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PubMed2003

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V.

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.

Amino Acid Sequence↗
PubMed2001

4',6-diamidino-2-phenylindole (DAPI) interacts with rare structures of GC polymers.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investigated using fluorescence techniques. We employed fluctuation correlation spectroscopy, which measures the diffusion coefficient of fluorescent particles, to demonstrate that the fluorescence was originating from relatively slowly diffusing entities. These entities display a very large heterogeneity of diffusing coefficients, indicating that molecular aggregation is extensive in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye coverage systematically. At very low coverage we observed a relatively bright fluorescent component with a lifetime value of approximately 4 ns. The stoichiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular aggregates. The amount and characteristics of this bright fluorescent component were different between the homo and the alternating polymer, indicating that the difference in sequence of the two polymers is responsible for the different aggregates which are then detected in the fluorescence experiment. At large GC polymer coverage we observed a relatively wide distribution of fluorescent species with short lifetime values, in the range between 0.12 and 0.2 ns. Given the stoichiometry of binding of this fluorescent component, we concluded that it could arise either from intercalative and/or non-specific binding to the DNA double-stranded molecules. We comment on the origin of the rare but brightly fluorescent binding sites and discuss the potential to detect such unusual DNA structures.

Binding Sites↗
PubMed2003

A phase II and pharmacokinetic study of pegylated liposomal doxorubicin in patients with advanced hepatocellular carcinoma.

PURPOSE: Chemotherapy of advanced hepatocellular carcinoma (HCC) is frequently limited by unacceptable toxicity. Long-circulating polyethylene glycol-coated (PEGylated) liposomal doxorubicin (PLD) has low systemic toxicity. Its safety and efficacy in patients with advanced HCC and the relationship between hepatic function and pharmacokinetics were investigated in this phase II study. METHODS: Patients were given 30 mg/m(2) PLD every 3 weeks and the dose was escalated to 45 mg/m(2) from the third course if the toxicity was deemed tolerable. The plasma level of doxorubicin was determined with fluorometry. RESULTS: A total of 40 patients were recruited into this phase II study. The toxicities were usually mild but unexpectedly, three cirrhotic patients died of infection without neutropenia. Four had a partial response (response rate 10%, confidence interval 0-20%). The median duration of response was 5.6 months. The median time to tumor progression and the median survival of all patients was only 2 and 3 months, respectively. Patients with advanced HCC had lower initial serum concentration, larger volume of distribution and more rapid clearance than patients with other malignancies and normal liver function. However, the pharmacokinetic parameters correlated with neither toxicity nor response. CONCLUSIONS: The disposition of PLD in patients with liver dysfunction was not hampered, but it did not exhibit higher activity compared with free drug, and the risk of infection must be watched closely especially in patients with liver cirrhosis.

Adolescent↗
PubMed2002

Lysophosphatidylcholine alters enterocyte monolayer permeability via a protein kinase C/Ca2+ mechanism.

The activity of phospholipase A(2) (PLA(2)) is elevated in the intestinal epithelia of patients with inflammatory bowel disease. We recently reported that PLA(2) mediates the hydrolysis of phosphatidylcholine (PC) to lysophosphatidylcholine (L-PC) when both are applied to the apical surface of cultures enterocyte monolayers, resulting in increased bacterial translocation (BT) and decreased transepithelial electrical resistance (TEER). However, the mechanism by which the converted L-PC affects tight-junction permeability (TJP) as reflected by decreased TEER is unknown. There are some reports that protein kinase C (PKC) or Ca(2+) mediate TJP in enterocyte monolayer models. To investigate whether the observed change in TJP was mediated via PKC or Ca(2+) in our Caco-2 monolayer model, human Caco-2 enterocytes were grown to confluence on porous filters in the apical chamber of a two-chamber cell culture system. The filters were then transferred to an Ussing chamber for precise, real-time resistance measurements. After 30 min equilibration, PC (0.1 or 1 mM) and L-PC (0.01, 0.1 or 1 mM), PMA 200 or 300 nM (phorbol 12-myristate 13-acetate, PCK activator), or staurosporine 12 nM (PKC inhibitor) were added to the apical chamber and TEER was measured every 20 s for 2 h. The concentration of intracellular free Ca(2+) in the monolayers before and after treatment with L-PC (1 mM) was measured by fluorometry of whole monolayers using the fluorescent calcium indicator fura-2. Neither PC at any dose nor the 0.01-mM L-PC dose had an effect on TEER. The 0.1-mM dose of L-PC had its greatest effect (47% +/- 3.5% reduction in TEER vs control) within 6 min following its addition, with TEER recovery to control levels (100%) at 2 h ( P < 0.05). The 1-mM dose of L-PC had its greatest effect (6% +/- 0.5% reduction in TEER vs control) within 3 min after its addition, but the TEER did not recover to control levels after 2 h of incubation ( P < 0.05). The addition of 200 or 300 nM PMA inhibited the observed recovery of TEER by L-PC. Conversely, the addition of 12 nM staurosporine enhanced TEER recovery to control levels. The 1-mM dose of L-PC increased the concentration of intracellular free Ca(2+) immediately after the addition of L-PC. These results suggest that L-PC alters TJP via a PKC/Ca(2+) interaction in our Caco-2 monolayer model.

