Tumor-induced osteomalacia: experience from a South American academic center.

The majority of tumor-induced osteomalacia cases have been reported in the Northern Hemisphere and Asia. In this first series of South American patients, we show that the clinical presentation and sensitivity of plasmatic fibroblast growth factor 23 and somatostatin analog-based imaging are similar to those described in other populations. INTRODUCTION: Describe the experience of clinical presentation, diagnostic study, and treatment of patients with tumor-induced osteomalacia (TIO) in a South American academic center in comparison to literature. METHODS: Analysis of the records of patients diagnosed with TIO. The clinical presentation, diagnostic studies, and treatment were analyzed. Fibroblast growth factor 23 (FGF23) was measured by ELISA. RESULTS: Six patients were diagnosed with TIO during the studied period. The patients' median age was 53 years (range 22-64). All patients presented with weakness and pain in the extremities. Four experienced fractures during their evolution. The median time to diagnosis was 4.5 years (1-20). Biochemical studies showed hypophosphatemia, median of 1.4 mg/dL (1.2-1.6), with low maximum rates of tubular reabsorption of phosphate adjusted for glomerular filtration rate. FGF23 was elevated in 4/6 patients and inappropriately normal in the other two. In three patients, the location of the tumor was clinically evident and confirmed with anatomical imaging. In the remaining patients, two tumors were located with 68Ga DOTATATE-PET/CT and one with OctreoScan. The causal tumors were located in the lower extremities in five patients and invading the frontal sinus in one patient. In all patients, tumors were successfully removed. Within 14 days, there was normalization of phosphate and FGF23 levels and resolution of clinical symptoms in all patients. In all cases, the histopathology was compatible with a phosphaturic mesenchymal tumor. CONCLUSIONS: The clinical presentation, delay time to diagnosis, FGF23 diagnostic sensitivity and histopathology in this first series of South American patients is similar to those described in other populations. The success of localization by somatostatin analog-based imaging, suggests this may the optimal imaging modality.

Overexpression of connexin 43 reduces melanoma proliferative and metastatic capacity.

BACKGROUND: Alterations in connexin 43 (Cx43) expression and/or gap junction (GJ)-mediated intercellular communication are implicated in cancer pathogenesis. Herein, we have investigated the role of Cx43 in melanoma cell proliferation and apoptosis sensitivity in vitro, as well as metastatic capability and tumour growth in vivo. METHODS: Connexin 43 expression levels, GJ coupling and proliferation rates were analysed in four different human melanoma cell lines. Furthermore, tumour growth and lung metastasis of high compared with low Cx43-expressing FMS cells were evaluated in vivo using a melanoma xenograft model. RESULTS: Specific inhibition of Cx43 channel activity accelerated melanoma cell proliferation, whereas overexpression of Cx43 increased GJ coupling and reduced cell growth. Moreover, Cx43 overexpression in FMS cells increased basal and tumour necrosis factor-α-induced apoptosis and resulted in decreased melanoma tumour growth and lower number and size of metastatic foci in vivo. CONCLUSIONS: Our findings reveal an important role for Cx43 in intrinsically controlling melanoma growth, death and metastasis, and emphasise the potential use of compounds that selectively enhance Cx43 expression on melanoma in the future chemotherapy and/or immunotherapy protocols.

Increased free fetal DNA levels in early pregnancy plasma of women who subsequently develop preeclampsia and intrauterine growth restriction.

