Capillary electrophoretic assay for nitrate levels in the vitreous of proliferative diabetic retinopathy

The determination of nitric oxide (NO) in human vitreous samples is complicated by the relatively short half-life of the analyte and the viscous, high salt and protein biological matrix. In this work, we developed a fast (<5min) and useful CE method to determine the stable metabolite, nitrate, from vitreous samples. This proposed method has been successfully applied to determine the nitrate levels from the vitreous humor of patients undergoing vitrectomy for a variety of conditions. A statistically significant increase (P=0.000001) of the mean level of nitrate was observed in vitreous humor of patients with proliferative diabetic retinopathy (41.17+/-4.09microM, n=27) versus controls (15.22+/-0.86microM, n=35). The elevated levels of nitrate in the vitreous of patients known to have diabetic retinopathy suggests that NO is involved with the pathology of this disease.     

Chemokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy

PURPOSE: To determine levels of the chemokines CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL7/MCP-3, CCL8/MCP-2, CXCL5/ENA-78, CXCL6/GCP-2, CXCL10/IP-10, and CXCL11/I-TAC in the vitreous humor and serum, from patients with proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and rhegmatogenous retinal detachment with no PVR (RD), and to investigate the expression of MCP-1, CXCL12/SDF-1, and the chemokine receptor CXCR3 in epiretinal membranes. METHODS: Paired vitreous humor and serum samples were obtained from patients undergoing vitrectomy for the treatment of RD (57 specimens), PVR (32 specimens), and PDR (88 specimens). The levels of chemokines were measured by enzyme-linked immunosorbent assays. Eighteen PDR and 5 PVR membranes were studied by immunohistochemical techniques. RESULTS: Of all the chemokines studied, only MCP-1 and IP-10 were detected in vitreous humor samples. MCP-1 levels in vitreous humor samples were significantly higher than in serum samples (p < 0.001). MCP-1 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0002). MCP-1 levels in vitreous humor samples from patients with active PDR were significantly higher than in inactive PDR cases (p = 0.0224). IP-10 levels in vitreous humor samples were significantly higher than in serum samples (p = 0.0035). IP-10 levels were significantly higher in vitreous humor samples from patients with PVR and PDR compared with RD (p = 0.0083). The incidence of IP-10 detection in vitreous humor samples was significantly higher in active PDR cases compared with inactive cases (p = 0.0214). There was a significant association between the incidence of IP-10 detection and increased levels of MCP-1 in vitreous humor samples from all patients, and patients with RD and PDR (p < 0.001 for all comparisons). MCP-1, and SDF-1 were localized in myofibroblasts in PVR and PDR membranes and in vascular endothelial cells in PDR membranes. CXCR3 was expressed by vascular endothelial cells in PDR membranes. CONCLUSION: MCP-1, IP-10 and SDF-1 may participate in pathogenesis of PVR and PDR. Myofibroblasts and vascular endothelial cells are the major cell types expressing MCP-1, SDF-1, and CXCR3 in epiretinal membranes.     

Determination of ofloxacin and moxifloxacin and their penetration in human aqueous and vitreous humor by using high-performance liquid chromatography fluorescence detection

Information on comparing the penetration of ofloxacin and moxifloxacin in the human eye is unavailable, although these two antibiotics are commonly used in ophthalmic surgery. There is a need for a rapid, reliable, and sensitive methodology for their determination in ocular fluids. We developed a robust HPLC procedure with fluorescence detection for simultaneous analysis of ofloxacin and moxifloxacin in human and rabbit aqueous and vitreous samples. The linearity of the method ranged from 10 ng/ml to 100 microg/ml with r(2) > 0.996. Most inter- and intrabatch imprecision was about 5% (range 1.6-7.6%), recoveries between 95 and 104%, and accuracies between 93 and 104% at 0.1 and 1 microg/ml. The detection limits of both compounds were 10 ng/ml (0.028 nmol/ml for ofloxacin and 0.023 nmol/ml for moxifloxacin). No sample treatment was necessary for aqueous humor and only acetonitrile precipitation was required for vitreous humor. The chromatographic time was short, 22 min. We applied this method to study penetrations of ofloxacin and moxifloxacin in aqueous and vitreous humors of human and rabbits. There was no significant difference of penetration between the two antibiotics into aqueous and vitreous but ofloxacin was found at significantly higher concentrations in aqueous than in vitreous. We also detected contralateral transfer of the antibiotics in rabbit eyes.     

