Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Factor V Leiden and pulmonary embolism in a young woman taking an oral contraceptive.

A 34-year-old woman with a history of asthma and oral contraceptive use died suddenly. Autopsy examination showed chronic pulmonary emboli with an acute pulmonary saddle embolus. An underlying congenital thrombophilic disorder was considered. Molecular studies on DNA isolated from blood using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis revealed coagulation factor V Leiden mutation. The incidence of venous thromboembolism in patients with factor V Leiden mutation and associated activated protein C (APC) resistance is discussed.

PIP: This article presents a case report of a 34-year-old woman with a known medical history of asthma and oral contraceptive use who died suddenly from a massive pulmonary embolus. This woman had no underlying malignancy, trauma, recent surgery, or other predisposing factors putting her at risk of venous thromboembolism except for her use of oral contraceptives. Upon autopsy, a large saddle embolus was found occluding the main pulmonary arteries with bilateral extension into the smaller arteries. An acute pulmonary embolus obliterating the lumen of the main pulmonary artery was found during the microscopic examination. Molecular studies of blood DNA using polymerase chain reaction and restriction fragment length polymorphism revealed a coagulation factor V Leiden mutation. Female carriers of factor V Leiden mutation who take oral contraceptives have a more than 30-fold increased risk of developing deep venous thrombosis. This case demonstrates that the woman had an underlying predisposition that was further potentiated by oral contraceptive use. The incidence of venous thromboembolism in patients with factor V Leiden mutation and associated activated protein C resistance is discussed.

Molecular cloning and structural analysis of the human gene encoding cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) appears to play a role in the vertebrate visual process as a substrate-routing protein, influencing the enzymatic partitioning of 11-cis-retinol at a key branch point in the visual cycle. Genomic clones spanning 29 kilobases and encompassing the human CRALBP gene have been isolated by screening two human genomic libraries and from polymerase chain reaction amplification of human leukocyte DNA. The sequence of 13,647 contiguous nucleotides has been determined, including 3130 and 516 bases from the 5'- and 3'-flanking regions, respectively. The human CRALBP gene exists as a single copy in the genome based on Southern analyses and localization to a single site on human chromosome 15 (Sparkes, R. S., Heinzmann, C., Goldflam, S., Kojis, T., Saari, J. C., Mohandes, T., Klisak, I., Bateman, J. B., and Crabb, J. W. (1992) Genomics 12, 58-62). The gene is composed of eight exons and seven introns with average lengths of 198 base pairs and 1.2 kilobases, respectively, and which exhibit conventional vertebrate splicing. Alu repetitive sequences exist in introns 4 and 5 as well as in the 5'- and 3'-flanking regions of the gene. RNase protection and primer extension analyses indicate that the human CRALBP gene transcription start site is 922 bases upstream of the initiation codon. The first exon is entirely untranslated and both exon 2 and exon 8 contain untranslated regions. The proximal 5'-flanking region lacks GC boxes and consensus TATA and CCAAT boxes at the usual positions. The 3'-untranslated region of CRALBP exon 8 is essentially identical to a partial cDNA clone reportedly isolated from a human hippocampus cDNA library, suggesting that the protein may be expressed in a wider spectrum of tissues than previously recognized. The human CRALBP genomic clones and structure provide valuable tools for studying the physiological role of the protein in vision and visual disorders.

Acidic fibroblast growth factor gene 5' non-coding exon and flanking region from hamster DDT1 cells: identification of the promoter region and transcriptional regulation by testosterone and aFGF protein.

Selected clones of Syrian hamster DDT1-MF2 cells are responsive to testosterone for growth. Heparin binding growth factor 1 (HBGF-1) or acidic fibroblast growth factor (aFGF) can replace testosterone (T) in the stimulation of growth in these cells. This phenomena is correlated with testosterone's ability to elevate aFGF mRNA two- to threefold in DDT1 cells. To better understand the possible mechanisms of regulation of aFGF mRNA by steroids and other growth factors, we isolated the aFGF 5' non-coding exon and its flanking region from a EMBL3 DDT1 genomic library, using a 5' non-coding exon 69 bp DDT1 aFGF cDNA probe. Clones spanning 30 kb of genomic DNA were isolated. After restriction mapping and DNA sequence analysis, the clones were shown to contain all of the 5' non-coding exon included in the cDNA and approximately 10 kb of 5' flanking region. RNase protection and primer extension assays confirmed that the 5' non-coding exon is included in the DDT1 aFGF mRNA and that a major transcription start site is approximately 136 bp upstream of the 5' non-coding splice junction of this exon. The 5' flanking region DNA was inserted into pBLCAT3 reporter gene and transfected into DDT1 cells. Chloramphenicol acetyltransferase (CAT) assays demonstrated that there are promoter elements in the -1645/-392 and -392/+131 regions of the aFGF gene in the context of DDT1 cells. NIH 3T3 cells, on the other hand, show no CAT activity with these aFGF-CAT plasmids. CAT assays also demonstrated that addition of testosterone (T) or aFGF to DDT1 cells increased CAT activity threefold. This activity was mapped to -1645 to -4 bp region of this DDT1 aFGF gene promoter.

Identification of Mycoplasma binding proteins utilizing a 100 kilodalton lung fibroblast receptor.

A 100 kilodalton glycoprotein receptor for Mycoplasma pneumoniae has been isolated from MRC-5 human lung fibroblasts. This receptor, as well as anti-receptor serum, were both capable of inhibiting the attachment of 14C-labelled M. pneumoniae to MRC-5 fibroblasts. The receptor was also capable of inhibiting the attachment of C-labelled M. gallisepticum and M. genitalium, but not M. pulmonis, to MRC-5 fibroblasts. This indicates that a common sequence may exist in these binding proteins of M. pneumoniae, M. genitalium, and M. gallisepticum. This receptor and anti-receptor serum were utilized to probe M. pneumoniae, M. genitalium, and M. gallisepticum for their corresponding binding proteins. A 32 kilodalton protein in M. pneumoniae, a 90 kilodalton protein in M. genitalium and a 139 kilodalton protein in M. gallisepticum were recognized.

Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum.

A 5.5 kilobase DNA fragment from an Eco RI digest of the Mycoplasma gallisepticum genome was specific for the detection of M. gallisepticum. This 5.5 kb fragment was initially cloned into bacteriophage lambda gt11 followed by subcloning into the plasmid vector pGEM-3Z. The incorporation of a biotin label was accomplished by utilizing biotin-11-dUTP in a nick translation reaction. This probe, designated pMg6, reacts specifically with M. gallisepticum when tested against various mycoplasma DNAs in Southern blot hybridization analysis. Spot-blot hybridization data indicate the pMg6 is capable of detecting 800 pg of M. gallisepticum DNA.

A constitutively transcribed actin gene is associated with the nuclear matrix in a Drosophila cell line.

The relationship between transcriptional activity and gene association with the nuclear matrix has been investigated in Drosophila melanogaster. The nuclear matrix of Schneider cell line 2 of Drosophila was isolated and observed to conform to expected dimensions in phase contrast and scanning electron microscopic preparations. This structure contains proteins that appear similar to the intact nucleus. High salt extracted nuclei digested with DNase I released 98% of the DNA, whereas digestion with Eco RI released a maximum of 80%. These and other nuclease digestions indicate that satellite DNA as well as some unique sequence DNA are bound to the nuclear matrix. A constitutively transcribed actin gene was enriched in the nuclear matrix bound DNA. Two other nontranscribed genes, a muscle-specific actin gene and the myosin heavy chain gene, showed no enrichment in nuclear matrix DNA.