Caco-2 Cells↗
PubMed1998

Matrix metalloproteinases, but not cathepsins B, H, and L or their inhibitors in peripheral blood of patients with rheumatoid arthritis are potentially useful markers of disease activity.

OBJECTIVE: The clinical activity of rheumatoid arthritis (RA) is not reliably reflected by laboratory measures. Proteolytic enzymes involved in the cascade of joint destruction are potentially useful parameters to monitor the extent of joint inflammation in RA. This study compares the validity of two classes of proteolytic enzymes, matrix metalloproteinases (MMP) and lysosomal cysteine proteinases (cathepsin B, H, and L) as well as their respective inhibitors to serve as parameters of RA disease activity. METHODS: The proteolytic activity of cathepsin B, H, and L was determined by fluorometry in sera of 20 patients with active RA and of 20 healthy donors. In addition, the concentrations of cathepsin B and L as well as of cathepsin inhibitors stefin A, stefin B, and cystatin C were measured by ELISA. The plasma concentrations of MMP-1 (collagenase), MMP-3 (stromelysin), tissue inhibitor of metalloproteinases 1 (TIMP-1), and of MMP-1/TIMP-1 complex (MT complex) were analyzed by ELISA as well. RESULTS: A significant increase of MMP-1, MMP-3, and MT complex was observed in RA plasma, compared to normal controls, whereas TIMP-1 concentrations did not differ. In contrast, neither serum activity nor protein concentration of any of the cathepsins or cathepsin inhibitors were elevated in RA. CONCLUSION: Despite ample evidence in the literature that cathepsin activity contributes to the pathogenesis of inflammatory joint disease, this is not reflected by the conditions in peripheral blood. In contrast to the cysteine proteinases, MMP-1 and MMP-3 as well as MT complex are elevated in RA. In the context of findings in the literature, this stresses the importance of MMP as disease activity markers, compared to cysteine proteinases or their inhibitors.

Aged↗
PubMed1998

Progression of retinopathy and alteration of the blood-retinal barrier in patients with type 2 diabetes: a 7-year prospective follow-up study.

BACKGROUND: The study was carried out to evaluate the correlation between blood-retinal barrier (BRB) permeability and the progression of diabetic retinopathy (DR), defined by development of "need for photocoagulation", over a 7-year period by means of vitreous fluorometry (VF). METHODS: Forty type 2 diabetic patients with minimal or no retinopathy, aged 40-65 years (mean 53.9 + 7.3 years), were followed up prospectively for 7 years. Investigations including standard ophthalmological examination, fundus photography, fluorescein angiography and VF were performed at entry and 1, 4, 5 and 7 years later. Only one eye per patient was included in the study. Need for photocoagulation was based on Early Treatment Diabetic Retinopathy Study protocols and decided by the attending ophthalmologist. RESULTS: After 7 years of follow-up a total of 22 of the 40 eyes had received photocoagulation. The eyes that needed photocoagulation were those that had higher VF values at the entry of the study and showed higher rates of deterioration (initial values 5.1 + 1.9 vs 2.8 + 1.5 x 10(-6) min-1, P < 0.001; annual increase in leakage for the first year, 1.5 + 0.8 vs 0.5 + 1.0 x 10(-6) min-1, P < 0.001,). The eyes that did not need photocoagulation during the 7 years of follow-up showed stable VF readings (-0.1 + 1.2 x 10(-6) min-1, difference between initial values and 7 years later). CONCLUSIONS: Abnormally high VF values and their rapid increase over time are good indicators of progression and worsening of the retinopathy in diabetes type 2.

