A reassessment of Holt's data on: "The question of lumbar discography"

This paper is a detailed critique of Holt's study entitled "The Question of Lumbar Discography." His finding of a 37% false positive rate in asymptomatic volunteers has been used to discredit lumbar discography as a valid diagnostic study in the investigation of low back pain patients with nonprolapsing disc disease of the lumbar spine. The authors show why Holt's paper should no longer be used as scientific and authoritative evidence against the use of discography by today's standards.     

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.     

The Complete Set of Predicted Genes from Saccharomyces cerevisiae in a Readily Usable Form

Nearly all of the open reading frames (ORFs) of the yeast Saccharomyces cerevisiae have been synthesized by PCR using a set of ~6000 primer pairs. Each of the forward primers has a common 22-base sequence at its 5′ end, and each of the back primers has a common 20-base sequence at its 5′ end. These common termini allow reamplification of the entire set of original PCR products using a single pair of longer primers—in our case, 70 bases. The resulting 70-base elements that flank each ORF can be used for rapid and efficient cloning into a linearized yeast vector that contains these same elements at its termini. This cloning by genetic recombination obviates the need for ligations or bacterial manipulations and should permit convenient global approaches to gene function that require the assay of each putative yeast gene.     

Tg cells in peripheral blood lymphocytes from patients with rheumatoid arthritis

Using combinations of methods for detecting Fc receptors and B lymphocytes, non-B, non-T lymphocytes and T lymphocytes we have shown T_G cells to be significantly increased in rheumatoid arthritis (RA) patients' peripheral blood lymphocytes (PBL). The possible significance of this finding to the disease process is discussed.     

The transformation-related protein p53 is not bound to the SV40 T antigen in BALB 3T12 cells expressing T antigen

In most murine cells transformed by the SV40 virus, virtually all of the cellular phosphoprotein p53 is in a complex with the SV40 T antigen. Here, we report that, in SV40-infected T-antigen-positive Balb 3T12 mouse cells, most (approximately 80%) of the p53 is not in complex. Complex formation was determined by measuring the amounts of [35S]methionine-labeled p53 which coprecipitated with T antigen when using monoclonal antibody to T antigen. The amount of complex formation was expressed as a percentage of total p53 present, measured by the amount of p53 precipitated with the monoclonal antibody to the p53. The values were confirmed by Western blotting procedure, in which the steady-state levels of the proteins were measured. In these measurements after complete precipitation with antibody to T antigen, the residual p53 in the supernatant was precipitated by antibody to p53, and this amount was denoted as free p53. There was no significant difference seen between the [35S]methionine-labeled tryptic peptides of complexed and the free p53 (or between complexed and free T antigens) as determined by two-dimensional gel electrophoresis and chromatography. Virus rescue experiments and retransformation by the rescued virus showed that there was no mutation in the SV40 DNA coding for the T antigen which could account for the lack of complex formation. Both p53 and T antigen were underphosphorylated in cells which exhibited reduced complex formation. Tumorigenicity in syngeneic mice and anchorage-independent cell growth in culture of various cloned mouse cells with or without T antigen expression was compared. The changes in the biologic properties were explainable solely on the basis of known or expected effects of expression of the T antigen and were independent of complex formation or of absence of complex formation between p53 and T antigen.     

Bone cell populations and histomorphometric correlates to function

A circadian context has been used to develop information about the proliferative and functional behavior of the cell populations that function to model the long bones of growing rats. We asked: Are the proliferating cells in the growth cartilages and diaphyseal bone of young adult growing rats distributed within single or multiple populations? Can cytomorphometry (TEM-C) be used to determine ultrastructural correlates to the well-defined circadian rhythm of matrix formation displayed by functionally synchronous populations of metaphyseal osteoblasts? Can TEM-C reveal changes in osteoclast ultrastructure that could index a biological rhythm for osteoclastic bone mineralysis/resorption? Kinetic results derived from multiple radiothymidine labeling (DNA synthesis) support the single population model for chondrocytes and diaphyseal osteoprogenitor cells. TEM-C studies at the midpoints of the daily light and dark spans show that osteoblast RER-membrane development and cysternal volumes are maximal at the recorded daytime peak of net collagen synthesis. The extent of metaphyseal osteoclast surface ruffling (mineralysis) is also twofold greater during the day than the night--an observation supporting the concept that bone formation and resorption activities are coupled.     

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 M GTP, 1 M VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.8-fold). Less pronounced effects of VIP were found in the medullary collecting tubule isolated from the outer stripe (2.5-fold) and in the granular portion of the distal convoluted tubule (2.0-fold). VIP stimulation of AC activity in these segments amounted to 25 to 40% of the effect elicited by other agonists (arginine vasopressin, calcitonin or parathyroid hormone) in their respective target segments. A low response to VIP was observed in the cortical thick ascending limb (1.8-fold) which represented less than 5% of the calcitonin-stimulated AC activity. In the thin descending limb VIP produced a slight and variable stimulation of AC. VIP was without effect upon AC in the convoluted and straight portions of the proximal tubule, the medullary thick ascending limb and the cortical collecting tubule. Halfmaximal stimulation of AC by VIP was observed at 26±10 nM (n=3) in OMCTi and at 19 nM (n=2) in DCTb. Related peptides glucagon, secretin and PHI gave lower stimulations of AC compared to VIP in OMCTi. Conversely for rat OMCTi, under identical conditions, glucagon was much more effective than VIP.     

Sensitivity of the Procleix HIV-1/HCV assay for detection of human immunodeficiency virus type 1 and hepatitis C virus RNA in a high-risk population

The Procleix HIV-1/HCV Assay is a high-throughput nucleic acid test for the simultaneous detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNA during blood donor screening. This study evaluated the clinical sensitivity of the Procleix assay and assessed the assay's ability to identify HIV-1- and HCV-infected individuals undetected by standard serologic tests. Plasma samples were obtained prospectively from 539 individuals at high risk for HIV-1 and HCV infection at seven clinics affiliated with Johns Hopkins University. Samples were tested in the Procleix HIV-1/HCV Assay and, if reactive, were then tested in the Procleix HIV-1 and HCV discriminatory assays to differentiate the source of viral RNA positivity. Of these 539 subjects, 287 (53.2%) tested reactive in the Procleix HIV-1/HCV Assay. In discriminatory assay testing, 12 of 287 subjects (4.2%) were reactive for HIV-1 RNA only, 260 (90.6%) were reactive for HCV RNA only, and 11 (3.8%) were coinfected with HIV-1 and HCV. The clinical sensitivity for samples tested neat was 100% for HIV-1 and 99.3% for HCV. Three subjects with Procleix HCV reactive/seronegative results seroconverted upon follow-up and were confirmed as Procleix HCV yield cases. The Procleix HIV-1/HCV Assay is a highly sensitive test that detects ongoing and early HIV-1 and HCV infection in a significant number of subjects at high risk for these diseases. Confirmation of Procleix yield cases upon follow-up demonstrated the ability of the Procleix HIV-1/HCV Assay to detect the presence of HIV-1 and HCV in blood earlier than standard serologic tests.