Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection.

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.

Nucleotide sequence of the gene coding for ovine corticotropin-releasing factor and regulation of its mRNA levels by glucocorticoids.

The ovine gene CRF, coding for corticotropin-releasing factor, has been isolated and the nucleotide sequence determined. The degree of nucleotide sequence homology between the ovine and human CRF genes is unusual, in that the 5' flanking regions are more highly conserved than the protein-coding regions. This striking degree of homology would indicate that a strong selective pressure is being exerted over an extensive area of the 5' flanking region, which could include transcriptional control elements. The 5' flanking region of the ovine CRF gene contains five elements which share homology with the glucocorticoid receptor DNA binding sequence. Also Northern blot analysis indicates that hypothalamic CRF mRNA levels are negatively regulated by glucocorticoids. Dexamethasone treatment halves the CRF mRNA content of the hypothalamus, whereas adrenalectomy causes a three- to four-fold increase in CRF mRNA levels.

The sequence and tissue expression of ovine renin.

The primary structure of the sheep renin precursor has been determined from its cDNA sequence. A library of cDNA clones was constructed from adrenalectomized sheep kidney poly(A)+ RNA and screened for sheep renin sequences with a cloned mouse renin cDNA probe. Of the 300,000 clones generated, 24 were hybridization positive and the nucleotide sequences of two of the longest clones were determined. These clones coded for the mature sheep renin protein and the 3'-untranslated sequence but did not extend to the amino-terminal region of preprorenin. Clones corresponding to the 5' region of renin mRNA were generated by the polymerase chain reaction and their nucleotide sequences determined. The sheep renin precursor consists of 400 amino acids with a putative leader sequence of 14 amino acids and a putative 45 or 53 amino acid prosegment. The mature sheep renin protein has a 73% sequence identity with human renin. Northern analysis demonstrated the presence of renin mRNA in the kidney but not in other tissues in the sheep. While sodium depletion of sheep caused a rise in renin mRNA in the kidney, adrenalectomy also led to a large increase in renal renin mRNA. Southern analysis of genomic DNA suggests that there is only one gene coding for renin in the sheep.

The function of conserved elements in the promoter of the mouse angiotensinogen gene.

The angiotensinogen gene encodes the precursor protein for the potent vasoconstrictor angiotensin II. Although the gene is expressed in several tissues, the liver is the major source of circulating protein. In previous in-vivo studies we have found that a mini-gene containing 750 bp of 5'-flanking sequence is transcribed in a manner which largely parallels the expression of the endogenous gene. In this report, we characterized conserved elements in the promoter region, in order to determine their role in the transcription of the angiotensinogen gene. Constructs fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into hepatocarcinoma Hep G2 cells as well as into nonhepatic cell lines. We found that 5'-deletion mutant constructs, containing sequences from +25 to -90 bp and -321 to -750 bp, were each able to activate transcription. These constructs contain the TATA box and core promoter sequences, including an Sp1-binding site, and two glucocorticoid responsive elements respectively. In the non-hepatic cell lines, HeLa and Jeg-3, we found that the constructs were transcribed at a much lower rate when compared with the expression of a plasmid containing the Rous sarcoma virus long terminal repeat fused to the CAT gene. Constructs which included sequence 5' to -244 were oestrogen inducible. An element which is conserved between rodent and human angiotensinogen promoters is contained within a sequence which is oestrogen responsive, while another binds the liver-enriched transcriptional activator hepatocyte nuclear factor 1. However, the role of this transactivator in the transcription of angiotensinogen remains uncertain.

Granular juxtaglomerular cells and prorenin synthesis in mice treated with enalapril.

