Bovine insulin-like growth factor binding protein-3: organization of the chromosomal gene and functional analysis of its promoter.

Insulin-like growth factor binding protein-3 (IGFBP-3), the major IGFBP in the circulation, is synthesized by the vascular endothelium in vivo and has been shown to be an important modulator of the physiological effects of IGF. IGFBP-3 is regulated by a number of growth factors/cytokines to which the vascular endothelium is exposed, including IGF-I stimulation and TGF-beta1 inhibition of IGFBP-3 in cultured endothelial cells. To understand the mechanisms of transcriptional regulation of IGFBP-3, we have cloned the bovine IGFBP-3 gene and begun the functional analysis of its promoter. Southern analysis indicated a single copy gene. The gene spanned approximately 10 kb and was divided into five exons, the fifth containing the 3' untranslated region. The transcription start site was 137 bp upstream of the initiation codon and a TATA box was located 26 bp 5' to this CAP site. No CAAT box was present but a GC rich sequence element, containing two overlapping putative AP-2 binding elements, was located 5' to the TATA box. Transient transfection studies with a series of 5' truncated luciferase reporter constructs were conducted in primary cultures of bovine aorta endothelial cells. Results of the transfection studies indicated that 1) nearly 80% of the maximal basal promoter activity was retained within the first 130 bp of the 5' flanking sequence; 2) this region responded to IGF-I, despite lacking the TTF-1/TTF-2 (thyroid specific transcription factors) binding elements that are required for IGF-I stimulation of thyroglobulin synthesis. These binding elements have also been suggested to be involved in IGF-I regulation of IGFBP-3 transcription, thus, implying the existence of novel cis-acting elements that mediate the IGF-I stimulation of bovine endothelial cell IGFBP-3 mRNA synthesis; 3) deletion of the GC rich sequence element resulted in a 60% reduction in basal promoter activity as well as loss of the IGF-1 stimulatory effect; 4) the TGF-beta1 mediated inhibition of IGFBP-3 transcription required sequence element(s) beyond 1.5 kb of its promoter.

Insulin-like growth factor binding protein production by bovine and human vascular smooth muscle cells: production of insulin-like growth factor binding protein-6 by human smooth muscle.

Insulin-like growth factor binding protein (IGFBP) secretory profiles were determined for vascular smooth muscle cells (VSMC) derived from bovine aorta and human aorta, pulmonary artery, and coronary artery. The bovine cells produced IGFBP-4, IGFBP-3, and an IGFBP-3 protease. IGF-I stimulated messenger RNA (mRNA) and media levels of IGFBP-3. The human cells produced IGFBP-3, IGFBP-4, and IGFBP-3 and IGFBP-4 proteases. The three human cells also produced a 30K IGFBP, shown to be IGFBP-6, based on increased affinity for IGF-II vs. IGF-I, size decrease when treated with O-glycanase, but not N-glycanase, reactivity with IGFBP-6 antiserum, presence of a 1.3-kilobase pair mRNA that hybridized to IGFBP-6 specific complementary DNA, and N-terminal amino acid sequence corresponding to IGFBP-6. In the human cells, IGF-I increased media levels of IGFBP-3 through stimulation of IGFBP-3 mRNA and dissociation of cell bound IGFBP-3, and decreased IGFBP-4 via potentiation of IGFBP-4 proteolysis. Neither the bovine nor the human aorta VSMC produced sufficient IGFBP-2 or IGFBP-2 mRNA to be detected by ligand blot and Northern analysis, as previously reported for porcine and rat aorta smooth muscle cells. The variable expression of IGFBPs and IGFBP proteases by VSMC are likely to contribute to differential vascular reactivity to the IGFs in larger arterial blood vessels.

Postmortem diagnosis of autochthonous acute chagasic myocarditis by polymerase chain reaction amplification of a species-specific DNA sequence of Trypanosoma cruzi.

