Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging.

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD+/NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD+/NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.

Gradient blue native polyacrylamide gel electrophoresis (BN-PAGE) is a well established and widely used technique for activity analysis of high-molecular-weight proteins, protein complexes, and protein-protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non-gradient BN-PAGE. The procedure, similar to that of non-gradient SDS-PAGE, is simple because there is no expensive gradient maker involved. The non-gradient BN-PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non-gradient BN-PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc.

Expression of the P2X2 receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse.

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X2 receptor (P2X2R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X2R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm²) and area profile (µm²) of P2X2R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X2R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X2R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm²) of P2X2R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (µm²) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X2R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.

Use of cytokeratin 8 immunohistochemistry for assessing cell death after radiofrequency ablation of breast cancers.

The effects of minimally invasive therapies such as radiofrequency ablation (RFA) and laser induced thermal therapy on breast carcinoma lesions usually is assessed by NADH diaphorase enzyme histochemistry for cell viability. NADH staining requires frozen material, however, with its associated poor morphology. We aimed to validate cytokeratin 8 (CK 8) immunohistochemistry as an alternative that works on paraffin sections. RFA was performed ex vivo on 20 breast resections after surgery and in vivo in eight patients who underwent general anesthesia followed by immediate resection. After treatment, specimens were lamellated and the tumors were divided into two equal parts. One part was fixed in neutral buffered formaldehyde for routine histopathological evaluation using hematoxylin and eosin (H & E) staining and CK 8 immunostaining. The other section was snap frozen and stored at -80° C for staining with NADH diaphorase. Both NADH diaphorase and CK 8 immunostaining demonstrated a clear and comparable demarcation between viable and nonviable tissues. The morphology of the CK 8 immunostained slides was much better, and fatty tissues could be judged readily by contrast to the NADH stained frozen sections, which had poor morphology and whose fatty parts were difficult to interpret. CK 8 immunohistochemistry seems to be well suited for assessing cell viability in breast tissue and for assessing the effects of RFA for breast cancer treatment. Because it can be applied to paraffin fixed material, it provides much better morphology than NADH staining and also can be applied to fatty tissues that usually are difficult to work up for frozen sections. Therefore, CK 8 immunohistochemistry may be preferred over NADH diaphorase staining for daily pathology practice for assessing the viability of breast carcinoma cells after RFA treatment.

Effects of short-duration electromagnetic radiation on early postnatal neurogenesis in rats: Fos and NADPH-d histochemical studies.

The immediate effects of whole body electromagnetic radiation (EMR) were used to study postnatal neurogenesis in the subventricular zone (SVZ) and rostral migratory stream (RMS) of Wistar rats of both sexes. Newborn postnatal day 7 (P7) and young adult rats (P28) were exposed to pulsed electromagnetic fields (EMF) at a frequency of 2.45 GHz and mean power density of 2.8 mW/cm(2) for 2 h. Post-irradiation changes were studied using immunohistochemical localization of Fos and NADPH-d. We found that short-duration exposure induces increased Fos immunoreactivity selectively in cells of the SVZ of P7 and P28 rats. There were no Fos positive cells visible within the RMS of irradiated rats. These findings indicate that some differences exist in prerequisites of proliferating cells between the SVZ and RMS regardless of the age of the rats. Short-duration exposure also caused praecox maturation of NADPH-d positive cells within the RMS of P7 rats. The NADPH-d positive cells appeared several days earlier than in age-matched controls, and their number and morphology showed characteristics of adult rats. On the other hand, in the young adult P28 rats, EMR induced morphological signs typical of early postnatal age. These findings indicate that EMR causes age-related changes in the production of nitric oxide (NO), which may lead to different courses of the proliferation cascade in newborn and young adult neurogenesis.

Histological, enzymohistochemical and biomechanical observation of skeletal muscle injury in rabbits.