Adult↗
PubMed1998

The effect of acetazolamide on passive and active transport of fluorescein across the blood-retina barrier in retinitis pigmentosa complicated by macular oedema.

BACKGROUND: The carbonic anhydrase inhibitor acetazolamide (AZM) reduces macular oedema in some patients with retinitis pigmentosa. To better understand the oedema-reducing effect of AZM, the effect of AZM on passive permeability and active transport of fluorescein across the blood-retina barrier was studied in patients with retinitis pigmentosa and varying degrees of macular oedema. METHOD: The selection of patients was based on an introductory examination including vitreous fluorometry for qualitative assessment of the vitreous. Macular oedema was graded by fluorescein angiographic leakage. The effect of AZM on the transport properties of the blood-retina barrier was determined by differential spectrofluorometry, in a randomised, double-masked, cross-over study, comprising 2 weeks' treatment with AZM (500 mg/day) and 2 weeks' treatment with placebo. The penetration ratio, defined as the ratio between vitreous concentration 3 mm in front of the retina and the plasma integral, was determined for fluorescein and its metabolite fluorescein glucuronide at 30-60 min and at 120 min after fluorescein injection. Passive permeability and unidirectional permeability in the direction vitreous to blood, due to outward active transport of fluorescein, were determined in those cases where the curves for vitreous concentration of fluorescein could be fitted to a mathematical model. Visual acuity was tested by use of ETDRS standard logarithmic charts. RESULTS: Twenty-two patients volunteered to participate in the study. Signs of significant vitreous detachment/liquefaction caused the exclusion of ten patients after the introductory examination. Nine patients with approximately intact vitreous and varying degrees of oedema completed the cross-over study. AZM treatment was related to a decrease in the penetration ratio of 21% for fluorescein (P=0.01) and of 22% for fluorescein glucuronide (P=0.004). Passive permeability and unidirectional permeability were determined in seven patients. AZM caused a decrease of 27% in the passive permeability of fluorescein (from 1.1 x 10(1) nm/s, P=0.031), and a 95% increase in unidirectional permeability of fluorescein (from 1.2 x 10(2) nm/s, P=0.047). AZM led to a reduction in the grade of macular oedema as determined by fluorescein angiography in three out of seven patients. Only small improvements (< or =5 letters) in visual acuity were noted. CONCLUSION: The present study indicates that the oedema-reducing effect of AZM is due to decreased leakage and stimulated active transport across the blood-retina barrier.

Acetazolamide↗
PubMed1999

Polyamine system in developing rat eye and an animal model of retinopathy of prematurity.

BACKGROUND: In experimental models of retinopathy of prematurity (ROP), a vasoproliferative disorder of the retina, retinal lesions are usually assessed by morphological examination. However, studies suggest that the polyamine system may be useful in monitoring proliferation processes. For this reason, polyamine concentrations in rat erythrocytes (RBC) and the regulation of polyamine system in rat eyes under the conditions relevant to ROP were investigated. METHODS: Newborn Wistar rats were reared in room air (control) or exposed first to hyperoxia (60% or 80% oxygen, 2 weeks) and then to normoxia (relative hypoxia, 1 or 2 weeks). Blood was collected from orbital vessels at 2 weeks of age and before death. Polyamine system-related enzyme activities were measured in retina and lens with radioassays. Polyamines were quantified by fluorometry after extraction, dansylation and HPLC separation. RESULTS: Oxygen (80% only) significantly decreased RBC polyamine concentrations, which then markedly increased after rats were transferred for a week to normal air, suggesting retardation of growth processes and compensatory stimulation, respectively. However, polyamine system changes in the rat eye were not so pronounced. Enzyme activities and polyamine concentrations tended to be lower in retina after hyperoxia and were only slightly higher, with the exception of ornithine decarboxylase, after a subsequent 1 week of normoxia. In litters subjected to normoxia for longer periods no changes were found. CONCLUSION: The transient and short-lived alteration in polyamine metabolism, especially in the eye, suggests that exposure of newborn rats to high oxygen supplementation followed by normoxia does not necessarily result in marked retinopathy.

