<Emphasis Type="Italic">ScreenMill</Emphasis>: A freely available software suite for growth measurement,analysis and visualization of high-throughput screen data

Background  

Many high-throughput genomic experiments, such as Synthetic Genetic Array and yeast two-hybrid, use colony growth on solid media as a screen metric. These experiments routinely generate over 100,000 data points, making data analysis a time consuming and painstaking process. Here we describe ScreenMill, a new software suite that automates image analysis and simplifies data review and analysis for high-throughput biological experiments.     

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.     

Ribosomal Ribonucleic Acid-Adenine (N6-) Methylase of Escherichia coli Strain B: Ionic and Substrate Site Requirements

These investigations are concerned with the ionic and substrate-site requirements of ribosomal ribonucleic acid (rRNA)-adenine (N(6)-) methylase of Escherichia coli B. The methylase was essentially inactive in solutions of low ionic strength. The addition of MgCl(2) (optimal at 5 mM) or; to a lesser degree, KCl (optimal at 45 mM) stimulated the rate of methylation; the combination of MgCl(2) and KCl stimulated methylation to an extent equivalent to the sum of the stimulation of each acting alone. The extent of nonspecific binding of the methylase to rRNA decreased as the ionic strength of the solution increased. In the absence of ions, dimethylsulfoxide (DMSO), a nucleic acid denaturing agent, had little influence on the rate of methylation; however, DMSO plus KCl synergistically increased both the rate and the extent of methylation to a greater degree than the combination of Mg(2+) plus K(+). NH(4) (+) was less effective than K(+), and the divalent Mg(2+) offered little stimulation. Monovalent anions (acetate, nitrate, and chloride) were equally effective, whereas divalent SO(4) (2-) was decidedly inhibitory. The appropriate ionic milieu of mono- and divalent cations was required to provide the appropriate conformation of the rRNA and to facilitate specific interactions of the methylase and its recognition sites in the rRNA, while decreasing nonspecific ionic binding of the methylase to rRNA. DMSO may facilitate methylation by increasing the number of substrate sites exposed in single-stranded regions of the rRNA. Nonmethylatable rRNA species served as competitive inhibitors, whereas the polyanions deoxyribonucleic acid, transfer RNA, and polyadenylic acid were inactive. Micrococcus lysodeikticus and Bacillus subtilis rRNA, methylated by the methylase, each contained two distinct heptanucleotides containing newly synthesized 6-methyladenine moieties. The data are consistent with the view that E. coli strain B possesses two species of rRNA-adenine (N(6)-) methylases, each of which recognizes a specific adenine moiety in a unique pentapurine nucleotide sequence in a single-stranded region of rRNA.     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells

The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.     

Limited effects of recombinant human and murine interleukin 1 and tumour necrosis factor on production of acute phase proteins by cultured rat hepatocytes

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.