OBJECTIVE: To determine if maternal plasma ffDNA is increased early in pregnancies which subsequently develop preeclampsia (PE) and intrauterine growth restriction (IUGR). METHODS: Blood was obtained at 11-14 weeks and plasma stored. Among those who delivered a male infant and had a birth weight under the tenth centile and/or PE, we divided them into those who delivered before 35 weeks (9) and those who delivered after this gestation (15). A third group with uncomplicated pregnancies was used as controls (24). Real time-polymerase chain reaction (RT-PCR) was carried out to detect the multi-copy Y chromosome associated DSY14 gene. RESULTS: There were no differences between the ffDNA levels in the group delivered after 35 weeks and the control group (2.23ge/mL-1.61ge/mL p = 0.39). However, the levels of ffDNA at 11-14 weeks were statistically, significantly higher in patients that delivered before 35 weeks (4.34ge/mL-1.61ge/mL p = 0.0018). A logistic regression analysis shows that for every unit (1ge/mL) in which ffDNA increases, the likelihood of having PE or a fetus growing under the tenth centile delivered before 35 weeks increases by 1.67 times (CI 1.13-2.47). CONCLUSION: The concentration of ffDNA is significantly higher even during early pregnancy, in patients who subsequently develop PE and/or IUGR and are delivered before 35 weeks.

ATP steal between cation pumps: a mechanism linking Na+ influx to the onset of necrotic Ca2+ overload.

We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.

p53 Protects renal inner medullary cells from hypertonic stress by restricting DNA replication.

We previously found that p53 upregulation by hypertonicity protected renal inner medullary collecting duct (mIMCD3) cells from apoptosis. The purpose of the present study was to investigate the mechanism by which p53 protects the cells. We now find that hypertonicity (NaCl added to a total of 500 mosmol) inhibits DNA replication and delays G(1)-S transition as concluded from analysis of cell cycle distributions and bromodeoxyuridine (BrDU) incorporation rates. Lowering of p53 with p53 antisense oligonucleotide attenuated such effects of hypertonicity, resulting in an increased number of apoptotic cells in S phase and cells with >4 N DNA. Results with synchronized cells are similar, showing that cells in the early S phase are more sensitive to hypertonicity. Immunocytochemistry revealed that p53 becomes phosphorylated on Ser(15) and translocates to the nucleus in S both in isotonic and hypertonic conditions. Caffeine (2 mM) greatly reduces the p53 level and Ser(15) phosphorylation, followed by a remarkable increase of DNA replication rate, by failure of hypertonicity to inhibit it, and by reduction of cell number during hypertonicity. Finally, inhibition of DNA replication by the DNA polymerase inhibitor aphidicolin significantly improves cell survival, confirming that keeping cells in G(1) and decreasing the rate of DNA replication is protective and that these actions of p53 most likely are what normally help protect cells against hypertonicity.

Direct toxicity of nonsteroidal antiinflammatory drugs for renal medullary cells.

Antipyretic analgesics, taken in large doses over a prolonged period, cause a specific form of kidney disease, characterized by papillary necrosis and interstitial scarring. Epidemiological evidence incriminated mixtures of drugs including aspirin (ASA), phenacetin, and caffeine. The mechanism of toxicity is unclear. We tested the effects of ASA, acetaminophen (APAF, the active metabolite of phenacetin), caffeine, and other related drugs individually and in combination on mouse inner medullary collecting duct cells (mIMCD3). The number of rapidly proliferating cells was reduced by approximately 50% by 0.5 mM ASA, salicylic acid, or APAF. The drugs had less effect on confluent cells, which proliferate slowly. Thus, the slow in vivo turnover of IMCD cells could explain why clinical toxicity requires very high doses of these drugs over a very long period. Caffeine greatly potentiated the effect of acetaminophen, pointing to a potential danger of the mixture. Cyclooxygenase (COX) inhibitors, indomethacin and NS-398, did not reduce cell number except at concentrations greatly in excess of those that inhibit COX. Therefore, COX inhibition alone is not toxic. APAF arrests most cells in late G(1) and S and produces a mixed form of cell death with both oncosis (swollen cells and nuclei) and apoptosis. APAF is known to inhibit the synthesis of DNA and cause chromosomal aberrations due to inhibition of ribonucleotide reductase. Such effects of APAF might account for renal medullary cell death in vivo and development of uroepithelial tumors from surviving cells that have chromosomal aberrations.

Reduced Na-K pump but increased Na-K-2Cl cotransporter in aorta of streptozotocin-induced diabetic rat.