Nitrophenylphosphate as a tool to characterize gill Na, K-ATPase activity in hyperregulating Crustacea

The kinetic properties of a gill Na(+), K(+)-ATPase from the freshwater shrimp Macrobrachium olfersii were studied using p-nitrophenylphosphate (PNPP) as a substrate. Sucrose gradient centrifugation of the microsomal fraction revealed a single protein fraction that hydrolyzed PNPP. The Na(+), K(+)-ATPase hydrolyzed PNPP (K(+)-phosphatase activity) obeying Michaelis-Menten kinetics with K(M)=1.72+/-0.06 mmol l(-1) and V(max)=259.1+/-11.6 U mg(-1). ATP was a competitive inhibitor of K(+)-phosphatase activity with a K(i)=50.1+/-2.5 micromol l(-1). A cooperative effect for the stimulation of the enzyme by potassium (K(0.5)=3.62+/-0.18 mmol l(-1); n(H)=1.5) and magnesium ions (K(0.5)=0.61+/-0.02 mmol l(-1), n(H)=1.3) was found. Sodium ions had no effect on K(+)-phosphatase activity up to 1.0 mmol l(-1), but above 80 mmol l(-1) inhibited the original activity by approximately 75%. In the range of 0-10 mmol l(-1), sodium ions did not affect stimulation of the K(+)-phosphatase activity by potassium ions. Ouabain (K(i)=762.4+/-26.7 micromol l(-1)) and orthovanadate (K(i)=0.25+/-0.01 micromol l(-1)) completely inhibited the K(+)-phosphatase activity, while thapsigargin, oligomycin, sodium azide and bafilomycin were without effect. These data demonstrate that the activity measured corresponds to that of the K(+)-phosphatase activity of the Na(+), K(+)-ATPase alone and suggest that the use of PNPP as a substrate to characterize K(+)-phosphatase activity may be a useful technique in comparative osmoregulatory studies of Na(+), K(+)-ATPase activities in crustacean gill tissues, and for consistent comparisons with well known mechanistic properties of the vertebrate enzyme.     

Synergic effect of salinity and zinc stress on growth and photosynthetic responses of the cordgrass, Spartina densiflora

Spartina densiflora is a C(4) halophytic species that has proved to have a high invasive potential which derives from its physiological plasticity to environmental factors, such as salinity. It is found in coastal marshes of south-west Spain, growing over sediments with between 1 mmol l(-1) and 70 mmol l(-1) zinc. A glasshouse experiment was designed to investigate the synergic effect of zinc from 0 mmol l(-1) to 60 mmol l(-1) at 0, 1, and 3% NaCl on the growth and the photosynthetic apparatus of S. densiflora by measuring chlorophyll fluorescence parameters and gas exchange, and its recovery after removing zinc. Antioxidant enzyme activities and total zinc, sodium, calcium, iron, magnesium, manganese, phosphorus, potassium, and nitrogen concentrations were also determined. Spartina densiflora showed the highest growth at 1 mmol l(-1) zinc and 1% NaCl after 90 d of treatment; this enhanced growth was supported by the measurements of net photosynthetic rate (A). Furthermore, there was a stimulatory effect of salinity on accumulation of zinc in tillers of this species. Zinc concentrations >1 mmol l(-1) reduced growth of S. densiflora, regardless of salinity treatments. This declining growth may be attributed to a decrease in A caused by diffusional limitation of photosynthesis, owing to the modification of the potassium/calcium ratio. Also, zinc and salinity had a marked overall effect on the photochemical (photosystem II) apparatus, partially mediated by the accumulation of H(2)O(2) and subsequent oxidative damage. However, salinity favoured the recovery of the photosynthetic apparatus to the toxic action of zinc, and enhanced the nutrient uptake.     

Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits

Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.     

