Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes.

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.

Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter.

Three different cell differentiation experimental model systems (human embryonic stem cells, mouse F9 cells, and human HL-60 promyelocytic cells) were used to determine the relationship between the reduction in telomerase activity after differentiation and the regulation of the promoter for the hTERT gene. Promoter constructs of three different lengths were subcloned into the PGL3-basic luciferase reporter vector. In all three experimental systems, all three promoter constructs drove high levels of reporter activity in the nondifferentiated state, with a marked and time-dependent reduction after the induction of differentiation. In all cases, the smallest core promoter construct (283 nt upstream of the ATG) gave the highest activity. Electrophoretic mobility shift assays revealed transcription factor binding to two E-box domains within the core promoter. There was also a marked time-dependent reduction in this binding with differentiation. In addition, a distinct and novel element was identified within the core promoter, which also underwent time-dependent reduction in transcription factor binding with differentiation. Site-directed mutagenesis of this novel element revealed a correlation between transcription factor binding and promoter activity. Taken together, the results indicate that regulation of overall telomerase activity with differentiation is mediated at least in part at the level of the TERT promoter and provides new information regarding details of the regulatory interactions that are involved in this process.

Human telomerase reverse transcriptase promoter regulation in normal and malignant human ovarian epithelial cells.

The telomerase RNA-protein complex responsible for maintenance of telomeric DNA at chromosome ends, is usually inactive in most primary somatic human cells, but is specifically activated with in vitro immortalization and during tumorigenesis. Although expression of the RNA component of telomerase appears to be constitutive, the expression pattern of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is correlated with measured enzyme activity. In particular, a >80% concordance has been reported between telomerase activity and hTERT mRNA expression in ovarian tumors. Accordingly, to learn more about the mechanism regulating hTERT gene expression in ovarian carcinoma, we have performed a detailed analysis of the 5'-flanking promoter region of the hTERT gene. We have reported previously the isolation and analysis of a 5.8-kb genomic fragment containing the human hTERT gene promoter (M. Tzukerman et al., Mol. Biol. Cell, 11: 4381-4391, 2000). Deletion analysis of this promoter was carried out using transient transfection of promoter-reporter constructs in four different telomerase-expressing, ovarian carcinoma-derived cell lines, the tumorigenic properties of which have been characterized, and was compared with telomerase-negative primary human fibroblasts and nontransformed ovarian epithelial cells. These assays have shown that the hTERT promoter is inactive in telomerase-negative cells and is active in telomerase-positive cell lines. A core promoter of 283 bp upstream of the transcription initiation site (TI) was found to be sufficient for maximum promoter activity, suggesting the presence of inhibitory elements within the larger promoter sequence. Gel shift analysis of the core promoter using nuclear extracts from the ovarian and control cell lines revealed specific transcription factor binding using extracts from telomerase-positive cells. Among the binding elements, we identified two E-boxes (CACGTG) as well as a novel element (MT-box), which we identified recently in a number of differentiation systems. Site-directed mutagenesis was used to introduce mutations into this novel transcription factor binding element. These mutations significantly affect the transcriptional activity of hTERT promoter in a cell type-specific manner and suggest that the transcription factors that bind to the E-box and the novel element cooperatively function as major determinants of hTERT expression and telomerase activity in ovarian cancer. Further comparison of promoter activity, telomerase activity, and telomere length among the different ovarian cancer cells indicated that a threshold level of telomerase activity is apparently sufficient to protect telomere integrity and permit the immortal state of the different ovarian cancer cell lines.

Promoter region of the murine fibroblast growth factor receptor 2 (bek/KGFR) gene.