The activities of Na-K-ATPase and Na-K-2Cl cotransporter (NKCC1) were studied in the aorta, heart, and skeletal muscle of streptozotocin (STZ)-induced diabetic rats and control rats. In the aortic rings of STZ rats, the Na-K-ATPase-dependent (86)Rb/K uptake was reduced to 60.0 +/- 5.5% of the control value (P < 0.01). However, Na-K-ATPase activity in soleus skeletal muscle fibers of STZ rats and paired control rats was similar, showing that the reduction of Na-K-ATPase activity in aortas of STZ rats is tissue specific. To functionally distinguish the contributions of ouabain-resistant (alpha(1)) and ouabain-sensitive (alpha(2) and alpha(3)) isoforms to the Na-K-ATPase activity in aortic rings, we used either a high (10(-3) M) or a low (10(-5) M) ouabain concentration during (86)Rb/K uptake. We found that the reduction in total Na-K-ATPase activity resulted from a dramatic decrement in ouabain-sensitive mediated (86)Rb/K uptake (26.0 +/- 3.9% of control, P < 0.01). Western blot analysis of membrane fractions from aortas of STZ rats demonstrated a significant reduction in protein levels of alpha(1)- and alpha(2)-catalytic isoforms (alpha(1) = 71.3 +/- 9.8% of control values, P < 0.05; alpha(2) = 44.5 +/- 11.3% of control, P < 0.01). In contrast, aortic rings from the STZ rats demonstrated an increase in NKCC1 activity (172.5 +/- 9.5%, P < 0.01); however, in heart tissue no difference in NKCC1 activity was seen between control and diabetic animals. Transport studies of endothelium-denuded or intact aortic rings demonstrated that the endothelium stimulates both Na-K-ATPase and Na-K-2Cl dependent (86)Rb/K uptake. The endothelium-dependent stimulation of Na-K-ATPase and Na-K-2Cl was not hampered by diabetes. We conclude that abnormal vascular vessel tone and function, reported in STZ-induced diabetic rats, may be related to ion transport abnormalities caused by changes in Na-K-ATPase and Na-K-2Cl activities.

Cell cycle delay and apoptosis in response to osmotic stress.

As part of the urinary concentrating mechanism, renal inner medulla cells may be exposed to extremely variable NaCl and urea concentrations that can reach very high levels. A number of studies, reviewed herein, aim to understand how such osmotic stress affects the cells and what protective mechanisms might exist. The majority of these studies are done on inner medullary epithelial cells that grow continuously in tissue culture (mIMCD3). Cells grown at 300 mosmol/kg survive increase to 500 mosmol/kg by adding NaCl or urea, but only after a growth arrest of approximately 24 h. At a higher osmolality (650-700 mosmol/kg) most cells die within hours by apoptosis. The cells both in vitro and in vivo adapt to high osmolality by a number of mechanisms, including accumulation of variety of organic osmolytes and induction of heat shock proteins. The cell cycle delay results from blocks at the G1 and G2/M checkpoints and slowing during S. After adding NaCl, but not urea, the amount and transcriptional activity of p53 (the tumor suppressor protein) increases. The p53 is phosphorylated on ser-15 and is transcriptionally active at 500 mosmol/kg (associated with cell survival), but not at 700 mosmol/kg (associated with apoptosis). Reduction of p53 expression by p53 antisense oligonucleotide increases sensitivity of renal cells in culture to hyperosmotic stress caused by NaCl. The possible mechanisms of the protection action of p53 against hypertonic stress are discussed.

Regulation of aquaporin-2 trafficking by vasopressin in the renal collecting duct. Roles of ryanodine-sensitive Ca2+ stores and calmodulin.