Effect of amiloride on cell volume regulation in renal straight proximal tubules

Amiloride has been shown to impair cell volume regulatory decrease in amphiuma red cells. The present study has been performed to test for the influence of amiloride on volume regulatory decrease and electrical properties in isolated perfused mouse straight proximal tubules. Replacement of 40 mmol/l NaCl with 80 mmol/l mannitol in bath perfusate does not appreciably affect the cell volume or the potential difference across the basolateral cell membrane. Reduction of osmolarity by omission of mannitol leads to cell swelling by 16.7 +/- 0.7% (n = 7), followed by volume regulatory decrease to 107.2 +/- 1.2% (n = 7) of original cell volume within 2 min. 1 mmol/l amiloride (but not 0.1 mmol/l amiloride) in the bath depolarizes the basolateral cell membrane from -63 +/- 1 mV (n = 24) by +16 +/- 1 mV (n = 16), decreases the apparent potassium transference number from 0.69 +/- 0.02 (n = 5) to 0.36 +/- 0.05 (n = 5), and significantly impairs volume regulatory decrease without appreciably modifying cell volume in isotonic solutions. 1 mmol/l amiloride in the luminal perfusate leads to a slight hyperpolarization of the basolateral cell membrane but does not interfere with volume regulatory decrease. Reduction of bath osmolarity depolarizes the basolateral cell membrane within 30 s by +7.8 +/- 0.8 mV (n = 18) in the absence and by +18 +/- 2 mV (n = 8) in the presence of amiloride. In the presence of reduced bath osmolarity and amiloride the potassium transference number amounts to 0.36 +/- 0.04 (n = 8). The hyperpolarization following luminal application of amiloride is most likely due to inhibition of luminal sodium channels, whereas bath amiloride depolarizes the basolateral cell membrane by reduction of basolateral potassium selectivity. As in amphiuma red cells amiloride impairs volume regulatory decrease in proximal straight renal tubules.     

Ion channels in human endothelial cells.

Ion channels were studied in human endothelial cells from umbilical cord by the patch clamp technique in the cell attached mode. Four different types of ion channels were recorded: i) potassium channel current that rectifies at positive potentials in symmetrical potassium solutions (inward rectifier); ii) low-conductance non-selective cation channel with a permeability ratio K:Na:Ca = 1:0.9:0.2; iii) high-conductance cation-selective channel that is about 100 times more permeable for calcium than for sodium or potassium; iv) high-conductance potassium channel with a permeability ratio K:Na = 1:0.05. The extrapolated reversal potential of the inwardly rectifying current was near to the potassium equilibrium potential. The slope conductance decreased from 27 pS in isotonic KCl solution to 7 pS with 5.4 mmol/l KCl and 140 mmol/l NaCl in the pipette but 140 mmol/l KCl in the bath. The low-conductance non-selective cation channel showed a single-channel conductance of 26 pS with 140 mmol/l Na outside, 28 pS with 140 mmol/l K outside, and rectified in inward direction in the presence of Ca (60 mmol/l Ca, 70 mmol/l Na, 2.7 mmol/l K in the pipette) at negative potentials. The current could be observed with either chloride or aspartate as anion. The high-conductance non-selective channel did not discriminate between Na and K. The single-channel conductance was about 50 pS. The extrapolated reversal potential was more positive than +40 mV (140 K or 140 Na with 5 Ca outside). Both the 26 and 50 pS channel showed a run-down, and they rapidly disappeared in excised patches. The high-conductance potassium channel with a single-channel conductance of 170 pS was observed only rarely. It reversed near the expected potassium equilibrium potential. The 26 pS channel could be stimulated with histamine and thrombin from outside in the cell-attached mode. Both the 26 pS as well as the 50 pS channel can mediate calcium flux into the endothelial cell.     

Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

Diabetes can lead to serious microvascular complications like proliferative diabetic retinopathy (PDR), which is the leading cause of blindness in adults. The proteomic changes that occur during PDR cannot be measured in the human retina for ethical reasons, but could be reflected by proteomic changes in vitreous humor. Thus, we considered that comparisons between the proteome profiles of the vitreous humors of PDR and nondiabetic controls could lead to the discovery of novel pathogenic proteins and clinical biomarkers. In this study, the authors used several proteomic methods to comprehensively examine vitreous humor proteomes of PDR patients and nondiabetic controls. These methods included immunoaffinity subtraction (IS)/2-DE/MALDI-MS, nano-LC-MALDI-MS/MS, and nano-LC-ESI-MS/MS. The identified proteins were subjected to the Trans-Proteomic Pipeline validation process. Resultantly, 531 proteins were identified, i.e., 415 and 346 proteins were identified in PDR and nondiabetic control vitreous humor samples, respectively, and of these 531 proteins, 240 were identified for the first time in this study. The PDR vitreous proteome was also found to contain many proteins possibly involved in the pathogenesis of PDR. The proteins described provide the most comprehensive proteome listing in the vitreous humor samples of PDR and nondiabetic control patients.     

Metabolism of angiotensin I by guinea pig aqueous humor

We investigated the degradation of angiotensin I (Ang I) by guinea pig aqueous humor at physiological pH (pH 7.4) and assessed the activity of responsible enzymes using various enzyme inhibitors. The aqueous humor was incubated with Ang I in the presence or absence of an enzyme inhibitor at 37 degrees C for the appropriate time period. The resulting peptides were analyzed by a Beckman HPLC system with a Waters microBondapak C18 analytical column using a 30-min increasing linear gradient of 10 to 40% acetonitrile containing 0.05% trifluoroacetic acid (TFA) and H2O containing 0.05% TFA at a flow rate of 1 mL/min. Detection was done by absorbance at 214 nm. Angiotensin II (Ang II) was a major product (39.3+/-4.10 nmol x h(-1) mL(-1), n = 5) of Ang I hydrolysis. Traces of angiotensin 1-9, angiotensin IV, and angiotensin 1-7 were also produced. Chymostatin (0.05 mmol/L), EDTA (1 mmol/L), enalaprilat (0.1 mmol/L), and ebelacton B (0.01 mmol/L) inhibited generation of Ang II from Ang I by guinea pig aqueous humor by 89+/-4.6, 56+/-7.6, 33+/-5.1, 20+/-6.5%, respectively. Our findings indicate that guinea pig aqueous humor contains several enzymes that can form Ang II. The chymostatin-sensitive type of enzyme was the most active one found in guinea pig aqueous humor. Angiotensin I converting enzyme, carboxypeptidase A, and deamidase may also contribute to angiotensin II formation in guinea pig ocular fluid.     

Transport and steady-state concentration of plasma proteins in the vitreous humor of the chicken embryo: implications for the mechanism of eye growth during early development

The nature and origin of the proteins of the vitreous humor were examined in chickens during embryonic and early posthatching stages. The major proteins of the vitreous humor were similar in electrophoretic mobility to plasma proteins at all ages examined. Earlier studies from our laboratory and experiments described below showed that plasma proteins continuously entered and left the eye throughout its development. From these data it was concluded that the majority of vitreous-humor proteins were derived from the blood. The protein concentration of the vitreous humor was 13% of that of the plasma from embryonic Days 6 through 15 (E6 through E15). After E15, the relative protein concentration in the vitreous humor declined with respect to the plasma and reached 4% of the plasma protein concentration at hatching. Several possibilities were considered to account for how proteins can rapidly enter and leave the eye, yet maintain a steady-state concentration in the vitreous humor that is less than one-seventh of that in the blood.     

Urinary prostanoid excretion in healthy women with different degrees of induced potassium depletion.