OBJECTIVE: To explore the pathophysiological and biomechanical features of skeletal muscular injury for providing a rational basis for its treatment, prevention and rehabilitation. METHODS: In 70 adult rabbits, the left tibialis anterior (TA) muscle was stretched to injury, while the right TA muscle served as control. Histological, enzymohistochemical and biomechanical changes were observed on days 0, 1, 2, 3, and 7 after injury. Cytochrome oxidase (CCO), acid phosphatase (ACP), ATPase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH), NADH-diaphorase (NADHD), glutamatedehydrogenase (GDH), alpha-glycerophosphate dehydrogenase (alpha-GPD) and lactate dehydrogenase (LDH) were measured. The examined biomechanical parameters included maximal contractile force, ultimate load, length, energy absorption, tangent stiffness, and rupture site. RESULTS: Partial or complete rupture of TA muscle occurred near the muscle-tendon junction. There was an intense inflammatory reaction on day 1 and 2 after injury. Endomysium fibrosis and myotube formation were observed on day 3, and developed further on day 7. The activity of cell oxidases (CCO, ATPase, MDH, alpha-GPD, SDH, NADHD and GDH) showed a significant drop from day 0 to 2, and resumed with different levels on day 3. The increment of enzymatic activities continued on day 7 and the levels of NADHD and alpha-GPD reached to the levels of control muscle. Maximal contractile force was 70.17%+/-3.82% of controls immediately after injury, 54.82%+/-3.09% at 1 day, 66.41%+/-4.36% at 2 days, 78.39%+/-4.90% at 3 days and 93.64%+/-5.02% at 7 days. Ultimate load was 85.78%+/-7.54% of controls at the moment of injury, 61.44%+/-5.91% at 1 day, 49.17%+/-4.26% at 2 days, 64.43%+/-5.02% at 3 days, and 76.71%+/-6.46% at 7 days. CONCLUSIONS: Endomysium fibrosis and scar formation at the injured site are responsible for frequent recurrence of skeletal muscle injury. Recovery of tensile load slower than that of maximal contractile force may be another cause. Whether the injured muscle returns to normal exercise is mainly determined by the tensility on which the muscle-tendon can bear rather than the maximal contractile force.

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE.

Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.

Nitric oxide in blastocyst implantation in the rhesus monkey.

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.

Age-related increase in 4-hydroxynonenal adduction to rat heart alpha-ketoglutarate dehydrogenase does not cause loss of its catalytic activity.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (p< or =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (p< or =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (p< or =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

Isozymes in Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids and Ae. kotschyi x S. cereale amphiploids in relation to their parents.

Seven enzymatic systems in F1 Aegilops kotschyi and Ae. biuncialis x Secale cereale hybrids, Aegilops kotschyi x S. cereale amphiploids and their parental species (Ae. kotschyi, Ae. biuncialis and S. cereale) were analysed by starch and polyacrylamide gel electrophoresis. Five of them (phosphoglucose isomerase, glutamic oxalacetic transaminase, esterase, acid phosphatase, and diaphorase) were polymorphic and two (malic dehydrogenase and superoxide dismutase) were monomorphic. Several isophorms of phosphoclucose isomerase, esterase, acid phosphatase, and diaphorase were detected in some hybrids and amphiploids, but absent in the parents. The role of regulators, translocations and recombination is discussed in relation to the origin of these new isophorms. Some parental isozymes were absent both in hybrids and amphiploids, probably as a result of the suppression of structural genes in new combinations of the three genomes.

Daily exercise normalizes the number of diaphorase (NOS) positive neurons in the hypothalamus of hypertensive rats.