In the renal collecting duct, vasopressin increases osmotic water permeability (P(f)) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca(2+) mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nm) and 8-(4-chlorophenylthio)-cAMP (0.1 mm) elicited marked increases in [Ca(2+)](i) (fluo-4). Vasopressin-induced Ca(2+) mobilization was completely blocked by preloading with the Ca(2+) chelator BAPTA. In parallel experiments, BAPTA completely blocked the vasopressin-induced increase in P(f) without affecting adenosine 3',5'-cyclic monophosphate (cAMP) production. Previously, we demonstrated the lack of activation of the phosphoinositide-signaling pathway by vasopressin in IMCD, suggesting an inositol 1,4,5-trisphosphate-independent mechanism of Ca(2+) release. Evidence for expression of the type 1 ryanodine receptor (RyR1) in IMCD was obtained by immunofluorescence, immunoblotting, and reverse transcription-polymerase chain reaction. Ryanodine (100 microm), a ryanodine receptor antagonist, blocked the arginine vasopressin-mediated increase in P(f) and blocked vasopressin-stimulated redistribution of aquaporin-2 to the plasma membrane domain in primary cultures of IMCD cells, as assessed by immunofluorescence immunocytochemistry. Calmodulin inhibitors (W7 and trifluoperazine) blocked the P(f) response to vasopressin and the vasopressin-stimulated redistribution of aquaporin-2. The results suggest that Ca(2+) release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.

Role of 11beta-hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries.

The aim of the present study was to demonstrate rapid effects of aldosterone on the Na(+)-H(+) exchanger in strips of human vascular vessels and to determine whether 11beta-hydroxysteroid dehydrogenase enzyme (11beta-HSD) could play a protective role in this response, such as that described for the classic type I mineralocorticoid receptor (MR). The activity of 11beta-HSD isoforms 1 and 2 were measured in fetal and adult arteries. Both isoforms are present in adult and fetal vessels. However, a significant difference in the proportion of each isoform was found. Isoform 1 activity (in pmol x min(-1) x 100 mg(-1) protein) was 42+/-5 in fetal vessels and 29+/-2 in adult arteries, and isoform 2 activity was 78+/-7 in fetal and 12+/-2 in adult tissue. The nongenomic effect of aldosterone on Na(+)-H(+) exchanger activity was measured in strips of chorionic and radial uterine arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein. Recordings of intracellular pH (pH(i)) were made by videofluorescence microscopy. Aldosterone (0.5 nmol/L) rapidly increased pH(i), with a half-maximal effect between 2 and 3 nmol/L in both fetal and adult vessels. Ethylisopropylamiloride, a specific inhibitor of the Na(+)-H(+) exchanger, inhibited this effect. The hormone-mediated increase in pH(i) was unaffected by spironolactone, a classic antagonist of MR, but was completely blocked by RU28318. Cortisol (up to 1 micromol/L) had no effect on pH(i), but when applied in the presence of carbenoxolone, a dramatic increase in Na(+)-H(+) exchanger activity was evident. The increments on pH(i) for each cortisol concentration were similar to those observed for aldosterone. These findings suggest that vascular 11beta-HSD plays an active role in maintaining the specificity of the rapid effects of aldosterone.

Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation.

Acute hypertonicity causes cell cycle delay and apoptosis in mouse renal inner medullary collecting duct cells (mIMCD3) and increases GADD45 expression. Because the tumor suppressor protein p53 may be involved in these effects, we have investigated the role of p53 in mIMCD3 response to hyperosmotic stress. Acute elevation of osmolality with NaCl addition from the control level of 320 mosmol/kg to 500-600 mosmol/kg greatly increased the levels of total and Ser(15)-phosphorylated p53 within 15 min. However, similar elevation of osmolality with urea did not increase p53 levels. Our studies indicate that induced p53 is transcriptionally active because NaCl addition to 500-600 mosmol/kg stimulated transcription of a luciferase reporter containing a p53 consensus element and appropriately altered mRNA levels of known transcriptional targets of p53, i.e. increased MDM-2 and decreased BCL-2 levels. Elevating NaCl further to 700-800 mosmol/kg rapidly killed most of the cells by apoptosis. At these higher NaCl concentrations, p53 levels were further increased although Ser(15) phosphorylation and transcriptional activity were significantly lower than levels at 500-600 mosmol/kg. At NaCl-induced 500 mosmol/kg, apoptosis was rare in the presence of control, nonspecific oligonucleotide but highly prevalent upon addition of p53 antisense oligonucleotide that substantially reduced p53 levels. We conclude that induction of active p53 in mIMCD3 cells by hypertonic stress contributes to cell survival.