We report a fatal case of vector-transmitted acute Chagas' myocarditis in a seven-month-old child in south Texas. This diagnosis was not suspected during the three days of hospitalization that preceded the child's death, which was caused by heart failure. A diagnosis of acute myocarditis, probably of viral origin, was listed as the cause of death after cardiac tissue was examined microscopically at autopsy. One year after the death of the patient, a diagnosis of Trypanosoma cruzi myocarditis, based solely on morphological grounds, was made after newly prepared slides of cardiac tissue were examined. Seven years later, we confirmed the diagnosis of T. cruzi infection by using the polymerase chain reaction to amplify a species-specific genomic repetitive DNA sequence of the parasite from fixed cardiac tissue.

Comparison of PCR and microscopic methods for detecting Trypanosoma cruzi.

The diagnosis of acute infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is generally made by detecting parasites by microscopic examination of fresh blood. Although highly specific, this approach often lacks sensitivity. Several years ago, PCR assays for the detection of T. cruzi were described, but the sensitivities and specificities of these tests have not yet been defined precisely. In the present study, we first compared the sensitivities of PCR methods that differ in sample processing as well as in the target sequences that are amplified. Then, we challenged eight mice with T. cruzi, and on 31 days over a 380-day period, we compared the ability of the PCR method with the highest sensitivity to detect parasites in blood with that of microscopic examination. During the acute phase of the infections, parasites were detected on average 3.9 days earlier by the PCR method than by microscopy. Furthermore, the infected mice were consistently positive by the PCR method during the chronic phase, while parasites were intermittently detected by microscopic examination during that period. Overall, among the 248 comparisons, in 84 the PCR method was positive and no parasites were seen by microscopic examination, whereas the reverse was true in only 1 case, a difference that is highly significant. These findings suggest that this approach should be in patients suspected of having acute Chagas' disease. Moreover, the higher sensitivity of the PCR method observed in both the acute and chronic phases of the T. cruzi infections in the mice that we studied indicates that this approach should be useful in evaluating experimental drugs in T. cruzi-infected laboratory animals.

Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-I and TGF-beta.

We have examined the regulation of endothelial IGFBP-3 production by IGF-I and TGF-beta, two growth factors thought to play a major roles in the complications of diabetes mellitus. In addition, we developed a sensitive method for IGFBP-3 mRNA quantitation by adapting the fluorescent modification of the competitive PCR strategy. Our results using both Northern analysis and the fluorescent competitive PCR method indicate that: (1) IGFBP-3 mRNA is increased 2- to 10-fold by IGF-I and maximally reduced to 20% of control by TGF-beta; (2) the changes in mRNA levels correlate with the levels of IGFBP-3 protein secreted into the media by these cells; (3) the induction of IGFBP-3 mRNA and protein by IGF-I analogs was directly related to their ability to bind to the type I IGF receptor, reflecting an IGF-I receptor-mediated process; and (4) steady state IGFBP-3 mRNA levels did not change significantly after a 6 h incubation with actinomycin D in the presence or absence of the growth factors suggesting that the observed IGF-I/TGF-beta effects occur at the level of gene transcription rather than mRNA stability.

Maxicircle genomic organization and editing of an ATPase subunit 6 RNA in Trypanosoma cruzi.

The DNA sequence of a 5736-nucleotide (nt) Trypanosoma cruzi maxicircle fragment was determined. Sequence comparisons indicate that its 5' terminus is the homologue of the downstream portion of the NADH dehydrogenase subunit 7 gene and that its 3' region is homologous to the maxicircle unidentified reading frame II gene. The region between these two gene segments contains six additional genes that encode mitochondrial proteins, including ATPase subunit 6 (A6). Comparison of the A6 maxicircle DNA sequence with that of an A6 cDNA indicates that the A6 RNA is extensively edited throughout its length. A 49-nt sequence that could serve as template for transcription of a guide RNA for editing a segment of the A6 RNA was found in one of 24 minicircle variable regions sequenced. Moreover, the presence of an RNA having this sequence was demonstrated in an RNAse protection assay. This is the first identification of a guide RNA template in a T. cruzi minicircle. Taken together, our findings suggest that T. cruzi and Trypanosoma brucei brucei are phylogenetically closer to each other than they are to Leishmania tarentolae, despite the relative similarity of the life cycles of the latter and T. cruzi.