It is well known that nitric oxide (NO), within the paraventricular nucleus (PVN) of the hypothalamus, mediates sympatho-inhibition via an inhibitory GABA-ergic mechanism. Furthermore, the inhibitory GABA-ergic mechanism is impaired in the spontaneously hypertensive rat (SHR). These data suggest that the NO system, within the PVN, may also be impaired in the SHR. In addition, previous studies have documented that daily exercise attenuates the development of tachycardia, hypertension and blood pressure related cardiovascular disease risk factors in SHR. These data suggest that daily exercise enhances the inhibitory GABA-ergic and/or NO systems. Therefore, this study was designed to test the hypothesis that hypertension, in the SHR, is associated with a lower number of NADPH-diaphorase (a commonly used marker for neuronal NOS activity) positive neurons within the PVN and that daily exercise increases the number of NOS positive neurons. Using a standard histochemical protocol, NOS positive neurons were measured in the PVN, supraoptic nucleus, median preoptic area, lateral hypothalamus, nucleus of the tractus solitarius and rostral ventrolateral medulla. Results document that SHR have significantly fewer NOS-positive neurons in the PVN than their genetic control, the Wistar-Kyoto (WKY) rats (110+/-11 versus 139+/-17). Furthermore, daily exercise increased the number of NOS positive neurons in the SHR to levels seen in the WKY rats. These data demonstrate that hypertension, in the SHR, is associated with a lower number of NOS positive neurons within the PVN and that daily exercise increases the number of NOS positive neurons within the PVN.

Incomplete renal tumor destruction using radio frequency interstitial ablation.

PURPOSE: We evaluate the efficacy of temperature based radio frequency ablation as a potential treatment modality for small (less than 3.5 cm.) renal tumors. MATERIALS AND METHODS: We treated 15 patients with a total of 20 tumors with radio frequency ablation through an open surgical approach immediately before partial nephrectomy. All tumors were biopsied before radio frequency ablation treatment. Tumors were heated to 90 to 110C for 6 to 16 minutes (mean 9.1). Tumor ablation was monitored by direct vision and ultrasound. Partial nephrectomy was performed in standard fashion. All specimens were stained with hematoxylin and eosin, and 5 specimens were stained for nicotinamide adenine dinucleotide (NADH) diaphorase activity. RESULTS: Tumors ranged from 1.5 to 3.5 cm. (mean 2.4) in greatest dimension. All 20 specimens had evidence of morphologically unchanged tumor and normal renal parenchyma on standard hematoxylin and eosin staining. Of the 5 specimens 4 stained positively for NADH in areas confirmed to be tumor in hematoxylin and eosin stained neighboring sections. There was 1 intraoperative renal pelvic thermal injury requiring pyeloplasty and 2 postoperative caliceal leaks requiring stent placement. CONCLUSIONS: In our series radio frequency therapy did not result in total tumor destruction when specimens were examined with hematoxylin and eosin or NADH staining. We believe that radio frequency interstitial tumor ablation of renal cell carcinoma without subsequent tissue resection should continue to be an investigational treatment modality for those who would otherwise undergo partial or radical nephrectomy.

Neurogenesis and gliogenesis in the spinal cord of turtles.

A 5-bromo-3'-deoxyuridine (BrdU) pulse administered to juvenile turtles resulted in cell labeling throughout the gray matter (GM) and white matter (WM) of the spinal cord. One and twenty-four hours postinjection, larger densities of BrdU-labeled nuclei (LN) occurred within the GM, with a density peak localized in the central region (CR). Seven days later, density differences between GM and WM disappeared, accompanying a more uniform distribution of LN in the GM (absence of the central peak). Multiple injection experiments also showed similar evolution in the distribution of LN. Morphometric studies revealed that the size of LN had undergone time-related increments: Larger nuclei appeared at protracted fixation time points. Double-labeling experiments indicated that BrdU-labeled cells expressed neuroactive substances, such as gamma-aminobutyric acid (GABA), neuron-specific nuclear protein (NeuN), and the cytoplasmic early postmitotic neuronal marker (TUC-4). Other BrdU-labeled cells expressed the glial-specific protein (GFAP). GABA-BrdU, TUC-4-BrdU, and GFAP-BrdU double-labeled cells were recognized 6 days after the first BrdU injection. NeuN-BrdU double-labeled cells were found at 50 days postinjection. Three-dimensional transmission electron microscopy revealed the presence of synapses and typical kinocilia in putative immature nerve cells. Kinocilia were also found in putative immature glial cells. In consideration of the scattered distribution pattern of BrdU-labeled cells, in animals fixed 1 hour postinjection, the existence of a single proliferating center was discarded. The CR, including the ependymal epithelium, showed the highest density of LN.

Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae.