Effect of casein-derived peptides on D-xylose absorption assessed by H2 breath test in normal volunteers.

Studies have shown a promoting effect of food on small intestinal absorption. Casein hydrolysate seems be more effective in increasing of D-xylose absorption in dogs than the whole protein and lactulose. The purpose this study was to analyze the effect of groups of peptides derived from casein hydrolysate on the absorption of D-Xylose and intestinal transit time in normal subjects. Seven normal volunteers participated in the study. Three peptide fractions were isolated from casein enzymatic hydrolysate by means of a preparative HPLC silica column. On separate days subjects drank test solutions containing lactulose, D-xylose, and D-xylose with one of three peptide groups. The hydrogen breath test was used to indirectly estimate D-xylose absorption and orocecal transit time. Two peptide fractions when added to D-xylose were followed by an increased absorption characterized by decreased H2 production. A nonstatistically significant increase of orocecal transit time was observed with these peptides.

Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells.

We investigated the effects of hyperosmolality on survival and proliferation of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. High NaCl and/or urea (but not glycerol) reduces the number of viable cells, as measured with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550-1,000 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G(2)M phase of the cell cycle within 8 h, as measured by flow cytometry. Up to 600 mosmol/kg the effect is only transient, and by 12 h at 550 mosmol/kg the effect reverses and most cells are in G(1). Flow cytometry with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the S phase of the cell cycle slows, depending on the concentrations of NaCl and/or urea, and that the duration of G(2)M increases greatly (from 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Addition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more also induces apoptosis, as demonstrated by characteristic electron microscopic morphological changes, appearance of a subdiploid peak in flow cytometry, and caspase-3 activation. The number of cells with subdiploid DNA and activated caspase-3 peaks at 8-12 h. Caspase-3 activation occurs in all phases of the cell cycle, but to a disproportionate degree in G(0)/G(1) and S phases. We conclude that elevated NaCl and/or urea reduces the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G(2)M and G(1), and by inducing apoptotic cell death.

Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralytic shellfish poison (PSP), in cats.

Saxitoxin (STX) was the first known and most studied toxic component of paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na+ channel. Although the toxin's mechanism of action is well known at the molecular level, there are still many unresolved questions about its pharmacokinetics and the PSP intoxication syndrome in mammals. Some of these questions are addressed in the present paper, which describes an experimental design which allowed us to follow the dynamics of STX poisoning in vivo. Adult cats were anaesthetized and permanently coupled to artificial ventilation, they were then intravenously injected with Low (2.7 microg of STX/kg) and high doses (10 microg of STX/kg) of toxin. Cardiovascular parameters such as blood pressure and electrocardiograms were recorded, urine and blood samples were collected during the four hours of experimental time. In order to quantify mass amount of STX, we used the post-column derivatization HPLC method. Urine and blood samples were cleansed using a C-18 Sep-Pack cartridge and ultrafree microcentrifuge filters. At the end of each experiment, the animals were killed and tissue samples from brain, liver, spleen and medulla oblongata were extracted to measure the amount of STX. As compared to control period, Low doses of STX made no difference in hemodynamics parameters. In contrast, high doses drastically reduced blood pressure, produced myocardial failure and finally cardiac arrest. Administration of 2.5 microg/kg x min of dobutamine restored hemodynamics parameters and allowed the animal to overcome the shock. With high doses, the calculated STX renal clearance in cats is 0.81 ml/min x kg(-1). This valued corresponds to 20.25% of the reported inulin renal clearance. Nevertheless with Low doses the STX renal clearance is 3.99 ml/min x kg(-1). This data suggest that in cats with normal cardiovascular parameters and diuresis, the STX excretion mainly involves glomerular filtration. During experimental time, no PSP toxins other than STX was detected in the body fluids and tissue samples analyzed, indicating that the mammals can not metabolize this molecule. STX was found in intensely irrigated organs such as the liver and spleen but also in the central nervous system (brain and medulla oblongata), showing that STX was capable of crossing the blood brain barrier.