Aging↗
PubMed2004

In vivo heat shock preconditioning mitigates calcium overload during ischaemia/reperfusion in the isolated, perfused rat heart.

Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca2+ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 degrees C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca2+i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45+/-11 vs. 64+/-22 mmHg; developed pressure: 72+/-12 vs. 41+/-20 mmHg; maximum rate of pressure increase (+dP/dtmax): 1,513+/-305 vs. 938+/-500 mmHg/s; maximum rate of pressure decrease (-dP/dtmax): -1,354+/-304 vs. -806+/-403 mmHg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca2+] ([Ca2+]i) after reinstallation of flow. The dynamic parameters of the Ca2+i transients, i.e. the maximum rate of increase/decrease (+/-dCa2+i/dtmax) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca2+i load, and most likely, preserved Ca2+ sensitivity of the myocardial contractile machinery.

Animals↗
PubMed1997

Role of anions in the regulation of cell volume and intracellular pH in perfused rat mandibular salivary gland.

To investigate the role of Cl- in the regulation of the basolateral transporters of salivary acinar cells, we have measured cell volume and intracellular pH (pHi) in perfused rat mandibular glands using proton NMR spectroscopy and BCECF fluorometry respectively. When perfusate Cl- was replaced by glucuronate, isethionate, methylsulphate, nitrate or thiocyanate, cell volume decreased slowly by about 15% over a 10-min period. Replacement with bromide, which substitutes for Cl- on the Na+-K+-2Cl- cotransporter, caused only a small (4%) reduction in cell volume. Replacement of Cl- by glucuronate, isethionate or methylsulphate evoked a biphasic increase in pHi consisting of a rapid initial increase followed by a slower secondary rise whose time course was similar to that of cell shrinkage. As judged by the effects of HCO3- omission, 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 1 mM amiloride, the initial rise in pHi was due to Cl-/HCO3- exchange while the secondary rise resulted from activation of Na+/H+ exchange. Although replacement of Cl- by nitrate or thiocyanate also caused cell shrinkage, these substituting anions were less effective in activating the exchanger. Therefore, while the upregulation of the exchanger following Cl- replacement may be due in part to cell shrinkage, there is also evidence for the involvement of an anion-sensitive regulatory mechanism. This would be consistent with the hypothesis that both changes in cell volume and in intracellular Cl- concentration contribute to the up-regulation of the exchanger following muscarinic stimulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Naloxone is protective against indomethacin-induced gastric damage in cholestatic rats.

We compared indomethacin-induced gastric damage in three groups of rats-bile duct-ligated, sham-operated, and unoperated-and evaluated the role of the opioid system by blocking the effects of endogenous opioids with naloxone. Indomethacin was administered orally in a dose-dependent manner at 10, 30, and 45 mg/ kg. Naloxone was administered intraperitoneally in several doses of 0.5 and 1 mg/kg, starting 30 min before indomethacin (10mg/kg) administration and continued every 30 min. The animals were killed 4h after indomethacin administration. Indomethacin induced more severe gastric damage in bile duct-ligated rats than in sham and unoperated animals, and administration of naloxone (1 mg/kg) every 30 min inhibited the potentiation of indomethacin-induced gastric damage in bile duct-ligated rats, but not in the control groups (sham-operated and unoperated rats). Plasma indomethacin level was also measured, by fluorometry, but showed no significant difference between the groups. Endogenous opioids have been reported to accumulate in plasma of cholestatic animals, and, considering the results of this study, we suggest the opioid system plays an important pathophysiologic role in the pathogenesis of peptic ulcers in cholestatic subjects.

Animals↗
PubMed1992

Effects of high pressure on polarity change of the water-liposome interface induced by volatile anesthetics.

