Augmentation of central arterial pressure in mild primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is associated with increased cardiovascular risk, although the mechanisms involved remain unclear. Recent evidence has shown increased pulse pressure to be a powerful predictor of cardiovascular events. As increases in pulse pressure are due largely to arterial stiffening, we measured arterial stiffness in 21 subjects with PHPT (18 women and 3 men; 46-71 yr old) and 21 age- and sex-matched healthy controls using pulse wave analysis, a technique that measures peripheral arterial pressure waveforms and generates corresponding central aortic waveforms. This allows determination of the augmentation of central pressure resulting from wave reflection and augmentation index, a measure of vessel stiffness. Metabolic parameters were also measured. The serum calcium level among PHPT subjects was (mean +/- SD) 2.74+/-0.14 mmol/L. pulse wave analysis showed that both augmentation and the augmentation index were significantly higher in the PHPT group vs. controls [16+/-5 vs. 10+/-4 mm Hg (P < 0.001) and 36+/-9% vs. 25+/-6% (P < 0.001)] despite comparable brachial systolic pressures between groups (136+/-13 vs. 134+/-18 mm Hg). Patients with PHPT had higher fasting serum insulin levels [median (range), 15.8 (7.4-39.4) vs. 11.6 (5.1-23) mU/L; P < 0.05] and triglyceride (1.6+/-0.6 vs. 1.2+/-0.4 mmol/L; P < 0.05), but lower high density lipoprotein cholesterol (1.4+/-0.4 vs. 1.6+/-0.3 mmol/L; P < 0.05). These data indicate that subjects with mild PHPT (calcium, <3.0 mmol/L) have increased arterial stiffness, as evidenced by higher augmentation of central aortic pressures. Enhanced vessel stiffness may arise from a combination of structural and functional vascular changes due to hypercalcemia and/or metabolic abnormalities. Increased vascular stiffness in subjects with PHPT may account in part for the increased cardiovascular risk in this group.

Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function.

DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c-type cytochromes in the latter compartment. Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains. The previously assigned translational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional alpha-helix at the N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm. The newly assigned 5' end of the dipZ gene was shown to be preceded by a functional ribosome-binding site. The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function.

Undetectable urinary free cortisol concentrations in a case of Cushing's disease.

Measurement of the 24-h urinary free cortisol is a valuable screening test of endogenous hypercortisolism and, although false positive results may occur in a few situations, for example endogenous depression, false negative results are unusual. We report a case of a 48-year-old lady with pituitary-dependent Cushing's disease, whose 24-h urinary free cortisol excretion was consistently undetectable in association with increased plasma and salivary cortisol concentrations and reduced dexamethasone suppressibility. The patient had chronic renal impairment (creatinine clearance 21 ml/min) as a consequence of hypertension, despite only modestly increased urea and creatinine concentrations. Urinary free cortisol measurements must be interpreted with caution in patients with renal impairment.

Molecular genetics of the genus Paracoccus: metabolically versatile bacteria with bioenergetic flexibility.

Paracoccus denitrificans and its near relative Paracoccus versutus (formerly known as Thiobacilllus versutus) have been attracting increasing attention because the aerobic respiratory system of P. denitrificans has long been regarded as a model for that of the mitochondrion, with which there are many components (e.g., cytochrome aa3 oxidase) in common. Members of the genus exhibit a great range of metabolic flexibility, particularly with respect to processes involving respiration. Prominent examples of flexibility are the use in denitrification of nitrate, nitrite, nitrous oxide, and nitric oxide as alternative electron acceptors to oxygen and the ability to use C1 compounds (e.g., methanol and methylamine) as electron donors to the respiratory chains. The proteins required for these respiratory processes are not constitutive, and the underlying complex regulatory systems that regulate their expression are beginning to be unraveled. There has been uncertainty about whether transcription in a member of the alpha-3 Proteobacteria such as P. denitrificans involves a conventional sigma70-type RNA polymerase, especially since canonical -35 and -10 DNA binding sites have not been readily identified. In this review, we argue that many genes, in particular those encoding constitutive proteins, may be under the control of a sigma70 RNA polymerase very closely related to that of Rhodobacter capsulatus. While the main focus is on the structure and regulation of genes coding for products involved in respiratory processes in Paracoccus, the current state of knowledge of the components of such respiratory pathways, and their biogenesis, is also reviewed.

Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria.

The biogenesis of bacterial c-type cytochromes generally involves many gene products--some of which may also have roles in other processes--and their interaction with the disulphide-bond-forming system of the bacterial periplasm. However, in some bacteria a simpler process appears to operate that might be related to the formation of c-type cytochromes in thylakoids of photosynthetic cells. The corresponding process in fungal mitochondria is distinct.

Paracoccus denitrificans CcmG is a periplasmic protein-disulphide oxidoreductase required for c- and aa3-type cytochrome biogenesis; evidence for a reductase role in vivo.