The short-term and long-term effects (for up to 98 days) of the angiotensin converting enzyme inhibitor enalapril were investigated in male and female BALB/c mice. In control animals, separate antisera to renin and its prosequence produced an identical pattern of staining in granular cells of the juxtaglomerular apparatus (JGA) a short distance from the glomerulus. After 1 day of the enalapril treatment there was a decrease in the number of JGA granular cells immunostained with antisera to both renin and its prosequence. Electron microscopy revealed degranulation of mature granules from JGA granular cells. Fusion of granules with the cell membrane was not observed, but numerous membrane-like structures (myelin figures) were identified in the cytoplasm and extracellular space, indicating possible secretion. In addition, the volume proportion of granulated cells in relation to the glomerular volume was decreased, as was renal renin content. With continuing enalapril treatment, separate antisera to renin and its prosequence stained the same granulated JGA cells with equal intensity. The cells so stained increased in number, extending down the wall of the afferent arteriole to cortical radial arteries (interlobular arteries) upstream from the glomerulus. Ultrastructural studies revealed a progressive development of cytoplasmic granulation in JGA granular cells and in smooth muscle cells extending into cortical radial arteries. Furthermore, the volume proportion of granulated cells in relation to the glomerular volume was significantly increased, as was renal renin content. Thus, short-term enalapril treatment in mice provoked rapid secretion of renin via degranulation of mature granules from JGA granular cells. In contrast, long-term enalapril treatment produced a continuing stimulus for renin synthesis, secretion and storage, resulting in an increased thickness of the afferent arteriolar wall. The mechanism for this change appears to be hypertrophy and hypergranulation of granular JGA cells and neogranulation of smooth muscle cells upstream from the glomerulus. Identification of the intrarenal mediators that induce these phenotypic changes presents an interesting challenge.

Renin gene expression in vessels of the ovine renal cortex.

Using hybridization histochemistry, a technique which localizes specific mRNA populations in tissue sections with a 700 base pair recombinant DNA probe which codes for ovine renin, we have localized renin gene expression in the afferent arteriole of the juxtaglomerular apparatus (JGA) in the sheep renal cortex. Specific labelling representing renin gene expression was also found at a distance from the glomerular tuft in the walls of the afferent arteriole and also in cells in the medial layer of larger vessels of the renal cortex, specifically the interlobular arteries. These observations provide morphological evidence of renin gene expression at these sites and, combined with ultrastructural and immunocytochemical evidence suggest that renin is synthesized and stored in the afferent arteriole either within the JGA or at a distance from the glomerulus, and in the smooth muscle coat of the interlobular arteries in the sheep kidney.

Characterization of a specific antibody to the rat angiotensin II AT1 receptor.

We raised a polyclonal antibody against a decapeptide corresponding to the carboxyl terminus of the rat angiotensin II AT1 receptor. This antibody was demonstrated to be specific for the rat receptor according to a number of approaches. These included (a) the ultrastructural localization of immunogold-labeled receptor on the surfaces of zona glomerulosa cells in the adrenal cortex, (b) the specific labeling of Chinese hamster ovarian (CHO) cells transfected with AT1 receptors, (c) the identification of a specific band on Western blots, (d) the immunocytochemical co-localization of angiotensin receptors on neurons in the lamina terminalis of the brain shown to be responsive to circulating angiotensin II, as shown by the expression of c-fos, and (e) the correlation between the expression of the mRNA of the AT1 receptor and AT1 receptor immunoreactivity.(J Histochem Cytochem 47:507-515, 1999)

Purification and cloning of a corpuscles of Stannius protein from Anguilla australis.

The kidneys of teleost fish are associated with tissues containing secretory granules--the corpuscles of Stannius (CS). Electron microscopy indicates that the granules are of a proteinaceous nature and may represent hormones or enzymes of unrecognized physiological and biochemical function. In the present study, two-dimensional gel electrophoresis and electroelution was used to purify the major protein to homogeneity; it is approximately 32,000 Da in the reduced form and glycosylated. From the partial NH2-terminal sequence, a 75-mer oligonucleotide probe was synthesized and used to isolate a cDNA clone from which the complete amino acid sequence of the major CS protein was deduced. Polyclonal antibodies raised against CS homogenates were specific for the CS proteins (confirmed by immunohistochemistry). Hybridization histochemistry was used to confirm the location of the mRNA encoding the isolated protein. Incubation of CS homogenate with eel plasma or ovine renin substrate did not result in any angiotensin-like peptides whereas kidney homogenate did.