The effects of high pressure on the interactions between volatile anesthetics and three kinds of phosphatidyl choline liposomes were investigated by fluorometry, using a thiacarbocyanine dye (3,3"-dioctadecyl-2,2"-thiacarbocyanine) which is sensitive to the environmental viscosity and dielectric constant. Seven general anesthetics, halothane, enflurane, isoflurane, methoxyflurane, sevoflurane, diethylether and chloroform were used. We have previously reported that these anesthetics decreased the phase transition temperature and increased the effective dielectric constant of the water-liposome interface using dipalmitoyl-phosphatidylcholine. In this study, it was confirmed that the effects of anesthetics on the effective dielectric constant were not altered by the use of a gel or liquidcrystal membrane, and were reversed by the application of high pressure (<800 bars). The increase of the effective dielectric constant was attributed to the perturbation of hydrogen bonds at the liposomal interface. High pressure is considered to promote hydration. Our results obtained under high pressure supported previous observations made at ambient pressure, which suggested that the perturbation of hydrogen bonds at the water-liposome interface correlates with the mechanism of anesthesia.

Journal Article↗
PubMed2006

Photosynthetic parameters as indicators of trinitrotoluene (TNT) inhibitory effect: change in chlorophyll a fluorescence induction upon exposure of lactuca sativa to TNT.

Trinitrotoluene, as a compound of conventional explosive, may cause inhibitory effect on terrestrial plants. When Lactuca sativa was exposed to different concentrations of trinitrotoluene (32-1000 mg/kg), photosynthetic process was investigated by using rapid chlorophyll fluorescence kinetic and pulse modulated fluorometry. The decrease of chlorophyll a variable fluorescence was seen to be caused by the deactivation of photosystem II reaction centers. We found for rapid variable fluorescence to be a useful indicator to evaluate the inhibitory effect of trinitrotoluene on photosystem II primary photochemistry and electron transport. The fluorescence parameters, related to the reduction state of photosystem II and to non-photochemical dissipation of light energy, showed a strong relation between the inhibitory effect of photosystem II activity and concentration of trinitrotoluene. The change of photosynthetic fluorescence parameters induced by trinitrotoluene was a reliable indication of the plant physiological state. We proposed for the reduction state of photosystem II and the non-photochemical energy dissipation to be a useful tool in bioassay toxicity testing of trinitrotoluene polluted soil.

Biomass↗
PubMed2004

Relationship between standard semen parameters, calcium, cholesterol contents, and mitochondrial activity in ejaculated spermatozoa from fertile and infertile males.

PURPOSE: To correlate levels of cholesterol (CH), calcium (Ca2+), and mitochondrial activity (MA) with the standard semen parameters and to compare them between fertile and infertile men. METHODS: We studied 151 semen samples from infertile (n = 60) or fertile (n = 91) males. Basic sperm parameters were analyzed. Ca2+ and CH concentrations on seminal plasma were determined by enzymoimmunoanalysis. Intracellular Ca2+ and CH concentrations in the sperm plasma membrane and mitochondrial activity by fluorometry. RESULTS: There was a significant positive correlation between sperm membrane CH and sperm morphology. Intracellular Ca2+ was lower in infertile patients compared to fertile. No differences were found regarding Ca2+ and CH concentrations in seminal plasma. MA is directly and strongly related with sperm motility. CONCLUSIONS: Intracellular concentrations of Ca2+ and the proportion of CH in the sperm membrane are two important markers of the sperm quality due to its direct relationship with sperm morphology and fertility potential.

Calcium↗
PubMed2005

Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts.

The protective effects of sarpogrelate (SG), a 5-HT2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5 x 10(-7) M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (TNO) level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. Meanwhile, mitochondrial Ca2+ uptake on acidification or Ca2+ content changes of the perfusate was suppressed by pre-treatment with SG or the KATP channel opener diazoxide, but not the KATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT2A receptor induced in ischemia-reperfusion plays an important role in the mitochondrial KATP channel of hearts in close relation with NO and active oxygen radicals.

Animals↗
PubMed2006

Association of hypoglutathionemia with reduced Na+/K+ ATPase activity in type 2 diabetes and microangiopathy.