Cloning and characterization of the cDNA encoding guinea-pig properdin: a comparison of properdin from three species.

The cDNA sequence encoding properdin was generated from guinea-pig spleen RNA by the reverse transcription-polymerase chain reaction. This sequence was approximately 75% homologous with human and 71% homologous with murine properdin at the nucleic acid level. Guinea-pig properdin had six thrombospondin repeat sequences consisting of about 60 amino acids, each with six cysteine and three tryptophan residues. Additionally, the Valine-Threonine-Cysteine-Glycine sequence, reported to have important cell adhesive properties in malarial circumsporozoite proteins and thrombospondin, was conserved in the properdin sequence of guinea-pigs. Finally, mouse spleen was also examined to complete the sequence determination of the leader peptide and the initial four residues of murine properdin. This allowed a thorough comparison of the primary structure of properdin from all three species. Like human and murine properdin cDNAs, the guinea pig sequence contained a region of unique, non-homologous sequence (18 base pairs in length) within the fifth thrombospondin repeat, the significance of which remains unclear.

Expression of mRNAs of multiple growth factors and receptors by astrocytes and glioma cells: detection with reverse transcription-polymerase chain reaction.

1. Although glial cells in culture are known to secrete growth factors and are also known to be responsive to some of them, detailed comparisons are difficult because the bulk of information was based on various animals of origin, developmental stages, growth properties, culture age, and culture conditions. 2. To present a unified picture of the growth factors and their receptors found in glial cells, we surveyed the expression of messenger RNAs of a panel of growth factors and receptors, using reverse transcription-polymerase chain reaction (RT-PCR), in three common glial cell types: rat astrocytes in primary culture, rat glioma line C6, and human glioma line A172. 3. We observed that normal and neoplastic glial cells in culture express multiple growth factors and also possess most of the receptors to the factors, suggesting multiple autocrine functions. In addition, glia produce growth factors known to be capable of acting on neurons, implicating paracrine function involving glia-neuron interaction. Glial cells also produce growth factors and receptors that are capable of communicating with hematopoietic cells, suggesting neuroimmunologic interaction. What is most interesting is that glial cells express receptors for growth factors previously thought to be acting on neurons only. 4. The current study demonstrates the feasibility of screening from a small sample a large number of growth factors and receptors. The method portends future clinical application to biopsy or necropsy samples from brain tumors or pathologic brains suffering from degenerative diseases such as Alzheimer's or Parkinson's disease.

Searching for a new paradigm in health care delivery.

The cartoon character Pogo, uttering his now famous line, "We have met the enemy and it is us," might well have been referring to the dilemmas that we face today in American health care. A major source of our current difficulties in solving these admittedly complex problems lies in our way of thinking about, or conceptualizing, health care and health care delivery. We are caught in an old paradigm that is simply not adequate for dealing with the health care delivery problems of the '90s, not to mention those of the 21st Century.

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.

A major estrogen-regulated protein secreted from the liver of Xenopus laevis is a member of the serpin superfamily. Nucleotide sequence of cDNA and hormonal induction of mRNA.