We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the alpha-ketoglutarate dehydrogenase complex (KGDC). alpha-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.

Distribution and effects of PACAP, VIP, nitric oxide and GABA in the gut of the African clawed frog Xenopus laevis.

The distribution and possible effects on gastrointestinal motility of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide and gamma-amino-butyric acid (GABA) were investigated in the African clawed frog (Xenopus laevis) using immunohistochemistry and in vitro strip preparations. PACAP- and VIP-immunoreactive nerve fibres were common in the myenteric plexus as well as in the longitudinal and circular muscle layers all along the gastrointestinal tract. Double labelling demonstrated a close correlation between PACAP and VIP immunoreactivities, indicating that the two neurotransmitters are colocalised within the enteric nervous system. Occasionally, PACAP- and VIP-positive nerve cell bodies were seen in the myenteric or submucous plexa. In addition, VIP immunoreactivity coexisted with helospectin immunoreactivity. Nitric oxide synthase (NOS)-immunoreactive nerve cells were found in the myenteric plexus at an average density for the whole gastrointestinal tract of 4584 +/- 540 cells cm(-2). The NOS-immunoreactive nerve cells were usually multipolar with an average size of 11.3 +/- 3.7 x 23.2 +/- 6.6 microm. Some NOS-immunoreactive nerve fibres were VIP-immunoreactive but not all VIP-positive fibres showed NOS immunoreactivity. GABA immunoreactivity was found in nerve fibres and nerve cells in the myenteric plexus of all regions of the gut. Few GABA-immunoreactive nerve fibres were VIP-immunoreactive. PACAP 27, VIP, sodium nitroprusside (a nitric oxide donor; NaNP) and GABA caused similar responses on spontaneously contracting circular preparations of the cardiac stomach of X. laevis. The mean force developed was decreased, mainly by a reduction in resting tension, while the amplitude of contractions was not necessarily affected. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) increased the mean force developed, indicating a nitrergic tone in the preparations. In contrast, PACAP 27, VIP, NaNP, GABA and L-NAME had no significant effect on longitudinal strip preparations from the duodenum. These results indicate that PACAP, VIP, nitric oxide and GABA, which are known to be important inhibitory neurotransmitters in other vertebrates, are widely spread in the enteric nervous system of Xenopus laevis and may be involved in the inhibitory control of gastric motility. Although no effect of PACAP, VIP, nitric oxide or GABA on the longitudinal strips of the duodenum was seen in this study, this does not rule out the possibility that they might play an important role in controlling intestinal motility as well.

Does methylene blue protect the kidney tissues from damage induced by ciclosporin A treatment?

Ciclosporin A (CsA) is the first-choice immunosuppressant universally used in allotransplantation and autoimmune diseases. However, it has been demonstrated that this drug produces negative side effects in several organs and in particular in the lymphoid organs and in the kidney. It has been suggested that the CsA causes deleterious effects because it increases the oxygen free radical production. Here we wanted to test whether antioxidants protect the kidney parenchyma from the toxicity induced by CsA. We used methylene blue (MB), because it inhibits the formation of oxygen free radicals. The study was carried out in four groups of Wistar rats. Group I animals were intraperitoneally injected with MB (1 mg/kg/day) for 21 days; group II animals were subcutaneously injected with CsA (15 mg/kg/day) for 21 days; group III animals were treated with CsA combined with MB at the same doses and for the same periods as groups I and II, and group IV animals were injected subcutaneously with olive oil for 21 days as controls. The kidneys and the thymuses were subsequently removed and examined by conventional morphological staining (hematoxylin-eosin and Masson's trichrome) and enzymatic (NADPH-diaphorase, cytochrome, c oxidase, and superoxide anion production) and immunoenzymatic (inducible nitric oxide synthase--iNOS, endothelial nitric oxide synthase--eNOS) techniques. The thymuses were used to check the persistence of CsA-immunosuppressive effects during MB administration. Group I, III, and IV animals showed a normal kidney architecture and low levels of NADPH-diaphorase and of superoxide anion in all structures studied (proximal and distal tubules, glomeruli and the Henle loops). The cytochrome c oxidase showed a strong activity in proximal tubules, a moderate activity in distal tubules, and a weak activity in glomeruli and in the Henle loops. The expression of iNOS was weak in the proximal tubular epithelial cells and negative in the glomeruli, while eNOS was found to be moderately positive in the glomeruli and in the interstitial arteries, but not in the tubules and in the Henle loops. Degenerative changes with tubulointerstitial injury in the cortex of CsA-treated kidneys (group II) and increases of NADPH-diaphorase levels, iNOS activity, and superoxide staining were found in all structures. The expression of eNOS did not change in group I, III and IV animals. MB combined with CsA prevented the degenerative changes caused by CsA, preserving the structural, enzymatic, and immunoenzymatic integrity of the renal parenchyma. The mechanism by which MB exerts its protective action is not yet clear, but it seems to be due to its ability to inhibit xanthine oxidase and to quench nitric oxide production. Moreover, these data have been also supported by the following: (1) the superoxide anion levels were very high after CsA treatment and reduced after CsA-MB treatment, and (2) the iNOS levels increased in CsA-treated rats and showed normal levels after CsA-MB treatment. Moreover we demonstrated that MB administration did no compromise the CsA immunosuppressive effects, since the thymus showed a cytoarchitecture like that observed in CsA-treated rats.

