Mechanism for calcium urolithiasis among patients with hyperuricosuria: supersaturation of urine with respect to monosodium urate.

Since monosodium urate (NaU) may play an important etiologic role in the formation of renal stones containing Ca in patients with hyperuricosuria, the current studies were undertaken to define some of the physiocochemical factors which determine the formation of NaU. In solutions containing Na, uric acid was rapidly transformed to NaU at pH greater than 6. The results indicated that NaU, and not uric acid, was the stable phase above this pH. A reliable and simple method for the calculation of the state of saturation of urine with respect to NaU was developed from the ratio of concentration products of Na and total dissolved urate (Upi) in the ambient fluid before and after incubation of urine with synthetic NaU. The concentration product ratio closely approximated the ratio of activity products of Na+ and acid urate ion. In contrast, the relative saturation ratio, or the ratio of activity product of original sample and the thermodynamic solubility product of NaU, often differed from the activity product ratio in the individual urine samples. With the concentration product rate, it was found in 45 urine samples that a critical determinant for the supersaturated state with respect to NaU was the high concentration of UT. At UT greater than 300 mg/liter, urine samples were invariably supersaturated with respect to NaU. These results suggest that the nidus of NaU could potentially form in the urine of patients with hyperuricosuria and Ca stones.

Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation.

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus. Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.

Renal oxalate excretion following oral oxalate loads in patients with ileal disease and with renal and absorptive hypercalciurias. Effect of calcium and magnesium.

Intestinal absorption of oxalate was assessed indirectly from the increase in renal oxalate excretion following the oral administration of 5 mmol of stable oxalate. When sodium oxalate alone was given without divalent cations to patients in the fasting state, the urinary oxalate increased promptly (within 2 hours). The increase was more prominent and sustained in those with ileal disease (ileal resection or jujunoileal bypass); thus, 35 per cent of the orally administered oxalate eventually appeared in the urine in the group with ileal disease, 8 per cent in the group with stones (renal and absorptive hypercalciurias) and 9 per cent in the control group. This hyperexcretion of oxalate could be largely, but not totally, ameliorated by the concurrent oral administration of divalent cations. Although urinary oxalate decreased significantly following the oral administration of calcium or magnesium, hyperoxaluria persisted in most patients. The results suggested that the hyperabsorption of oxalate in ileal disease cannot be accounted for solely by an increased absorbable oxalate pool associated with calcium-fatty acid complexation. Moreover, although urinary oxalate decreased, urinary calcium increased concurrently when either calcium or magnesium was given. Thus, there was no significant change or increase in the urinary state of saturation with respect to calcium oxalate.

Selective effects of thiazide on intestinal absorption of calcium and adsorptive and renal hypercalciurias.

The effect of long-term thiazide therapy on the intestinal Ca absorption was measured in 10 well-defined cases of absorptive hypercalciuria with intestinal hyperabsorption of Ca and 8 with renal hypercalciuria ("renal leak" of Ca), many of whom had hyperabsorption of Ca. In most cases of absorptive hypercalciuria, the intestinal hyperabsorption of Ca persisted during treatment, despite restoration of normal urinary Ca. In contrast, the intestinal Ca absorption decreased significantly during thiazide therapy in 7 of 8 patients with renal hypercalciuria commensurate with the "correction" of the renal leak of Ca and secondary hyperparathyroidism. The results support the hypothesis that the intestinal hyperabsorption of Ca in absorptive hypercalciuria may be primary, whereas that in renal hypercalciuria may be associated with the hyperparathyroid state.

Juxtarenal aortic aneurysm with hostile neck anatomy: midterm results of minilaparotomy versus f-EVAR.

AIM: The aim of this study was to compare the results of complex aneurysm (hostile neck anatomies) repair in high-risk patients with two minimally invasive techniques, fenestrated endografting (f-EVAR where EVAR stands for endovascular aneurysm repair) and minilaparotomy. METHODS: All high-risk patients (N.=50, group 1) with hostile neck abdominal aortic aneurysms (AAAs) operated in the vascular surgery department of the "Policlinico Universitario G. Martino" of Messina (Italy) during a 5-year period (January 2006-December 2010) were cross-matched with 50 similar patients (group 2) treated in the Vascular Surgery Department of the "Hopital Cardiologique" University of Lille (France) with similar anatomies, comorbidities and risk factors. The patients in group 1 underwent open minilaparotomy surgery, and the patients in group 2 were treated with f-EVAR. The aim of our study was to compare perioperative complications, survival and reintervention rates. RESULTS: Perioperative cardiac complications occurred in 5 patients (10%) in group 1, and 1 patient (2%) in group 2 (P<0.092). Renal impairment not requiring permanent hemodialysis was significantly higher in group 1 (14% vs. 2% P<0.027), as well as respiratory complications (32% vs. 2% P<0.0001). Five patients (10%) in group 1 underwent reintervention vs. 4 patients in group 2 (P<0.7268). There was no statistically significant difference for survival rates at 30 days (92% in group 1 and 96% in group 2; P=0.399); at six months (90% vs. 96%; P=0.239); at one year (90% vs. 96%; P=0.239); and at two years (84% vs. 94%; P=0.110). However, we observed statistically significant differences in survival rates at three years (74% vs. 94%; P<0.006); at four years (70% vs. 86%; P<0.005); and at five years (65% vs. 68%; P<0.003). CONCLUSION: Our results showed that both techniques are effective in the treatment of AAA with hostile neck in high-risk patients. Although operative mortality rate was not statistically different, f-EVAR showed better results in terms of early complications and late survival.