Plasma renin activity (PRA), urinary excretions of PGE2, 6-keto-PGF1 alpha (6KPGF), TXB2 and renal function were determined in healthy women both in normal potassium balance (N, n = 14) and in experimental potassium depletion (KD). KD was induced by natriuretic treatment--associated to replacement of net NaCl and water losses--in the presence of either normal (congruent to 50 mmol/d) or low (less than or equal to 10 mmol/d) dietary potassium intake. By using different depletive patterns, three groups with estimated cumulative potassium deficit (mean +/- SEM) of 124 +/- 38 (KD0, n = 8), 160 +/- 43 (KD1, n = 8) and 198 +/- 22 mmol (KD2, n = 6), respectively, were obtained. Renal function by the clearance (cl.) method and urinary prostanoid concentrations by the RIA method were estimated during hypotonic polyuria (oral water load) and subsequent moderate antidiuresis induced by a low-dose infusion of lysine-8-vasopressin. 1. In KD0 group the potassium depletive treatment was inefficacious in significantly reducing either the plasma potassium concentration (PK) or the urinary potassium excretion (UKV). The reductions of PK and UKV as well as the enhancement of PRA became significant in KD1 and KD2 groups. 2. The urinary prostanoid excretions were not significantly changed in the KD0 and KD1 groups while in the KD2 group they were reduced, mainly concerning the urinary 6KPGF excretion. 3. Furthermore in the KD2 group, with larger potassium depletion, some of the typical hypokalemic renal dysfunctions appeared. The data suggest that a pathophysiologically critical degree of potassium depletion is associated with an inhibited renal prostanoid synthesis as well as an increased renin secretion.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.     

Effect of phosphate buffer concentration on the batch xylitol production by Candida guilliermondii

AIMS: To evaluate the effect of phosphate buffer concentration on growth and xylitol production by Candida guilliermondii FTI 20037. METHODS AND RESULTS: Fermentations runs were carried out in batch mode employing semisynthetic medium supplemented with phosphate buffer at different concentrations (from 200 to 600 mmol l(-1)). The xylitol yield (Y(P/S)) and volumetric productivity (Q(P)) were improved when the fermentation medium was supplemented with phosphate buffer at concentration of 600 mmol l(-1). Under this condition (Y(P/S)) and (Q(P)) values were 0.75 g g(-1) and 0.66 g l(-1) h(-1), respectively, whereas in the absence of the phosphate buffer these values decreased to 0.52 g g(-1) and 0.44 g l(-1)h(-1) respectively. CONCLUSIONS: The use of phosphate buffer at 600 mmol l(-1) promoted an easier pH control during shake flasks fermentation of C. guilliermondii. In addition the xylitol yield and productivity were significantly improved in response to the supplementation of potassium phosphate in the medium. The increase in these parameters could be related to both osmotic effect and pH control. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provided a method for improving the xylitol production from semisynthetic medium by C. guilliermondii, being possible their use as a simple strategy to achieve efficient fermentation processes employing complex medium such as lignocellulosic hydrolysates.     

Dopamine-induced epithelial K(+) and Na(+) movements in the salivary ducts of Periplaneta americana

K(+)- and Na(+)-selective double-barrelled microelectrodes were used for intracellular and luminal measurements in salivary ducts of Periplaneta americana. The salivary ducts were stimulated with dopamine (10(-6) mol l(-1)). Dopamine decreased intracellular [K(+)] from 112+/-17 mmol l(-1) to 40+/-13 mmol l(-1) (n=6) and increased intracellular [Na(+)] from 22+/-19 mmol l(-1) to 92+/-4 mmol l(-1) (n=6). Luminal [K(+)] was 15+/-3 mmol l(-1) in the unstimulated salivary ducts and increased to 26+/-11 mmol l(-1) upon stimulation with dopamine (n=10). Luminal [Na(+)] was insignificantly increased from 105+/-25 mmol l(-1) to 116+/-22 mmol l(-1) (n=12) by stimulation with dopamine. The potential difference across the basolateral membrane (PD(b)) was depolarized from -65+/-6 mV to -31+/-13 mV (n=12) and the transepithelial potential difference (PD(t)) was hyperpolarized from -13+/-6 mV to -22+/-7 mV (n=22, lumen negative) upon stimulation with dopamine. The re-establishment of prestimulus values of intracellular [K(+)] and [Na(+)] and PD(b) was inhibited by basolateral addition of ouabain (10(-4) mol l(-1)). Furosemide (10(-4) mol l(-1)) in the bath inhibited the dopamine-induced increase in intracellular [Na(+)], the decrease in intracellular [K(+)] and the depolarization of PD(b). We propose a model for dopamine-stimulated ion transport in the salivary ducts involving basolateral Na(+)-K(+)-2Cl(-) cotransport and active extrusion of K(+) via the apical membrane.     