Adrenal-dependent modulation of the catalytic subunit isoforms of the Na+-K+-ATPase in aorta.

Na+-K+-ATPase gene expression and activity were studied in aortas from adrenalectomized (ADX) rats and ADX rats with deoxycorticosterone supplement (ADX-DOCA). Northern analysis of RNA from ADX rats revealed a significant decrease in alpha2-mRNA levels (38.5 +/- 8.3% of control, P < 0.01) that was prevented by DOCA (P < 0.05). A decrease to 55.8 +/- 7.7% in alpha2-isoform protein was observed 8 days after adrenal removal (P < 0.05); DOCA reversed this effect (90.8 +/- 10.5%). Adrenalectomy induced a decrease of 68.5 +/- 4.5% in beta1-mRNA (P < 0.01) and 52.7 +/- 8.3% in ADX-DOCA rats (P < 0.01). Also, a reduction in beta1-isoform protein that was not prevented by DOCA was detected after adrenalectomy (47.1 +/- 11%, P < 0.01). In contrast, no differences in alpha1-mRNA or -protein levels were observed. Vascular sodium pump activity was reduced to 59.8 +/- 4.6% of control values after adrenalectomy (P < 0.01); this reduction was reversed by DOCA. Our data indicate that corticosteroids regulate Na+-K+-ATPase isoform expression and activity in vascular tissue in vivo, suggesting a mineralocorticoid-dependent modulation of alpha2-Na+-K+-ATPase gene expression in aorta, with beta1-isoform expression dependent on the presence of glucocorticoids.

[Effect of casein derived peptides on D-xylose absorption and small intestinal motility in dogs]. / Efecto de fracciones pépticas derivadas de la caseína en la absorción de D-xilosa y motilidad intestinal en el perro. Estudio preliminar.

BACKGROUND: The presence of food in the intestinal lumen increases absorption from an isolated intestinal loop, the mechanisms involved are unknown. Casein, and its respective hydrolysate, increased D-xylose absorption in both normal volunteers and experimental animals; this effect was associated with prolonged small intestinal transit time and a decrease of motor activity. AIMS: To separate from casein hydrolysate, groups of peptides and to investigate their effects on both D-xylose absorption and small intestinal motility. MATERIAL AND METHODS: Studies were performed on five dogs with a surgically implanted duodenal cannula. Three groups of peptides were separated by means of a Silica Gel 60 column and were continuously infused through the duodenal cannula. After 15 min, 5 g of D-xylose were injected in the duodenum, plasma levels were measured, and the area under the curve was estimated. Motility was recorded by means of infused catheters and external transducers. RESULTS: Plasma levels of D-xylose were significantly increased during the infusion of one group of peptides compared to the others. In addition, the area under the curve: 3366 +/- 885 mg x min-1 observed with this group was significantly greater than the other two groups: 1432 +/- 183 mg x min-1 and 1137 +/- 280 mg x min-1 respectively. No statistically significant differences in motor activity were observed between the different groups of peptides. CONCLUSIONS: A group of peptides derived from casein was characterized by increasing D-xylose absorption. The presence of beta casomorphines might be the possible mechanism involved.

[Comparison of extra renal potassium management in hypertensive, diabetic and normal subjects]. / Estudio comparativo del manejo extrarrenal de potasio en sujetos controles, pacientes hipertensos y diabéticos.