Fibroblast growth factors (FGFs) and their receptors play an important role in cell growth, angiogenesis and embryonal development. Four distinct genes encoding fibroblast growth factor receptors (FGFRs) were identified: flg, encoding FGFR1, bek encoding FGFR2, and the genes for FGFR3 and FGFR4. Both FGFR2 and keratinocyte growth factor receptor (KGFR) are encoded by the same gene, bek. To study the regulation of expression of the FGF receptors we analysed the DNA sequence flanking the 5' region of the cDNA of murine FGFR2 to seek elements that control its transcription. A 5-kbp fragment containing the 5' end of the cDNA was isolated from mouse genomic library and used to map the promoter region. We found that the sequence encoding the 5' non-translated region of the FGFR2/KGFR cDNA contains an intron located 210 bp upstream from the translation start site. Using RNAase protection and primer extension, we identified the mRNA start 37 bp upstream from the beginning of the bek cDNA. The promoter activity was found to reside in a 1.3-kbp fragment upstream from the cDNA, and deletion mapping further localized the promoter to a 0.7-kbp fragment. The sequence of this region shows high G+C content (62%), which is particularly emphasized in the 200 bp upstream from the mRNA start (80% G+C). This region contains the CCGCCC, GGGCGG AND GGAGG motifs also found in promoters of other growth factor receptors. Neither TATA nor CAAT boxes were found near the RNA start site. The characterization of this promoter will allow studies of the regulation of expression of the FGFR2 during development and in pathophysiological states. The differences between the promoter sequence of the gene for FGFR2 (bek) and FGFR1 (flg) may explain their differential expression during development.

RAS is required for epidermal growth factor-stimulated arachidonic acid release in rat-1 fibroblasts.

Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.

Isolation of promoter for cytosolic phospholipase A2 (cPLA2).

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

Interindividual heterogeneity in the hypoxic regulation of VEGF: significance for the development of the coronary artery collateral circulation.

BACKGROUND: The coronary artery collateral circulation may be beneficial in protecting against myocardial ischemia and necrosis. However, there is a tremendous interindividual variability in the degree of new collateral formation in patients with coronary artery disease. The basis for this interindividual heterogeneity is not understood. In this study we test the hypothesis that failure to generate collateral vessels is associated with a failure to appropriately induce with hypoxia or ischemia the angiogenic factor, vascular endothelial growth factor (VEGF). METHODS AND RESULTS: We correlated the VEGF response to hypoxia in the monocytes harvested from patients with coronary artery disease with the presence of collaterals visualized during routine angiography. We found that there was a highly significant difference in the hypoxic induction of VEGF in patients with no collaterals compared with patients with some collaterals (mean fold induction 1.9+/-0.2 versus 3.2+/-0.3, P<0.0001). After subjecting the data to ANCOVA, using as covariates a number of factors that might influence the amount of collateral formation (ie, age, sex, diabetes, smoking, hypercholesterolemia), patients with no collaterals still have a significantly lower hypoxic induction of VEGF than patients with collaterals. CONCLUSIONS: This study provides evidence in support of the hypothesis that the ability to respond to progressive coronary artery stenosis is strongly associated with the ability to induce VEGF in response to hypoxia. The observed interindividual heterogeneity in this response may be due to environmental, epigenetic, or genetic causes. This interindividual heterogeneity may also help to explain the variable angiogenic responses seen in other conditions such as diabetic retinopathy and solid tumors.

Insulin production by human embryonic stem cells.

Type 1 diabetes generally results from autoimmune destruction of pancreatic islet beta-cells, with consequent absolute insulin deficiency and complete dependence on exogenous insulin treatment. The relative paucity of donations for pancreas or islet allograft transplantation has prompted the search for alternative sources for beta-cell replacement therapy. In the current study, we used pluripotent undifferentiated human embryonic stem (hES) cells as a model system for lineage-specific differentiation. Using hES cells in both adherent and suspension culture conditions, we observed spontaneous in vitro differentiation that included the generation of cells with characteristics of insulin-producing beta-cells. Immunohistochemical staining for insulin was observed in a surprisingly high percentage of cells. Secretion of insulin into the medium was observed in a differentiation-dependent manner and was associated with the appearance of other beta-cell markers. These findings validate the hES cell model system as a potential basis for enrichment of human beta-cells or their precursors, as a possible future source for cell replacement therapy in diabetes.

Expression of the multiple drug resistance gene in human renal cell carcinoma depends on tumor histology, grade, and stage.