Nitric oxide system is involved in glomerular hyperfiltration in Japanese normo- and micro-albuminuric patients with type 2 diabetes.

Glomerular hyperfiltration plays a pathogenic role in the early stages of diabetic nephropathy. Experimental studies in laboratory animals suggest that nitric oxide (NO) might be involved in the pathogenesis of glomerular hyperfiltration. We performed a cross-sectional study to determine the relationship between diabetic glomerular hyperfiltration and the NO system. Normoalbuminuric (n=41), microalbuminuric (n=25), and macroalbuminuric (n=16) patients with type 2 diabetes were recruited in this study and compared with age-matched 84 non-diabetic control subjects. Creatinine clearance and urinary NO(2)(-)/NO(3)(-) excretion (urinary NOx) were measured, and the expression of endothelial cell nitric oxide synthase (ecNOS) was evaluated in human renal tissues. Glomerular hyperfiltration was present in 19 (37.5%) and nine (36.6%) of normoalbuminuric and microalbuminuric type 2 diabetic patients, respectively. The urinary NOx was significantly higher in normoalbuminuric patients compared with normal subjects. Creatinine clearance correlated significantly with urinary NOx in normoalbuminuric and microalbuminuric patients. Immunohistochemical staining intensities for ecNOS were significantly increased in glomerular endothelial cells of microalbuminuric type 2 diabetic patients as compared with the control subjects. These results suggest that NO may contribute to the pathogenesis of glomerular hyperfiltration in Japanese type 2 diabetic patients.

Standardized measurements and differential spectroscopy in microplates.

Microplates (MPs) are excellent devices for the parallel processing of multiple samples for the spectroscopic analysis of chromophores and turbidity, for luminometric measurements, for cell culture applications, or simply for sample storage, library organization, and other high-throughput (HTP) processes. Disadvantages include an ill-defined pathlength, meniscus formation, evaporation, and cross-contamination. Here, we have developed a novel MP and lid system which can serve to minimize these drawbacks. Cup-like lids are inserted into MP wells. Thereby, liquid is pushed aside. The flat bottoms of the cup-like lids guarantee a planar interface and a defined pathlength. In addition, the devised MP system allows for differential spectroscopic analysis of multiple samples comparable to measurements in tandem cuvettes. This was shown by the investigation of the binding of reduced nicotinamide adenine dinucleotide to dihydrolipoamide dehydrogenase. The MP lid system described offers a low-cost solution for standardized spectrophotometric quantitations in any solvent compatible with the MP/lid material. In addition to the system's suitability for routine MP application, it should be advantageous as a simple and noninvasive method, i.e., no labeling and immobilization of analytes is required for detection of the interaction of molecules, for various HTP applications and drug screening purposes.

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems.

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.