BACKGROUND: Sodium and potassium ions are involved in the regulation of blood pressure and the genesis of hypertension. AIM: To assess internal potassium balance, as a measure of sodium pump activity, in subjects with essential hypertension and diabetic patients. PATIENTS AND METHODS: Eleven hypertensive subjects, 5 non-insulin-dependent diabetics and 16 age matched controls were studied. An acute oral load of 0.8 mEq/Kg body weight of KCl was administered and blood samples were drawn every 30 min thereafter, until 120 min, to measure plasma K+ levels. Urinary K+ excretion during this period was also measured. In eight hypertensive patients, the test was repeated after two week of supplementation with 60 mEq/day of KCl. The maximal increase in plasma potassium levels and the time required to achieve the maximum concentration was recorded. RESULTS: All patients had normal serum creatinine levels. Mean fasting blood glucose of diabetic patients was 133 +/- 15.1 mg/dl. No difference between patients and controls in maximal increase plasma potassium increase, was observed. In hypertensive patients the lapse to achieve the maximal potassium concentration was longer than in controls. After the period of potassium supplementation in hypertensive patients, there was a significant increase in basal plasma K+ levels and the temporal pattern of plasma potassium increase was similar to that of controls. Between 63 and 68% of retained K+ load was translocated to the intracellular space at 120 min in all study groups. CONCLUSIONS: Internal potassium balance is not significantly altered in subjects with essential hypertension or in non-insulin-dependent diabetics.

Biochemical evidence for adhesion-promoting role of major intrinsic protein isolated from both normal and cataractous human lenses.

In this study, we tested the adhesion-promoting role of major intrinsic protein from both normal human (cadaver) and senile cataractous lenses. Junctional membrane solubilized proteins and pure major intrinsic protein obtained from both type of lenses were reconstituted in neutral phosphatidylcholine liposomes. The interaction of these liposomes with phosphatidylserine vesicles was studied by resonance energy transfer. Our results show that normal human lens junction solubilized proteins and pure major intrinsic protein isolated from them promote adhesion. No quenching effect was observed when major intrinsic protein was omitted in the vesicle reconstitution, no other intrinsic protein of normal human junctional membrane provoked the adhesive effect. In contrast, major intrinsic protein isolated from human senile cataractous lens fails to induce adhesion. The proteolytic cleavages that in vitro originate major intrinsic protein 22,000 Da did not blunt its adhesive capability, suggesting that the proteolytic modifications that major intrinsic protein undergoes in senile cataract were not related with the incompetence of cataractous lens junctions to induce adhesion. Cataractous lens junctional membranes showed protein aggregates. These membranes were treated with sodium hydroxide and reconstituted into liposomes. The sodium hydroxide treatment removed the protein aggregates and restored the adhesive capability. Furthermore, the supernatant obtained after the sodium hydroxide treatment of cataractous junctional membranes, inhibited the adhesive effect of vesicles reconstituted with bovine solubilized proteins. These experiments prove that the failure to induce adhesion of human senile cataractous lens junction proteins is due to the interaction with protein aggregates, which can be removed by sodium hydroxide.

Lens major intrinsic protein (MIP) promotes adhesion when reconstituted into large unilamellar liposomes.

The vertebrate lens behaves like a syncytium, and it is formed mainly by cells called lens fibers. Between the fibers are extensive networks of membrane junctions. The major intrinsic protein (MIP) constitutes about 50-60% of the intrinsic membrane proteins found in lens fiber junctions. The role of MIP is unknown. Nevertheless, it has been proposed that it is the protein responsible for the adhesion between the plasmatic membranes of the lens fibers. The aim of our studies was to test the adhesion-promoting role of MIP. We reconstituted MIP into large unilamellar vesicles (LUV) of phosphatidylcholine (PC) and studied the vesicle aggregation between MIP-reconstituted LUV (PC-MIP) and phosphatidylserine (PS) vesicles. The aggregation process was monitored using methods based on resonance energy transfer (RET) and turbidity measurements. Neither RET nor an increase in turbidity occurred in any combination except in the presence of both MIP and PS. The liposomes thus aggregate through protein-lipid interactions. These results show that MIP promotes adhesion with negatively charged membranes, indicating that the adhesion is electrostatic in nature. Aggregation was fastest at pH 6.0. The aggregation effect was abolished with pronase treatment. Preincubation of PC-MIP vesicles with anti-MIP polyclonal serum also inhibited the aggregation. These studies are the first experimental evidence supporting the hypothesis of an adhesive role for MIP.