Levels of mRNA expressed by the multidrug resistance gene MDR1 were examined in 23 renal cell carcinoma samples and adjacent normal kidney cortex using reverse-transcription PCR. Comparison of MDR1 levels between histological types revealed that there was on average significantly more MDR1 in clear cell tumors than in oncocytomas (0. 89 +/- 0.10 versus 0.28 +/- 0.20, ratio of MDR1 in tumor cells to the drug-resistant cell line KB-8, P < 0.05). The mean MDR1 level of all of the non-oncocytoma tumors was not significantly different from the mean MDR1 level of normal adjacent kidney (0.89 +/- 0.10 versus 1.11 +/- 0.12, P = 0.07). However, the mean MDR1 level of the more undifferentiated clear cell tumors was significantly lower than the mean MDR1 level of adjacent normal kidney (0.74 +/- 0.10 versus 1.11 +/- 0.12, P < 0.05). MDR1 levels in early stage, clear cell tumors (n = 14) were lower than in tumors that had spread into perinephric tissue or had metastasized (n = 6) (0.77 +/- 0.08 versus 1.24 +/- 0.30, P < 0.05). In conclusion, MDR1 expression decreases in the more undifferentiated tumors, but still remains at levels high enough to be drug resistant. Higher MDR1 expression in the invasive tumors compared with noninvasive tumors suggests that MDR1 expression and invasiveness may be linked.

Hypoxic induction of vascular endothelial growth factor is markedly decreased in diabetic individuals who do not develop retinopathy.

OBJECTIVE: A small percentage of patients do not develop any evidence of diabetic retinopathy (DR) even after many years of diabetes. Vascular endothelial growth factor (VEGF) has been implicated in the development of DR. Therefore, we sought to determine if the regulation of VEGF differs in those patients who develop DR after many years of diabetes compared with those who do not develop DR. RESEARCH DESIGN AND METHODS: We performed standard 7-field stereoscopic fundus photography on 95 consecutive patients with type 1 and type 2 diabetes. Patients were categorized into 3 groups according to the presence or absence of DR and the duration of diabetes: group 1 was defined by presence of DR, group 2 was defined by absence of DR after >10 years duration of diabetes, and group 3 was defined by absence of DR with long-standing diabetes (> or =20 years for type 1 diabetes and > or =15 years for type 2 diabetes). Monocytes from 40 ml peripheral blood were isolated from all patients, and the hypoxic induction of VEGF was determined in vitro. RESULTS: We found no significant difference in the basal level of VEGF in patients with and without DR. However, we did find a markedly significant difference in the hypoxic induction of VEGF between patients from group 1 and group 3 (4.35+/-0.55 vs. 1.87+/-0.3, P<0.00013). CONCLUSIONS: This study suggests that 1 mechanism for the absence of DR in patients with long-standing diabetes is a decreased hypoxic induction of VEGF.

Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

Insulin blunts the natriuretic action of atrial natriuretic peptide in hypertension.

Hyperinsulinemia and insulin resistance are implicated in the etiology of hypertension, but the mechanisms involved have not been established. The objectives of this study were to determine whether untreated essential hypertensive patients are more sensitive to the antinatriuretic action of insulin and more resistant to the counteracting natriuretic effect of atrial natriuretic peptide in contrast to age- and sex-matched normotensive control subjects. Urinary sodium excretion was measured at baseline, during hyperinsulinemic euglycemic clamp, and during coadministration of insulin and atrial natriuretic peptide. Baseline urinary sodium excretion was not significantly different in the normotensive subjects (415 +/- 47 mumol/min, n = 12) and hypertensive patients (381 +/- 18 mumol/min, n = 10); with the institution of insulin infusion, there was a similar and significant decline from baseline (P < .001) to 289 +/- 35 mumol/min in normotensive subjects and 235 +/- 17 mumol/min in hypertensive patients. Atrial natriuretic peptide was able to oppose the antinatriuretic action of insulin in normotensive subjects, increasing urinary sodium excretion significantly to a mean level of 352 +/- 31 mumol/min (P < .05), which did not differ significantly from baseline. In the hypertensive group, atrial natriuretic peptide infusion had no effect on urinary sodium excretion (238 +/- 18 mumol/min), and the difference from baseline remained highly significant (P < .001). The hypertensive patients were significantly less insulin sensitive than their normotensive counterparts, as reflected by a lower glucose utilization rate and higher mean baseline plasma insulin level (P < .05 for each).(ABSTRACT TRUNCATED AT 250 WORDS)

Body fluid homeostasis in congestive heart failure and cirrhosis with ascites.