OBJECTIVE: Although recent studies link altered cellular redox state to protein dysfunction in various disease-states, such associations are least studied in clinical diabetes. Therefore, this study assessed the levels of reduced glutathione (GSH) and Na(+)/K(+) ATPase activities in type 2 diabetic patients with and without microangiopathy. METHODS: The study group comprised of a total of 160 subjects, which included non-diabetic healthy controls (n = 40) and type 2 diabetic patients without (n = 60) and with microangiopathy (n = 60), defined as presence of retinopathy with or without nephropathy. Erythrocyte Na(+)/K(+) ATPase activity and GSH levels were estimated spectrophotometrically and fluorometry was used to determine the plasma thiobarbituric acid reactive substances (TBARS) and serum advanced glycation end products (AGEs). RESULTS: GSH levels in diabetic subjects without (4.8+/- 0.15 mumol/g Hb) and with microangiopathy (5.2+/- 0.14 micromol/g Hb) were significantly lower (p < 0.001) compared to control subjects (6.3 +/- 0.14 mumol/g Hb). Erythrocyte Na(+)/K(+) ATPase activity was significantly reduced (p < 0.001) in diabetes subjects with (272 +/- 7 nmol Pi/mg protein/h) and without microangiopathy (304 +/- 8) compared to control (374 +/- 6) subjects. TBARS were significantly higher (p < 0.001) in diabetes subjects with (10.65 +/- 0.81 nM/ml) and without microangiopathy (9.90 +/- 0.5 nM/ml) compared to control subjects (5.18 +/- 0.18 nM/ml). Advanced glycation end product levels were also significantly (p < 0.001) elevated in diabetic subjects with microangiopathy (8.2+/- 1.8 AU) when compared to diabetes subjects without microangiopathy (7.0 +/- 2.0 AU) and control subjects (4.6 +/- 1.9 AU). On multivariate regression analysis, GSH levels showed a positive association with the Na(+)/K(+) ATPase activity and negative association with TBARS and AGE levels. CONCLUSION: Hypoglutathionemia and increased oxidative stress appears to be early biochemical aberrations in diabetes, and through protein alterations, oxidative stress and redox modifications may contribute to pathogenesis of diabetic microangiopathy.

Diabetes Mellitus, Type 2↗
PubMed2006

Regulation of exogenous gene expression by superoxide.

PURPOSE: Regulation of gene expression after gene introduction is a problematic aspect of gene therapy. Transcription regulates gene-specific transcriptional factors, which bind to regulatory regions in the promoter. The cytomegalovirus long terminal repeat (CMV-LTR) has a TPA response element (TRE) as a binding site for activator protein 1 (AP-1), which is induced by oxidative stress. The purpose of this study was to regulate exogenous gene expression in a vector with CMV-LTR using oxygen radicals. METHODS: We used two plasmids (1) pQBI25 encoding CMV-LTR and red-shift green fluorescent protein (rsGFP) cDNA and (2) pRc/CMV-SOD encoding CMV-LTR and human Cu, Zn-superoxide dismutase (SOD) cDNA. FR cells were transfected with pQBI25 (FR-pQBI25 cells), and L2 cells were transfected with pRc/CMV-SOD (L2-pRc/CMV-SOD cells). Each type of cell was exposed to oxygen radicals using paraquat for 24 h. Levels of c-fos, c-jun and rsGFP mRNAs were determined using reverse transcription polymerase chain reaction (RT-PCR). Levels of rsGFP protein were measured by fluorometry. Total SOD activity was measured using the nitrite method. RESULTS: Levels of c-fos, c-jun (AP-1 composition protein) and rsGFP mRNA were induced significantly by oxygen radical exposure in FR-pQBI25 cells. A positive correlation was observed between levels of c-fos mRNA and rsGFP mRNA and also between levels of c-jun mRNA and rsGFP mRNA. Levels of rsGFP protein were also induced significantly. Total SOD activity was induced significantly by oxygen radical exposure in L2-pRc/CMV-SOD cells. CONCLUSIONS: This study suggests that gene expression driven by the CMV- LTR promoter may be regulated by oxygen radicals.

Animals↗
PubMed2005

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence.

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (DeltaF proportional, variant Osm-1; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1-4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl2, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of EA=4.9 kcal mol-1, in the absence of HgCl2, and EA=11.9 kcal mol-1 in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg2+ ions and low activation energy.

Biological Transport↗