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein-disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG-PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c-type cytochromes, but also loss of spectroscopically detectable cytochrome aa3, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa3 and cytochrome c oxidase activity, although it did not restore c-type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein-disulphide bonds in certain protein substrates including c-type cytochrome polypeptides and/or polypeptides involved in c-type cytochrome biogenesis.

Development of acromegaly during treatment of hyperprolactinemia with bromocriptine: an unusual acidophil stem cell adenoma.

We present clinical details of a patient with a 20-yr history of amennorhea, a pituitary tumor, elevated PRL levels, and initially undetectable GH. Bromocriptine failed to fully suppress PRL, and there was no tumor shrinkage. Within 7 months of starting bromocriptine treatment, the patient developed clinical and biochemical signs of acromegaly. At surgery, a stem cell adenoma was excised. The mechanisms by which bromocriptine may have resulted in the development of acromegaly in this patient are discussed.

Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.

The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.

Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product is required for assembly of all c-type cytochromes, including cytochrome c1.

A transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c-type cytochromes and accumulated periplasmic apocytochrome c550, but the membrane-bound holocytochromes c1 and c552 were present at 5-10% of the levels observed in wild-type cells. DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild-type DNA library. A hybridizing 3.05 kb BamHI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DP108 and the B. japonicum cycH mutant COX3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.

Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop.

Three genes, narH, narJ and narI, of the membrane-bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (beta, putative delta and gamma) subunits of the two membrane-bound nitrate reductases of the enteric bacterium Escherichia coli. All iron-sulphur cluster ligands proposed for the E. coli beta subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly alpha-helical structure. Comparison of T. pantotropha NarI with the b-haem-binding integral membrane subunits of the E. coli enzymes allows assignment of His-53, His-63, His-186 and His-204 (T. pantotropha NarI numbering) as b-haem axial ligands and the construction of a three-dimensional model of this subunit. This model, in which the two b-haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane-bound uptake hydrogenases and membrane-bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate-reducing bacteria has structural resemblance to NarI.

Clinical evaluation for colonic polyps: usefulness of signs and symptoms in diagnosis.

This article reports a prospective cross-sectional study of the relationship between various signs and symptoms commonly ascribed to colorectal cancer and the presence or absence of colonic polyps found by colonoscopic examination. Of the 166 patients who participated in the study, 96 were positive for colonic polyps and 70 were negative. There was a significant increase in risk for adenomas polyps as age increased among males. For females, the step-wise logistic regression indicated that an absence of abdominal pain/cramping (p < .01), a change in stool shape (p < .01), and a history of colorectal polyps (undifferentiated) in first-degree relatives (p < .05) were associated with adenomatous polyps.

Differential reduction in soluble and membrane-bound c-type cytochrome contents in a Paracoccus denitrificans mutant partially deficient in 5-aminolevulinate synthase activity.

A mutant of Paracoccus denitrificans, DP104, unable to grow anaerobically with nitrate as the terminal electron acceptor or aerobically with methanol as the electron donor and staining negatively in the dimethylphenylene diamine oxidation (Nadi) test, was isolated by transposon Tn5::phoA mutagenesis. P. denitrificans DP104 grown aerobically with succinate or choline had very low levels (2 to 3% of the wild-type levels) of spectroscopically detectable soluble c-type cytochromes. In contrast, membrane cytochromes of the a, b, and c types were present at 50% of the levels found in the wild type. The apo form of cytochrome c550, at an approximately 1:1 molar ratio with the holo form, was found in the periplasm of DP104. The TnphoA element was shown to be inserted immediately upstream of the translational start of hemA, the gene coding for 5-aminolevulinate synthase, which was sequenced. Low-level expression of this gene, driven off an incidental promoter provided by TnphoA-cointegrated suicide vector DNA, is the basis of the phenotype which could be complemented by the addition of 5-aminolevulinate to growth media. Disruption of the hemA gene generated a P. denitrificans strain auxotrophic for 5-aminolevulinate, establishing that there is no hemA-independent pathway of heme synthesis in this organism. The differential deficiency in periplasmic c-type cytochromes relative to membrane cytochromes in DP104 is suggested to arise from unequal competition for the restricted supply of heme which results from the effects of the transposon insertion.

Characterisation and amino acid sequence of cytochrome c-550 from Thiosphaera pantotropha.

A cytochrome c-550, with mid-point potential +265 mV, has been purified from Thiosphaera pantotropha. The cytochrome was recognised by antibodies to Paracoccus denitrificans cytochrome c-550, but the two proteins were not immunologically identical. Amino acid sequencing of the cytochrome c-550 showed 85.9% and 95.5% identities, respectively, with the cytochromes c-550 of P. denitrificans and Thiobacillus versutus; these are amongst the highest values reported for similarities between class I c-type cytochromes of the c2 group. These similarities are consistent with the published values of 85% for the overall DNA similarity of P. denitrificans and T. pantotropha, but contrast with published 16S rRNA analyses which indicate identity between T. pantotropha and P. denitrificans and 97.5% similarity of T. versutus with these two organisms. Analysis by plasma-desorption mass spectrometry of the peptide containing the haem-binding motif isolated from the apocytochrome has shown that an Hg atom binds to one or both of the two thiol groups.