The urinary excretion of salt and water in man is regulated by a variety of renal and extrarenal mechanisms that respond to changes in dietary sodium intake as well as to alterations in the holding capacity of the vascular and interstitial compartments. Changes in extracellular fluid volume are detected by volume sensors located in the intrathoracic vascular bed, the kidney and other organs. These sensing mechanisms gauge the adequacy of intravascular volume relative to capacitance at various sites within the circulation. Congestive heart failure and cirrhosis with ascites are two disease states of man in which a hemodynamic disturbance within a given circulatory subcompartment is perceived by these sensing mechanisms and results in renal sodium retention. While the primary disturbance in both of these conditions originates outside the kidney, a variety of renal effector mechanisms respond to the perceived circulatory disturbance and result in enhanced tubule reabsorption of salt and water. These effector mechanisms involve physical adjustments in renal microvascular hemodynamics, tubule fluid composition and flow rate and transtubular ion gradients. These in turn are partially regulated by a variety of neural and humoral pathways including the renin-angiotensin-aldosterone axis, prostaglandins, and kinins.

Body fluid homeostasis in man. A contemporary overview.

In the steady state, urinary excretion of sodium is closely matched to dietary salt intake. Given rigorous defense of extracellular fluid osmolality, it is the quantity of sodium in the extracellular fluid that determines the volume of this compartment. Changes in extracellular fluid volume are detected by volume sensors located in the intrathoracic vascular bed, kidney and other organs. These mechanoreceptors gauge the adequacy of intravascular volume, relative to capacitance, at various sites within the circulation. The perception of a change in the normal relationship between intravascular volume and circulatory capacity evokes a host of renal effector mechanisms that lead ultimately to physiologically appropriate changes in urinary sodium excretion. These effector mechanisms involve physical adjustments in the glomerular filtration rate, renal microvascular hemodynamics and peritubular capillary Starling forces, tubule fluid composition, flow rate and transtubular ion gradients. Neural and humoral pathways are also involved and, among the latter, angiotensin II, aldosterone, prostaglandins and kinins have been studied extensively. The continuous interaction between these sensor and effector mechanisms serves to ensure near-constancy of the extracellular fluid volume, a condition essential for optimal circulatory performance.

Muscle sympathetic nerve activity and renal responsiveness to atrial natriuretic factor during the development of hepatic ascites.