Structural complexity in isoprenoid glycerol dialkyl glycerol tetraether lipid cores of Sulfolobus and other archaea revealed by liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometry of membrane lipid cores from Sulfolobus species reveals isomeric forms of ring-containing isoprenoid glycerol dialkyl glycerol tetraether components not previously recognised via the use of NMR and liquid chromatography-mass spectrometry techniques. Equivalent isomerism was confirmed for the components in other hyperthermophilic genera and in sediments which contain the lipids of mesophilic archaea. The recognition of the isomeric structures in distinct archaeal clades suggests that profiles of tetraether lipids reported previously may have oversimplified the true lipid complexity in archaeal cultures and natural environments. Accordingly, the extent of variation in tetraether structures revealed by the work should direct more informative interpretations of lipid profiles in the future. Moreover, the results emphasise that tandem mass spectrometry provides a unique capability for assigning the structures of intact tetraether lipid cores for co-eluting species during chromatographic separation.

The sensibility and specificity of cerebral oximetry, measured by INVOS - 4100, in patients undergoing carotid endarterectomy compared with awake testing.

BACKGROUND: Selective shunting during carotid endarterectomy (CEA) is advocated to reduce shunt-related stroke. Cerebral monitoring is essential for temporary carotid shunting. Many techniques are available for cerebral monitoring; however, none is superior to monitoring the patient's neurological status (awake testing) while performing the procedure under local anesthesia (LA). Cerebral oximetry (CO) has previously been used to show the adequacy of cerebral circulation in patients undergoing CEA. This investigation was designed to compare the performance of the INVOS-4100 cerebral oximeter and the neurologic functions, by means of detecting cerebral ischemia induced by carotid cross-clamping, in patients undergoing CEA under LA, namely cervical plexus block. METHODS: Patients scheduled for CEA under LA were included. Patients converted to general anesthesia (GA) or other types of operations other than CEA were excluded from this study. We enrolled 100 consecutive patients from January 2009 to December 2010. Bilateral regional cerebrovascular oxygen saturation (rSO(2)) was monitored in all patients, in addition to the awake testing. Changes in rSO(2) following carotid artery clamping were recorded. A drop greater than 20% was considered as an indicator of cerebral ischemia that might predict the need for carotid shunting. Patients were only shunted based on the awake testing. RESULTS: Of the 100 patients undergoing CEA under LA, 9 showed a significant drop in rSO(2) (range: 22.6-32.8%, mean: 26.4%): only three of them required shunting, while the remaining 6 had no changes in consciousness after internal carotid artery (ICA) cross-clamping and it was not necessary to place a shunt (false positive). Compared to the preclamping values, a significant decrease in rSO(2) was found on the hemisphere of the operated side, while no significant change was observed contralaterally. Ninety-one patients had no significant changes of CO values: in 89 of them there was no consciousness deterioration, so we didn't place a shunt (true negative), but 2 patients showing a non-significant post-clamping decline in CO saturation (1.5% and 18.2%) required shunting based on the awake testing (2 false negative). In the current study, the median drop in rSO(2) was 19% (range: 1.5-26.4%) in the 5 patients that required shunting. This represents a sensitivity of 60% and a specificity of 25% for CO in comparison to the awake testing. CONCLUSION: The results of this study suggest that the usefulness of CO in predicting cerebral ischemia is modest. Cerebral monitoring with INVOS-4100 has a high negative predictive value, but the positive predictive value is low.

Coupling of flagellin gene transcription to flagellar assembly in Bacillus subtilis.

The regulation of flagellin gene expression in Bacillus subtilis was examined in vivo by means of a lacZ translational fusion to the flagellin structural gene (hag). We have tested the effects of two known mutations (flaA4 and flaA15) in the major flagellar operon and of three deletions. One deletion was in frame in the fliI cistron, one was out of frame in the fliK cistron, and the last spanned about 21 kb of the flaA operon. In all instances, the expression of the flagellin gene was defective. Flagellin gene expression was restored in the strain with the 21-kb deletion by overexpression of the sigD gene under control of the isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible spac promoter. These results indicate that transcription of the flagellin gene is dependent on the formation of the flagellar basal body but that such a requirement can be bypassed by overexpression of sigD. Lack of expression of hag was observed in the presence of flaD1, flaD2, and delta sin mutations as well.

Pitfalls in parathyroid evaluation in patients with calcium urolithiasis.