Growth hormone releasing hormone 1-44 NH2 and 1-40 OH levels in normal subjects during growth hormone stimulation tests.

OBJECTIVE: Little is known about the relative circulating concentrations of growth hormone releasing hormone (GHRH) 1-44 NH2 and 1-40 OH in response to dynamic GH stimulation. We therefore studied the concentrations of growth hormone-releasing hormone (GHRH) 1-44 NH2 and 1-40 OH in the peripheral plasma of normal male subjects during GH stimulation tests. DESIGN: Tests were performed at 0900 h after an overnight fast. Stimulation tests, commenced at 0 minutes, included alpha-adrenergic activation with adrenaline (10 micrograms/min from 0 to 30 minutes) following beta-blockade with propranolol (1.5 mg/min from -10 to 0 minutes), alpha 2-adrenergic activation with clonidine 150 micrograms i.v., insulin hypoglycaemia (0.15 U/kg soluble insulin), L-arginine infusion (30 g from 0 to 30 minutes), L-dopa (500 mg orally) and oral glucose (100 g). SUBJECTS: Groups of healthy male volunteers aged 20-42 years, all within 10% of ideal body weight. MEASUREMENTS: Serum GH and plasma GHRH 1-44 NH2 and 1-40 OH were measured at intervals for between 60 and 390 minutes, depending on the stimulation test. RESULTS: There were no significant changes in either GHRH 1-44 or 1-40 following alpha-adrenergic activation with propranolol/adrenaline infusion, alpha 2-adrenergic activation with i.v. clonidine, insulin-induced hypoglycaemia or arginine infusion despite the expected rise in GH levels. After oral glucose, GH was initially suppressed with a late rise. There were no changes in GHRH 1-44 or 1-40 levels during either phase of this response. After L-dopa GH levels peaked at 90 minutes, 24.5 +/- 11.0 mU/l (mean +/- SEM). At 0 minutes GHRH 1-44 and 1-40 levels were 3.25 +/- 0.89 and 4.93 +/- 1.28 pmol/l respectively and rose in both cases, peaking at 60 minutes at 4.23 +/- 1.01 and 7.55 +/- 1.80 pmol/l (P < 0.05). At no time was there any evidence of differential secretion of GHRH 1-44 or 1-40. CONCLUSIONS: We have confirmed previous studies demonstrating a small rise in GHRH before the GH response to L-dopa. However, in all other situations of pharmacological stimulation of GH release we were unable to detect any significant changes in GHRH 1-44 or 1-40 levels. It seems most likely that peripheral GHRH does not reflect hypothalamic secretion. As yet there is no evidence for differential release of GHRH 1-44 and 1-40.

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c-type cytochrome biogenesis synthesise the methylamine-dehydrogenase polypeptides but cannot assemble the tryptophan-tryptophylquinone group.

Five mutants of Methylobacterium extorquens and four mutants of Paracoccus denitrificans that have a general defect in c-type cytochrome synthesis also failed to assemble an active methylamine dehydrogenase. In all cases methanol dehydrogenase, another periplasmic enzyme, was fully active. All nine mutant strains accumulated both the heavy and light subunits of methylamine dehydrogenase to essentially wild-type levels. In all nine mutants, the heavy-subunit and light-subunit polypeptides were proteolytically processed, suggesting that translocation to the periplasm had occurred; in the case of the P. denitrificans mutants, a periplasmic location for the heavy and light subunits was confirmed experimentally. While specific quinone staining of the methylamine dehydrogenase light subunit in wild-type M. extorquens and P. denitrificans strains could readily be demonstrated, the light subunit polypeptides accumulated by the mutants did not quinone stain, indicating that the methylamine dehydrogenase prosthetic group, tryptophan tryptophylquinone, is not assembled in the absence of functional c-type cytochromes.

Insertion of transposon Tn5 into a structural gene of the membrane-bound nitrate reductase of Thiosphaera pantotropha results in anaerobic overexpression of periplasmic nitrate reductase activity.

Chlorate-resistant mutants of the denitrifying bacterium Thiosphaera pantotropha were generated by transposon Tn5 mutagenesis. One class was deficient in membrane-bound nitrate reductase activity but retained a periplasmic nitrate reductase activity. Using transposon marker rescue it was shown that in one such mutant, M-6, the transposon was inserted in the membrane-bound nitrate reductase beta subunit structural gene (termed narH in order to be consistent with the nomenclature of the Escherichia coli major nitrate reductase operon). The translated sequence (total of 106 amino acids) from around the point of transposon insertion showed approximately 90% amino acid identity with the beta subunits of the E. coli nitrate reductases. Under anaerobic growth conditions M-6 overproduced the periplasmic nitrate reductase activity allowing anaerobic growth with nitrate as electron acceptor. A regulatory link was inferred between the presence of the membrane-bound nitrate reductase and expression of the periplasmic nitrate reductase. This is the first demonstration of full denitrification in an organism possessing only a periplasmic nitrate reductase.