PURPOSE: Sodium retention in cirrhosis has been attributed to an imbalance between vasoconstrictive antinatriuretic forces such as the sympathetic nervous system and vasodilatory natriuretic agents such as atrial natriuretic factor (ANF). With the development of refractory ascites, cirrhotic patients become unresponsive to the natriuretic effect of ANF. Animal data suggest that the sympathetic nervous system plays a key role in mediating the refractoriness to ANF. We therefore studied the relationship between sympathetic nerve activity (SNA) and the natriuretic response to ANF in normal subjects and cirrhotic patients. We also attempted to localize the intrarenal site of refractoriness to ANF by lithium clearance. PATIENTS AND METHODS: Twenty-six patients with biopsy-proven cirrhosis and seven age- and sex-matched normal volunteers were studied after a week of 20 mmol/day sodium intake and no diuretics. Muscle SNA was recorded from the peroneal nerve (microneurography) and correlated with responsiveness to a 2-hour ANF infusion. Lithium clearance was used as a marker of sodium reabsorption proximal to the intramedullary collecting duct, the main site of ANF action. Plasma norepinephrine, renin, and aldosterone levels were also determined. Patients were categorized into three groups: nine patients free of ascites (by ultrasonography), five ascitic patients who responded to a 2-hour ANF infusion (i.e., had a natriuretic response to ANF above 0.83 mmol/hour), and 12 ascitic patients who did not respond. RESULTS: Muscle SNA was greatly increased in the ascitic nonresponder patients compared with the normal subjects (64 +/- 4 versus 27 +/- 7 bursts/minute, p less than 0.001), moderately increased in ascitic responders (47 +/- 6 bursts/minute, p less than 0.05), but not significantly increased in nonascitic patients with cirrhosis (34 +/- 5 bursts/minute). SNA was positively correlated with plasma norepinephrine levels (r = 0.69; p less than 0.005) and inversely correlated with peak sodium excretion during the ANF infusion (r = -0.63; p less than 0.001). Plasma renin activity and aldosterone were markedly elevated in ascitic nonresponders, and normal in ascitic responders and nonascitic patients. Lithium clearance was reduced in ascitic patients compared with nonascitic patients, did not change after the ANF infusion, and correlated inversely with SNA (r = -0.61; p less than 0.01). CONCLUSION: These results support the concept that the sympathetic nervous system is a factor in renal sodium handling in cirrhosis, especially in the initiation of sodium retention and the development of refractory ascites. Refractoriness to ANF might be explained, at least in part, by increased neurally mediated sodium reabsorption proximal to the intramedullary collecting duct, the main site of ANF action.

Role of atrial natriuretic peptide in the natriuretic response to central volume expansion induced by head-out water immersion in sodium-retaining cirrhotic subjects.

PURPOSE: It is possible that abnormalities in atrial natriuretic peptide may be involved in the pathogenesis of sodium retention in edema states. We performed a study in a group of 12 sodium-retaining cirrhotic subjects to determine the role of this peptide in mediating differences in the natriuretic response to central volume expansion induced by head-out water immersion. PATIENTS AND METHODS: Each patient was maintained for seven days on a 20-mmol sodium intake, and then studied on both control and immersion days. On each day, measurements of the following were obtained: plasma atrial natriuretic peptide, hematocrit, electrolytes, creatinine, plasma renin activity, serum aldosterone, urinary cyclic guanosine monophosphate (cGMP), blood pressure, and pulse rate. RESULTS: In six subjects, immersion resulted in a marked natriuresis sufficient to induce negative sodium balance by the third hour, and these subjects were termed "responders." In these six patients, baseline pre-immersion levels of plasma renin activity and serum aldosterone were all below 3 ng/liter/second and 4 nmol/liter, respectively. In the other six subjects, the natriuretic response to immersion was markedly blunted and insufficient to induce negative sodium balance, and these subjects were termed "non-responders." In these subjects, baseline pre-immersion levels of plasma renin activity and aldosterone were all above 3.5 ng/liter/second and 5 nmol/liter, respectively, and were significantly elevated compared with the responders, and compared with the normal range for control subjects consuming the same sodium intake. In both groups of cirrhotic subjects, baseline levels of plasma atrial natriuretic peptide and cGMP excretion were significantly and comparably elevated compared with the normal range for control subjects ingesting the same sodium intake. Despite the marked difference in the natriuretic response to immersion in both responders and non-responders, there was a significant and comparable further elevation of plasma atrial natriuretic peptide and urinary cGMP excretion during immersion, compared with the control day. CONCLUSION: These results suggest that the relative resistance to the natriuretic action of atrial natriuretic peptide in the non-responders compared with the responders is mediated by anti-natriuretic factors acting at a level parallel with or beyond atrial natriuretic peptide release or coupling to its cGMP-linked receptors.

Acute effects of peritoneovenous shunting on plasma atrial natriuretic peptide in cirrhotic patients with massive refractory ascites.