Adrenaline cells of the rat adrenal cortex and medulla contain renin and prorenin.

The distribution and content of renin in Sprague-Dawley (SD) and transgenic (mREN-2)27 rats (TG) were compared to further define the cellular basis and function of the adrenal renin-angiotensin system. Antibody binding (to rat and mouse renin protein and prosequence) was visualised in serial paraffin sections using an avidin-biotin peroxidase technique. Chromaffin and adrenaline cells were identified by tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase immunoreactivity, respectively. In SD zona glomerulosa (ZG), renin and its prosequence localised to small steroid cells while in homozygous (receiving lisinopril) and heterozygous (untreated) TG, steroid cells labelled in all cortical zones. In addition, throughout the cortex of each strain, large polyhedral adrenaline chromaffin cells occurring singly or in small groups and occasionally in rays labelled for renin and prosequence. Similar large adrenaline cells immunolabelled for all antisera in medulla while other cells were only TH-positive. Total adrenal renin content was 53 times higher in heterozygous transgenics than SD rats and was mainly (74%) prorenin. In SD, 37% of cortical renin was prorenin but in adrenal medulla only active renin was detected. Thus, from present and previous work both renin and prorenin occur not only in mitochondrial dense bodies of the ZG, but also in secretory granules of adrenaline chromaffin cells in both cortex and medulla implying in situ synthesis and paracrine functions.

Is CRF a ganglionic transmitter or modulator in the cat sudomotor pathway?

The presence of corticotrophin releasing factor (CRF)-like immunoreactivity distinguishes a subset of cat sympathetic preganglionic neurons which supplies sweat glands. It is abundant in their terminals in the stellate ganglion. We sought first to determine whether this immunoreactivity is due to true CRF, then to test whether CRF plays any role in ganglionic transmission in the cat sudomotor pathway. CRF-immunoreactive material extracted from cat stellate ganglia and hypothalamus were eluted on HPLC at equivalent retention times, slightly less than that of standard sheep CRF. In chloralose-anaesthetised cats, sheep CRF (0.13, 1.3 and 13 micrograms/kg, i.v.) raised plasma immunoreactive ACTH levels by between 3- and 300-fold. Submaximal stimulus trains delivered to pre- or postganglionic nerves of the right stellate ganglion evoked electrodermal responses (EDR, a measure of sweat gland activity) in the right forepaw pad as well as increases in heart rate and blood pressure. Exogenous sheep CRF (dose range 130 ng/kg to 13 mg/kg) given close-arterially to the stellate ganglion in 5 chloralose-anaesthetised cats had no consistent effect on either baseline or preganglionically-evoked EDR. Given i.v. at 13 micrograms/kg to four further cats, sheep CRF caused no significant change in mean baseline or mean preganglionically-evoked EDP (P > 0.05; CUSUM test). Hexamethonium (10 or 30 mg/kg i.v.) abolished the EDR to preganglionic nerve stimulation (7/7 cats). Increasing preganglionic stimulus voltage, frequency and train duration failed to show any hexamethonium-resistant component of the EDR, although such effects were evident in the cardioaccelerator pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Malate dehydrogenase: isolation from E. coli and comparison with the eukaryotic mitochondrial and cytoplasmic forms.

Escherichia coli malate dehydrogenase has been isolated in homogeneous form by a procedure employing chromatography on DEAE-cellulose, 5-'AMP-Sepharose, and Sephacryl-200. It is composed of two identical polypeptide chains each of Mr = 32 500. Like porcine mitochondrial malate dehydrogenase, it is devoid of tryptophan, but otherwise it is not particularly more similar in composition to one of the eukaryotic isozymes than to the other. However, amino-terminal sequence analysis of the first 36 residues shows remarkable similarity of the bacterial and mitochondrial enzymes (69% identical residues) in contrast to the cytoplasmic form (27%). The two porcine heart enzymes are identical in 24% of the positions compared. These results clearly establish that all three forms of malate dehydrogenase have evolved from a common precursor and that the prokaryotic and mitochondrial forms have retained sequences that are much closer to the ancestral one than the cytoplasmic enzyme. These findings appear to further substantiate the endosymbiotic hypothesis for the origin of the mitochondrion.