Estrogen treatment of Xenopus frogs causes four mRNAs to become highly abundant in the liver. Three of these mRNAs have been previously identified as coding for vitellogenin, ferritin, and serum retinol binding protein. We show here that the fourth abundant liver messenger RNA comprises about 1500 nucleotides and codes for a 45-kDa secreted protein, designated Ep45. A clone complementary to Ep45 mRNA was isolated, and its identity was confirmed by hybridization selection of mRNA that translated in vitro into the Ep45 precursor. Nucleotide sequence analysis of the nearly full length cDNA revealed a total length of 1454 base pairs consisting of: 36 nucleotides of the 5' noncoding region, 1308 base pairs encoding an open reading frame of 436 amino acids, and 110 nucleotides of the 3' untranslated region. Ep45 mRNA may originate from as many as four closely spaced transcription start sites, which are 15 to 21 bases upstream of the first nucleotide of the cDNA clone. The Xenopus laevis genome appears to contain a single Ep45 gene. The deduced amino acid sequence indicates that Ep45 has features typical of a secreted protein, including a signal peptide of 16 amino acids and three potential sites for N-linked glycosylation, and is related to the serine protease inhibitors, a large family of proteins with very diverse physiological functions. Ep45 mRNA was absent in the liver of normal male frogs and increased at least 100-fold in response to estradiol-17 beta. Thus, both Ep45 and vitellogenin mRNAs are switched from undetectable to very high levels, a pattern of expression not found for any other mRNAs in Xenopus liver.

Molecular cloning of cDNA for the B beta subunit of Xenopus fibrinogen, the product of a coordinately-regulated gene family.

Fibrinogen, the principal blood-clotting protein, is made up of three different subunits synthesized in the liver. In vitro administration of glucocorticoids to liver cells from the frog Xenopus laevis causes a dramatic increase in fibrinogen synthesis. Investigations of molecular mechanisms underlying this hormonal stimulation at the mRNA level require cDNA clones complementary to the mRNAs coding for the three fibrinogen subunits, called A alpha, B beta, and gamma. We describe here the isolation and characterization of cDNA clones for the B beta subunit of Xenopus fibrinogen. cDNA libraries in both plasmid (pBR322) and phage (lambda gt10) cloning vectors were constructed from frog liver mRNA and screened with a rat B beta cDNA. Clones thus isolated hybridized to two Xenopus liver mRNAs 2500 and 1800 bases long, the previously-determined sizes for B beta mRNAs. The identity of the plasmid clone B beta-27 was confirmed by hybridization-selection of complementary mRNA which translated in vitro into the B beta polypeptide, as determined by size and susceptibility to thrombin cleavage. lambda/B beta 10, a clone representing nearly all of the 2500-base B beta mRNA, was isolated from the phage cDNA library. The 3'-end of this clone includes a polyadenylation signal about 20 residues upstream of a stretch of 34 adenosine residues, which probably represents the 3'-poly(A) tail of the messenger RNA. lambda/B beta 10 lacks only 20 nucleotides of full-length B beta mRNA at the 5'-end and there is one major start site of transcription. The 2500-base B beta mRNA has a 700-base extension at the 3'-end that is not present in the 1800-base mRNA. The Xenopus laevis genome contains two or three genes for the B beta fibrinogen subunit. Using the cDNA clone as a probe, B beta mRNA was shown to be induced at least 20-fold by glucocorticoid treatment of purified parenchymal cells of Xenopus liver maintained in primary culture.

Fetal development and neuronal/glial cell specificity of cAMP-dependent protein kinase subunit mRNAs in rat brain.

All known actions of cAMP in the brain require cAMP-dependent protein kinase (cAMPdPK), which consists of regulatory (R) and catalytic (C) subunits (R2C2). Using homologous rat cDNAs for all known cAMPdPK subunit isoforms found in the brain (RI alpha, RI beta, RII alpha, RII beta, C alpha, C beta) we observe that, in the fetal rat brain from 12 days of gestation to birth, while alpha subunit (RI alpha, RII alpha, C alpha) mRNA levels are abundant, beta subunit (RI beta, RII beta, C beta) mRNA levels increase from undetectable or very low levels to abundant levels. Furthermore, while alpha subunit mRNA levels are abundant in both primary neuronal and primary glial cultures, beta subunit mRNA levels are very low (C beta) or undetectable (RI beta, RII beta) in primary glial cultures, but are abundant in primary neuronal cultures. Thus, prior to about 12 days of gestation, cAMP in the brain may act only via the alpha cAMPdPK subunits in neuronal and glial precursor cells. After 12 days of gestation, coincident with the onset of final cell division in neurons, beta cAMPdPK subunits may also mediate the effects of cAMP predominantly in neurons.

