferricytochrome c oxidoreductase (D and L-LCR), which stereospecifically oxidize D- and L-lactate to pyruvate. These enzymes are nuclearly encoded and localized in mitochondria. In the yeast Kluyveromyces lactis, a mutant devoid of D- and L-LCR activities and unable to grow on racemic lactate was isolated. Transformation of the mutant with a K. lactis genomic library allowed the isolation of the KlCYB2 gene, restoring the growth on lactate and the L-LCR activity. The KlCYB2 gene and its flanking regions were sequenced (Accession No. AJ243324; EMBL/GenBank databases). The deduced amino acid sequence is highly homologous to the corresponding Saccharomyces cerevisiae and Hansenula anomala protein sequences previously characterized. The homology is missed in the N-terminal region, corresponding to the presequence cleaved during import into mitochondria. Analysis of KlCYB2 gene expression indicated that, in contrast to S. cerevisiae, the major regulatory feature is induction by lactate.

Current awareness on yeast.

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals - search completed 16th Feb 2000)

A Klaac null mutant of Kluyveromyces lactis is complemented by a single copy of the Saccharomyces cerevisiae AAC1 gene.

The KlAAC gene, encoding the ADP/ATP carrier in Kluveromyces lactis, has previously been cloned by complementation of the op1(aac2) mutation of Saccharomyces cerevisiae. We examined the effect of a null mutation of this gene on the phenotype of K. lactis. The consequence of this mutation was found to be multiple. The mutant was respiratory deficient, had an undetectable level of cytochrome a-a3 and b and did not grow on glycerol. The mitochondrial D-lactate ferricytochrome c oxidoreductase activity, as well as the lactate-induced transcription of its gene, KlDLD, was severely reduced. Furthermore, the mutant was unable to grow on galactose, maltose and raffinose. Transcript analysis showed that KlAAC was the only ADP/ATP carrier gene present in K. lactis. The Klaac mutation was fully complemented not only by AAC2, the major gene for the ADP/ATP carrier in S. cerevisiae, but also by AAC1, a gene which is poorly expressed in S. cerevisiae. AAC1 introduced in K. lactis was transcribed to a high level consistent with normal growth on glycerol being restored in the transformed mutant. KlAAC was not subject to control by KlHap2, in contrast to AAC2 which is regulated by the Hap2 complex in S. cerevisiae.

Regulation of the Saccharomyces cerevisiae DLD1 gene encoding the mitochondrial protein D-lactate ferricytochrome c oxidoreductase by HAP1 and HAP2/3/4/5.

Expression of the nuclear gene encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase (D-LCR) was investigated in Saccharomyces cerevisiae. This gene (DLD1) was found to be subject to several regulatory controls at the transcriptional level: synthesis of DLD1 mRNA is repressed by glucose, is derepressed in ethanol or lactate and is heme dependent. We therefore examined the role of the heme-dependent transcriptional activator Hap1p and the carbon source-dependent Hap2/3/4/5 complex. We found that the Hap2/3/4/5 complex and Hap1p have additive effects on the activation of DLD1 transcription: the Hap2/3/4/5 complex is necessary for DLD1 derepression following a shift from fermentable to non-fermentable carbon sources, while the Hap1p effect was independent of the carbon sources tested. An upstream region required for expression and regulation of the DLD1 gene was identified. Within this region the binding sites for both the Hap2/3/4/5 complex and Hap1p were defined by gel retardation experiments and site-directed mutagenesis. Comparison between sequences recognized by Hap1p in different promoters showed that the Hap1p binding site in the DLD1 promoter diverges from the consensus Hap1p binding site.

Transcriptional regulation of the KlDLD gene, encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase in Kluyveromyces lactis: effect of Klhap2 and fog mutations.

Expression of the Kluyveromyces lactis KlDLD gene, encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase (D-LCR), is subject to two metabolic controls at the transcriptional level: induction by lactate, the substrate of the D-LCR enzyme, and repression by glucose. By Northern analysis we determined the kinetics of the two regulatory processes and, by measurement of the expression of LacZ gene fused to the KlDLD promoter, we identified cis-elements involved in glucose repression and lactate induction. The effect of trans-acting factors on the transcription of KlDLD has been analyzed. The KlDLD gene is controlled by the products of the FOG1 and FOG2 genes, previously identified as involved in glucose de-repression. Moreover, the KlDLD gene is regulated by the product of KlHAP2, homologous to the HAP2 gene which in Saccharomyces cerevisiae is required for the induction of genes encoding mitochondrial components, upon shifting from a fermentable to a non-fermentable carbon source. We have demonstated that the KlHAP2 gene is necessary both for the lactate induction of KlDLD mRNA synthesis and for growth on this oxidative carbon source.

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein.

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

Characterization of a promoter mutation in the CYP3 gene of Saccharomyces cerevisiae which cancels regulation by Cyp1p (Hap1p) without affecting its binding site.

Cyp1p (Hap1p) activates, among others, the two structural genes, CYC1 and CYP3 (CYC7) which encode isocytochromes c in Saccharomyces cerevisiae. This activation is believed to occur through the binding of the protein to the dissimilar upstream activation sequences (UASs), UAS1 and UAS', present upstream of CYC1 and CYP3, respectively. In this paper, we describe a novel promoter mutation, CYP3-5, which results from a 39-bp deletion located about 160 bp upstream of the well-characterized CYP3 UAS. This deletion includes a sequence identical to the 3' moiety of the CYC1 UAS1. Strikingly, a sequence identical to the 5' part of the CYC1 UAS1 is also present 60 bp downstream of the 3' half in the wild-type gene, suggesting that a spatial organization of the promoter might lead to the reconstitution in vivo of an active UAS1-like sequence. Interestingly, we find that in the presence of the CYP3-5 mutation, which disrupts this potential UAS1, the CYP-UAS' complex is importantly diminished and the transcription of CYP3 is insensitive to the wild-type CYP1-activating protein.

FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of saccharomyces cerevisiae.

The fog1 and fog2 mutants of the yeast Kluyveromyces lactis were identified by inability to grow on a number of both fermentable and non-fermentable carbon sources. Genetic and physiological evidences suggest a role for FOG1 and FOG2 in the regulation of glucose-repressible gene expression in response to a glucose limitation. The regulatory effect appears to be at the transcriptional level, at least for beta-galactosidase. Both genes have been cloned by complementation and sequenced. FOG1 is a unique gene homologous to GAL83, SIP1 and SIP2, a family of regulatory genes affecting glucose repression of the GAL system in Saccharomyces cerevisiae. However, major differences exist between fog1 and gal83 mutants. FOG2 is structurally and functionally homologous to SNF1 of S. cerevisiae and shares with SNF1 a role also in sporulation.

Are non-specific reactions to tuberculin common?

Three thousand four hundred eighty-five BCG scar negative school children were given tuberculin test. Results showed very little non-specific reactions, suggesting BCG should produce high levels of protection in our population.

IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae.

Two mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, delta T1, which encodes a protein lacking the last 26 C-terminal amino acids, and delta T2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.