Ionic composition of endolymph and perilymph in the inner ear of the oyster toadfish, Opsanus tau

The concentrations of free Na+, K+, Ca(+, and Cl(-)in endolymph and perilymph from the inner ear of the oyster toadfish, Opsanus tau, were measured in vivo using double-barreled ion-selective electrodes. Perilymph concentrations were similar to those measured in other species, while endolymph concentrations were similar to those measured previously in elasmobranch fish, though significantly different from concentrations reported in mammals. Perilymph concentrations (mean +/- std. dev.) were as follows: Na+, 129 mmol l(-1) +/- 20; K+, 4.96 mmol l(-1) +/- 2.67; Ca2+, 1.83 mmol l(-1) +/- 0.27; and Cl(-), 171 mmol l(-1) +/- 20. Saccular endolymph concentrations were Na+, 166 mmol l(-1) +/- 22; K+, 51.4 mmol l(-1) +/- 16.7; Ca2+, 2.88 mmol l(-1) +/- 0.27; and Cl(-), 170 mmol l(-1) +/- 12; and semicircular canal (utricular vestibule) endolymph concentrations were Na+, 122 mmol l(-1) +/- 15; K+, 47.7 mmol l(-1) +/- 13.2; Ca2+, 1.78 mmol l(-1) +/- 0.48; Cl(-), 176 mmol l(-1) +/- 27. The relatively high concentrations of Ca2+ and Na+ in the endolymph may have significant implications for the physiological function of the mechanoelectrical transduction channels in the vestibular hair cells of fish compared to those of their mammalian counterparts.     

Calcium in the toad (Bufo marinus) cornea: binding by the stroma

1. The Ca concentration in the toad (Bufo marinus) cornea was 2.6 mmol/kg wet wt compared at 1.0 mmol/l in the bathing aqueous humor and 2.8 mmol/kg wet wt in the separated corneal stromal layer. Cell Ca content was calculated to be about 1.8 mmol/kg wet wt. 2. About 80% of the total Ca appears to be sequestered or bound to tissue components most of which (68% of the total) is associated with the stroma (2.2 mmol/kg wet wt stroma). 3. About 85-90% of the Ca in the stroma is readily exchangeable with external 45Ca. 4. The loss of accumulated 45Ca from the stroma was measured in vitro. This efflux of the isotope was enhanced by multivalent ions and was greatest when Ca2+ or La3+ was present in the external media. Other alkaline earth metal ions were not as effective. The relative effectiveness of this displacement of 45Ca was Ca = La greater than Sr greater than Ba greater than Mg. 5. The results suggest that the Ca2+ is bound by the amphibian stroma at sites that have a preference or specificity for this divalent ion as compared to the other alkaline earth metals. 6. The possible functional role of this bound Ca is discussed.     

A method of sodium and potassium determination in red cells with a simple cell separation technique

Heparinized blood was centrifuged repeatedly in Eppendorf's test tubes at 7,500 g in the Unipan microcentrifuge type 320. Packed red cells were hemolysed, then sodium and potassium were determined by means of the flame photometer. The percentage of trapped plasma determined with indocyanine green amounted to on average 1 per cent. There was a good precision of the method controlled on 20 aliquots of the same blood sample. Results of red cell sodium and potassium in 80 healthy volunteers were 10.42 +/- 1.56 mmol/l and 87.8 +/- 4.03 mmol/l respectively. No significant changes in the red cell sodium and potassium concentration were observed in heparinized blood during 5 hours storage at room temperature. The method cannot be used interchangeably with the method of Helbock and Brown, since the correlation coefficients were too low in parallel examinations.     

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.     

Vitreous humour as a potential DNA source for postmortem human identification

PURPOSE: The aim of this study was the assessment of vitreous humor as a potential DNA for forensic human postmortem identification. MATERIAL AND METHODS: Vitreous humor samples were collected using two alternative approaches from 25 corpses of either sex during autopsies. DNA was extracted by standard organic method. Recovered DNA was quantitiated fluorometrically. AmpFlSTR SGM Plus kit and ABI 310 Genetic Analyzer (Applera) were used to obtain genetic profiles. RESULTS: Different DNA yields were quantitated in vitreous body depending on cause of death and sampling approach. CONCLUSION: Vitreous humor is a potential DNA for forensic human postmortem identification depending on a sampling method used.