Primary hyperparathyroidism is a major cause of calcium urolithiasis and is easily recognised when it is classically manifested. However, subtle presentations of primary hyperparathyroidism may cause confusion with other causes of calcium stone disease or cause diagnostic difficulty. Several pitfalls of parathyroid evaluation and treatment are illustrated by four cases of calcium urolithiasis. Cases 1 and 2 represent ineffective or useless parathyroid surgery rendered for renal hypercalciuria and absorptive hypercalciuria, respectively. Cases 3 and 4 had mild or intermittent hypercalcaemia. The correct diagnosis of primary hyperparathyroidism was made in Case 3 by parathyroid venous sampling and bone densitometry. In Case 4, the thiazide provocative test was used to establish the diagnosis of primary hyperparathyroidism.

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

An exaggerated augmentation of renal calcium excretion after oral glucose ingestion in patients with renal hypercalciuria.

The calciuric response to an oral glucose load (100 g) was determined in 16 patients with calcium oxalate urolithiasis (seven with renal hypercalciuria and nine with absorptive hypercalciuria) and seven normal subjects. The rates of renal calcium excretion increased significantly after glucose ingestion in all three groups. The calciuric response in patients with absorptive hypercalciuria and intestinal hyperabsorption of calcium was indistinguishable from that of normal subjects. However, the calcium excretions were significantly higher during 1 hr preceding and 3 hr after glucose ingestion in patients with renal hypercalciuria (with presumed "renal leak" of calcium) than in normal subjects. The increment in the calcium excretion rate was also higher in patients with renal hyperacalciuria, particularly during the 2nd hour of glucose ingestion. The results provide a further support for the concept of different etiologies of renal and absorptive hypercalciurias.

Replacement of vegetative sigmaA by sporulation-specific sigmaF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis.

Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigmaF is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, sigmaF must compete with sigmaA, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for sigmaA and sigmaF, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for sigmaF was found to be about 25-fold lower than that for sigmaA. We used quantitative Western blotting to estimate the concentrations of E, sigmaA, and sigmaF in sporulating cells. The cellular concentrations of E and sigmaA were both about 7.5 microM, and neither changed significantly during the first 3 h of sporulation. The concentration of sigmaF was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of sigmaF was some twofold higher than that of sigmaA. This difference in concentration cannot adequately account for the replacement of sigmaA holoenzyme by sigmaF holoenzyme in the prespore, and it seems that some further mechanism-perhaps the synthesis or activation of an anti-sigmaA factor-must be responsible for this replacement.

Genotype, phenotype, and protein structure in a regulator of sporulation: effects of mutations in the spoIIAA gene of Bacillus subtilis.

SpoIIAA, a phosphorylatable protein, is essential to the regulation of sigmaF, the first sporulation-specific transcription factor of Bacillus subtilis. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding B. subtilis mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.

T-cell large granular lymphocytic leukemia following orthotopic liver transplantation.

Aggressive T-cell neoplasms are an infrequent complication of allogeneic organ and bone marrow transplantation. To date, chronic T-cell lymphoproliferative malignancies have not been described. The present case documents the occurrence of a T-cell large granular lymphocytic leukemia (T-LGL) in a patient following orthotopic liver transplantation. Genotype studies showed a clonal T-cell receptor beta-chain gene rearrangement. A unique feature was the detection of a specific chromosomal deletion at 1p32 involving the tal-1 gene, an abnormality previously described only in aggressive T-cell neoplasms.

Recombinant pyruvate kinase type I from Escherichia coli: overproduction and revised C-terminus of the polypeptide.

The gene encoding pyruvate kinase type I (PKI) of Escherichia coli was amplified by PCR, cloned and sequenced. The gene product was overexpressed in E. coli, using an inducible T7 RNA polymerase-based expression system. The transformed cells contained sixtyfold the enzyme activity of the reference cells and enabled the purification of 30 mg of highly active PKI from 1 liter of culture. The gene sequence was determined and found to be different from the one previously reported, i.e., the T nucleotide at position 1351 was missing. This resulted in a downstream shift of the stop codon, thus the deduced polypeptide was 470 amino acids long instead of 462. In addition the twelve C-terminal amino acids of the former sequence were changed.

Effect of phenytoin on bone and vitamin D metabolism.

Calcium and vitamin D metabolism were evaluated in 5 adult epileptic patients before and during treatment with phenytoin. Significant decreases occurred in serum concentrations of calcium, albumin, and 25-hydroxy-cholecalciferol. The decreases in serum calcium paralleled those in serum albumin. Significant increases occurred in serum alkaline phosphatase and 1 alpha, 25-dihydroxycholecalciferol, in urinary hydroxyproline, and in the fractional gastrointestinal absorption of calcium. Urinary cyclic adenosine monophosphate and serum parathyroid hormone did not change. The results suggest that the bone disease resulting from phenytoin therapy may be associated with a deficiency of 25-hydroxycholecalciferol and not of 1 alpha, 25-dihydroxycholecalciferol, and that reduced gastrointestinal absorption of calcium or changes in parathyroid function may not be necessary for the development of bone disease.