Plasma immunoreactive alpha-human atrial natriuretic peptide (ANP) was measured in six cirrhotic patients with massive refractory ascites, under strict metabolic conditions, while they were receiving a 20-meq sodium diet, both before and at two-hour intervals for eight hours following peritoneovenous shunting (PVS). The mean preoperative level of ANP was 75 +/- 18 pg/ml, which was found to be significantly higher than the normal range for this laboratory (8 to 24 pg/ml) (p less than 0.05). This value was also significantly higher than the value of 21 +/- 5 pg/ml (p less than 0.05) obtained in six patients with cirrhosis but without ascites. Following shunt insertion, an immediate natriuresis and diuresis were observed in five of the six cirrhotic patients with refractory ascites. In these five, right atrial pressure and ANP rose immediately, followed by a rise in the level of urinary cyclic guanosine monophosphate. The sixth subject had a delayed rise in right atrial pressure, and correspondingly the rise in ANP, the diuresis, and natriuresis were delayed. The changes in ANP following PVS were positively correlated with changes in right atrial pressure (p less than 0.05), urinary cyclic guanosine monophosphate (p less than 0.05), urinary sodium excretion (p less than 0.05), and urine volume (p less than 0.01). These results suggest that ANP may be important in mediating the acute response to PVS.

The effects of cyclosporine and cyclosporine metabolites in experimental small intestinal transplantation.

Cyclosporine metabolites (CM) were compared with cyclosporine for their in vitro and in vivo immunosuppressive, nephrotoxic, and hepatotoxic effects using (A) in vitro mixed lymphocyte induction of monocyte/macrophage procoagulant activity (PCA), an accurate marker of allograft rejection; (B) in vitro toxic effects on renal cells in culture; and (C) a unidirectional rejection model of rat small intestinal transplantation (SIT). CM were composed of OL1, OL17, OL18, and two additional peaks C and H, (peak C: mass = 1235, 15.3% of total CM, peak H: mass = 1205, 6.3% of total CM). In vitro, CM fully suppressed the one-way mixed lymphocyte culture-induced PCA from 52.5 +/- 8.2 mU/10(6) PBM to the basal level 22.3 +/- 6.6 mU/10(6) PBM (P less than 0.01), which was comparable to CsA (21.3 +/- 5.5 mU/10(6) PBM). Lewis rats that had received Lewis-Brown Norway F1 hybrid intestinal allografts when treated with CM, demonstrated near-normal histology with minimal signs of rejection as compared with the fulminant clinical and histological rejection observed in the control (untreated and Cremaphor/NaCl treated) animals. PCA was markedly elevated in the control animals, 278 +/- 172 (untreated) and 160 +/- 98 mU/10(6) PBM (Cremaphor/normal saline treated), whereas CsA-treated allogeneic transplants expressed only basal levels of PCA (14.0 +/- 4 mU/10(6) PBM) (P less than 0.01), associated with normal histology. CM-treated animals expressed PCA levels of 27.0 +/- 10 mU/10(6) PBM, which was significantly different from both control and CsA-treated animals (P less than 0.01). In contrast to CsA-treated animals, CM-treated allogeneic transplants demonstrated no apparent renal or hepatic toxicity, as measured by blood urea nitrogen (25.3 +/- 9.5 vs. 10.0 +/- 5.3 mg/dl), alkaline phosphatase (160.7 +/- 29.3 vs. 100.3 +/- 19.5 U/L), and aspartate transaminase (96.7 +/- 23.7 vs. 61.7 +/- 11.7 U/L) (P less than 0.01). Similarly, in contrast to CsA, CM had minimal or no toxicity in renal epithelial and mesangial cells in culture, as measured by minimal or no inhibition of DNA, RNA, and protein synthesis. These results suggest that CM have potent immunosuppressive properties with no apparent nephrotoxicity and hepatotoxicity in vitro and in vivo.

Genetic variant near cytosolic phospholipase A2 associated with schizophrenia.

Two studies were undertaken to determine a possible genetic basis for alterations in phospholipid metabolism in schizophrenia. Initial results demonstrated an association in 65 schizophrenics compared with a matched normal control population. A follow-up haplotype relative risk study of 44 triads (mother, father, affected offspring), confirmed the results seen in the association study. Results suggest that a genetic variant near the promotor region of the gene for cytosolic phospholipase A2, the rate-limiting enzyme in the synthesis of prostaglandins from arachindonic acid, is associated with schizophrenia.