Human ORFeome Version 1.1: A Platform for Reverse Proteomics

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.     

Epidemiological study of pap genes among diarrheagenic or septicemic Escherichia coli strains producing CS31A and F17 adhesins and characterization of Pap(31A) fimbriae

The association of the pap operon with the CS31A and F17 adhesins was studied with 255 Escherichia coli strains isolated from calves, lambs, or humans with diarrhea. The three classes of PapG adhesin with different receptor binding preferences were also screened. The pap operon was associated with 50 and 36% of human strains that produced CS31A and ovine strains that produced F17, respectively. Among the bovine isolates, the pap operon was detected in 61% of the CS31A-positive isolates and 72% of the strains that produce both CS31A and F17. The class II adhesin gene was present in bovine (20%) and ovine (71%) isolates. Both class II and III adhesins were genetically associated with 36% of the human strains. The highest prevalence of the pap operon was observed among E. coli strains that produce additional adhesins involved in the binding of bacteria to intestinal cells. Among the bovine isolates, the reference strain for CS31A and F17c was found to be positive for the pap operon. Phenotypic and genotypic characterizations were undertaken. Pap(31A) appeared as fine and flexible fimbriae surrounding the bacteria but did not mediate adhesion to calf intestinal villi. Pap(31A) production was optimal with bacteria cultured on minimal growth media and repressed by addition of exogenous leucine. The deduced amino acid sequence of the PapA(31A) structural subunit showed 57 to 97% identity with the different P-related structural subunits produced by E. coli strains isolated from pigs with septicemia or humans with urinary tract infections. None of the three papG allelic variants was detected, but a homologous papG gene was present in the chromosome of strain 31A.     

Interleukin 17 modulates the immune response to vaccinia virus infection

Interleukin 17 (IL-17) is a newly identified cytokine that has a homolog in herpesvirus saimiri. We inserted murine IL-17 into vaccinia virus to study the role of IL-17 in viral infection. Vaccinia virus expressing IL-17 (vv-IL17) and its parental control virus (vv-pRB) grew to similar titers in vitro; however, vv-IL17 was more virulent in mice with a threefold lower LD(50) than for vv-pRB, and mean time to death of 2.8 days versus 4.5 days. Mice infected with vv-IL17 had higher titers of virus in the ovaries (P < 0.02) and showed a decrease in NK cell cytotoxicity (P < 0.02) on day 3 after infection. No significant difference was found in CTL activity. In addition, a significant decrease in IgG2a (P < 0.01) and increases in IgG1, IgG3, and IgA (P < 0.03) vaccinia virus-specific antibody titers were observed in mice infected with vv-IL17 versus vv-pRB, suggesting a Th-2-like response to infection. These data indicate that IL-17 modulates the immune response during virus infection.     

Protection from insulin-dependent diabetes mellitus is linked to a peptide transporter gene

HLA class II association with insulin-dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult-onset and 51 childhood-onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for theTAP2*0201 allele [relative risk (RR)=0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR=3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR=0.06, pc<0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB 1*0302 haplotypes predisposing effect (RR=1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by x² analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to β cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.     

Oocyte insemination with spermatozoa precursors

Since the use of testicular spermatozoa in programs of assisted fertilization proved very successful, attention was focussed on the use of spermatids also carrying 23 chromosomes. Several difficulties became obvious; the first one concerned the recognition of round spermatids. This is a problem which does not concern elongating and elongated cells. The intra-cytoplasmic injection of elongated spermatids resulted in several pregnancies but this is not so for the round ones. Although, in the group of patients in whom only round spermatids are found at the time of the attempt, is to be divided into two categories; patients in whom previous research allowed to find spermatozoa, however few, and patients who never produced spermatozoa at all. This last group is no longer an indication for intracytoplasmic sperm injection procedure unless in the future new culture media allow a maturation into elongated forms.     

Treatment of the postenucleation socket syndrome with a new hydroxyapatite tricalcium phosphate ceramic implant.

PURPOSE: Surgical correction of the postenucleation socket syndrome (PESS) is challenging. Various biomaterials are used for reconstruction of the anophthalmic orbit, often with unsatisfactory long-term results. Implants have been placed between the periorbital and the orbital floor. The authors describe a new material composed of hydroxyapatite tricalcium phosphate (HA-TCP) in the form of ceramic blocks, to be placed into the orbital fat as a new surgical site. METHODS: Ten patients with PESS underwent surgery to compensate the volume deficit of the anophthalmic orbit. Blocks of HA-TCP were created by fragmentation of a larger piece, tailored as needed, and implanted in the orbital fat. The patients were monitored regularly with clinical and radiologic examinations to evaluate the behavior of the implants. RESULTS: The volume of the HA-TCP was measured in 5 cases (mean, 2.95 +/- 1.08 ml). A significant reduction of enophthalmos was obtained: mean Hertel exophthalmometry measured 12.7 +/- 2.5 mm preoperatively and 14 +/- 2.4 mm postoperatively (p < 0.05). The average prosthesis volume was significantly reduced in the cases measured: 2.7 ml +/- 0.94 ml preoperatively and 1.8 +/- 0.7 ml postoperatively (p < 0.02). There was a negative correlation between the HA-TCP implant volume and postoperative prosthesis volume (correlation coefficient = -0.925; p < 0.05). According to photographic evaluation, correction of the enophthalmos and of the superior sulcus depression were obtained in 70% and 90% of cases, respectively. Magnetic resonance imaging seems to demonstrate that the blocks become well integrated into the surrounding orbital tissue. CONCLUSION: The HA-TCP blocks correct some anomalies of PESS. Placement of the blocks directly into the orbital fat is a promising alternative to the traditional subperiosteal location.     

Pharmacokinetics of tiaprofenic acid in children after a single oral dose

Summary. Twelve healthy children in three age groups anaesthetized for minor surgery were given a single oral dose of tiaprofenic acid (3 mg · kg^–1) (TA). Seven blood samples and zero to 8 and 8 to 24 h urines were collected. TA concentrations in plasma and urine were measured by HPLC.No significant difference was found between the age groups in the kinetic parameters of TA and no correlation was found between these parameters and age; t_max=2.12h, C_max=8.78mg · l^–1, AUC_{(08 h)} 33.9mg · h · l^–1, AUC=39.3 mg · h · l^–1, t_1/2=2.35 h, V_z=0.319 l · kg^–1, CL=0.094 l · h^–1 · kg^–1. Renal clearance was 14 ml · h^–1. kg^–1. 33% of the TA dose was recovered in the 24 h urine, 48% of which was conjugated, whereas in adults, TA is only found in urine as conjugates.The apparent plasma clearance was significantly higher (56%) than in 12 healthy adults given 1.5 mg · kg^–1 TA. Volume of distribution and t_1/2 did not significantly differ between children and adults. Since no relationship has been established between plasma TA and either efficacy or toxicity, a different dose regimen cannot be recommended in 3–11 year-old children from that in adults.     

On the dynamical basis of the classification of normal galaxies

Some realistic galaxy models have been found to support discrete unstable spiral modes. Here, through the study of the relevant physical mechanisms and an extensive numerical investigation of the properties of the dominant modes in a wide class of galactic equilibria, we show how spiral structures are excited with different morphological features, depending on the properties of the equilibrium model. We identify the basic dynamical parameters and mechanisms and compare the resulting morphology of spiral modes with the actual classification of galaxies. The present study suggests a dynamical basis for the transition among various types and subclasses of normal and barred spiral galaxies.