Isolation and characterization of cDNA clones for the gamma subunit of Xenopus fibrinogen, the product of a coordinately regulated gene family.

Fibrinogen, the major structural protein involved in blood coagulation, is synthesized and secreted by the liver. In the frog Xenopus laevis, fibrinogen production is dramatically induced by glucocorticoids. The hormonal stimulation requires synthesis of three separate subunits, designated A alpha, B beta, and gamma. For investigation of the molecular mechanisms underlying this coordinate induction, we have isolated cDNA clones for the subunits of Xenopus fibrinogen. In this communication we describe the identification of clones for the gamma chain. Initially, a Xenopus liver cDNA library in pBR322 was screened with a rat gamma chain cDNA and a clone representing half of the 1600-base frog gamma mRNA was identified. This clone was shown to be complementary to gamma mRNA by hybrid selection of mRNA that translated in vitro into the gamma polypeptide. A clone about 1460 base pairs in length was then isolated from a Xenopus liver lambda gt10 cDNA library and subcloned into Bluescript SK-. This clone, designated X1 gamma 3, contains the entire 3'-end and lacks 38 bases at the 5'-end of gamma mRNA. The deduced amino acid sequence at the N-terminal is compatible with a signal peptide of 20-23 amino acids, in agreement with the calculated size of the frog gamma chain signal peptide. Following the signal sequence is a region of highly conserved amino acids that participate in disulfide bond formation critical for the maintenance of tertiary structure in mammalian fibrinogen. The gamma cDNA clone was used to measure gamma mRNA in purified Xenopus liver cells treated with glucocorticoids in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.

Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.

The polymerase chain reaction was used to amplify a 188-base pair (bp) segment of the repetitive 195-bp nuclear DNA sequence of Trypanosoma cruzi that is the most abundant sequence in this organism. The reaction amplified this repetitive element in four T. cruzi isolates from widely separated geographic regions. No amplification of the 188-bp fragment occurred when DNAs extracted from Leishmania spp., African trypanosomes, or blood samples from mice and humans were used. Amplification of one-half of the DNA from a single T. cruzi parasite produced an amount of the 188-bp element that was readily visible in a gel stained with ethidium bromide. Hybridization of a radiolabeled probe to membrane-bound amplification products increased the sensitivity to a level at which 1/200 of the DNA in a single parasite could be detected. T. cruzi DNA was readily detected in DNA extracted from the abdominal contents of infected insect vectors reared in the laboratory. No parasite DNA was detected in the blood samples of two individuals known to be infected with T. cruzi, possibly because in such patients the number of circulating parasites are extremely low or because parasitemias are intermittent. These results represent a considerable increase in sensitivity over previously reported methods for the detection of T. cruzi infections. Polymerase chain reaction amplification can be used to evaluate large numbers of samples in a single day and thus should be useful in large-scale studies of the prevalence of T. cruzi in both insect vectors and mammalian hosts.

Trypanosoma cruzi expresses diverse repetitive protein antigens.

We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship.

cis-acting mutations that affect rop protein control of plasmid copy number.

A number of pMB1 derivatives provide a trans-acting function that can suppress lethal runaway replication of a temperature-sensitive copy-number mutant of NTP1. Deletion analysis indicates that the region of the pMB1 genome that contains the rop gene is required for this suppression. Mutant derivatives of the temperature-sensitive copy-number mutant plasmid whose conditional lethal phenotype is not suppressed in trans by the region encoding the rop gene have been isolated. These rop-insensitive derivatives contain single nucleotide changes within the RNA I coding region.