Comparative response of the immature and mature ovine fetus to corticotrophin-releasing hormone (CRH).

The aim of this study was to address the possibility that the low concentrations of adrenocorticotrophin (ACTH) seen in the ovine fetus between 90 and 120 days of gestation could be attributed to an alteration in the sensitivity or responsiveness of the fetal pituitary to corticotrophin-releasing hormone (CRH), a key regulator of ACTH secretion. Chronically cannulated ovine fetuses at Days 104-108 (n = 11, representing fetuses from this 90-120-day period) and Days 138-142 (n = 6) of pregnancy received graded doses of ovine CRH (0.8, 1.6, 3.8 and 7.6 micrograms h-1 for 60 min each, given consecutively and in ascending order) or isotonic saline (n = 4 at both age groups studied). Arterial blood samples were taken concurrently for analysis of plasma immunoreactive CRH, ACTH and cortisol throughout the infusion to assess the pituitary-adrenal response. Regression lines describing the relationship between log.PCRH and log.PACTH were calculated for both age group studied. A significant (P < 0.001) rightward shift in the log.PCRH/log.PACTH regression line for the Day 104-108 group was found, suggesting that the ovine fetus at this age is less sensitive or responsive to exogenous oCRH than the mature Day 138-142 fetus. This decreased responsiveness could explain the low concentrations of endogenous ACTH seen during the 90-120-day period.

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR.

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10(4) cells are required for the analysis.

Radioimmunoassay for salivary carbonic anhydrase in human parotid saliva.

A specific radioimmunoassay for human carbonic anhydrase (CA) VI has been developed and used to determine the concentrations of the enzyme in saliva. The assay detected as little as 200 pg of CA VI and the antibody used did not cross-react with CA II or other salivary proteins. The method showed an intra-assay variation of 8.5% and an inter-assay variation of 16.9%. The concentration in parotid saliva varied over a wide range (from 9.7 micrograms/ml to 121 micrograms/ml) with an average value of 47.0 +/- 39.2 (SD) micrograms/ml (n = 50). The mean secretion rate of CA VI from the combined parotid glands was 42.8 +/- 37.9 micrograms/min. CA VI represented about 3% of the total protein in parotid saliva.

Equine angiotensin converting enzyme: a zinc metalloenzyme.

1. Angiotensin I converting enzyme from horse plasma has been extensively purified and shown to be homogeneous by disc-gel electrophoresis. 2. The metal ion involved in the catalytic reaction of the enzyme has been identified for the first time as zinc by atomic absorption spectrometry. 3. A number of other physicochemical properties of the enzyme are described and compared with results obtained by other investigators. The molecular weight was determined by gel filtration to be 113 000 daltons. The pH maximum was found to be 7-4. The chloride activation of the enzyme appears to act by facilitation of substrate binding to the enzyme. 4. By use of enzyme inhibitors, tyrosine has been implicated as a functional residue at the active site of the enzyme. 5. The enzyme shows a fairly high degree of specificity towards its substrates.

Complete amino acid sequence of ovine salivary carbonic anhydrase.

The primary structure of the secreted carbonic anhydrase from ovine salivary glands has been determined by automated Edman sequence analysis of peptides generated by cyanogen bromide and tryptic cleavage of the protein and Staphylococcus aureus V8 protease, trypsin, and alpha-chymotrypsin subdigests of the large cyanogen bromide peptides. The enzyme is a single polypeptide chain comprising 307 amino acids and contains two apparent sites of carbohydrate attachment at Asn-50 and Asn-239. The protein contains two half-cystine residues at 25 and 207 which appear to form an intramolecular disulfide bond. Salivary carbonic anhydrase shows 33% sequence identity with the ovine cytoplasmic carbonic anhydrase II enzyme, with residues involved in the active site highly conserved. Compared to the cytoplasmic carbonic anhydrases, the secreted enzyme has a carboxyl-terminal extension of 45 amino acids. This is the first report of the complete amino acid sequence of a secreted carbonic anhydrase (CA VI).