Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Increases in corticospinal responsiveness during a sustained submaximal plantar flexion.

Studying the responsiveness of specific central nervous system pathways to electrical or magnetic stimulation can provide important information regarding fatigue processes in the central nervous system. We investigated the changes in corticospinal responsiveness during a sustained submaximal contraction of the triceps surae. Comparisons were made between the size of motor-evoked potentials (MEPs) elicited by motor cortical stimulation and cervicomedullary motor-evoked potentials (CMEPs) elicited by magnetic stimulation of the descending tracts to determine the site of any change in corticospinal responsiveness. Participants maintained an isometric contraction of triceps surae at 30% of maximal voluntary contraction (MVC) for as long as possible on two occasions. Stimulation was applied to the motor cortex or the cervicomedullary junction at 1-min intervals during contraction until task failure. Peripheral nerve stimulation was also applied to evoke maximal M waves (M(max)) and a superimposed twitch. Additionally, MEPs and CMEPs were evoked during brief contractions at 80%, 90%, and 100% of MVC as a nonfatigue control. During the sustained contractions, MEP amplitude increased significantly in soleus (113%) and medial gastrocnemius (108%) muscles and, at task failure, matched MEP amplitude in the prefatigue MVC ( approximately 20-25% M(max)). In contrast, CMEP amplitude increased significantly in medial gastrocnemius (51%), but not in soleus (63%) muscle and, at task failure, was significantly smaller than during prefatigue MVC (5-6% M(max) vs. 11-13% M(max)). The data indicate that cortical processes contribute substantially to the increase in corticospinal responsiveness during sustained submaximal contraction of triceps surae.

Corticospinal-evoked responses in lower limb muscles during voluntary contractions at varying strengths.

This study investigated corticospinal-evoked responses in lower limb muscles during voluntary contractions at varying strengths. Similar investigations have been made on upper limb muscles, where evoked responses have been shown to increase up to approximately 50% of maximal force and then decline. We elicited motor-evoked potentials (MEPs) and cervicomedullary motor-evoked potentials (CMEPs) in the soleus (Sol) and medial gastrocnemius (MG) muscles using magnetic stimulation over the motor cortex and cervicomedullary junction during voluntary plantar flexions with the torque ranging from 0 to 100% of a maximal voluntary contraction. Differences between the MEP and CMEP were also investigated to assess whether any changes were occurring at the cortical or spinal levels. In both Sol and MG, MEP and CMEP amplitudes [normalized to maximal M wave (Mmax)] showed an increase, followed by a plateau, over the greater part of the contraction range with responses increasing from approximately 0.2 to approximately 6% of Mmax for Sol and from approximately 0.3 to approximately 10% of Mmax for MG. Because both MEPs and CMEPs changed in a similar manner, the observed increase and lack of decrease at high force levels are likely related to underlying changes occurring at the spinal level. The evoked responses in the Sol and MG increase over a greater range of contraction strengths than for upper limb muscles, probably due to differences in the pattern of motor unit recruitment and rate coding for these muscles and the strength of the corticospinal input.

Evidence for reduced efficacy of the Ia-pathway during shortening plantar flexions with increasing effort.

To determine whether the soleus (SOL) H-reflex is modulated during shortening contractions in a manner that has been observed for isometric contractions, SOL H-reflexes and M-waves were elicited via percutaneous electrical stimulation to the tibial nerve at an intensity that evoked an H-reflex at 50% of its maximum in 11 healthy subjects. Paired electrical stimuli were delivered as the ankle angle passed through 90 degrees at an interval of 400 ms while the subject performed shortening contractions at levels of plantar flexion torque ranging between 2 and 30% of that during a maximal voluntary contraction (MVC). H-reflexes were also recorded during the performance of isomeric contractions of plantar flexors at similar levels of plantar flexion torque and at the same joint angle (muscle length) in an additional five healthy subjects. Correlations were examined between the peak-to-peak amplitude of the first H-reflexes, M-waves and plantar flexion torques in both protocols. It was revealed that no significant correlation was found between the SOL H-reflex and increasing plantar flexion torque during shortening contractions (rho = -0.07, P = 0.15), while a strong positive correlation was observed for the isometric conditions (rho = 0.99, P < 0.01). No significant change was observed in the SOL M-wave for either contraction type. Furthermore, the H-reflexes elicited via paired stimuli with the same background activity in voluntary shortening contractions showed almost identical amplitudes, suggesting that the level of homosynaptic post-activation depression did not change in response to the varying levels of activation in voluntary shortening contractions. Therefore, the lack of increase in the H-reflex during shortening contractions at increasing intensities is possibly due to a centrally regulated increase in presynaptic inhibition. Such a downward modulation of the reflex suggests that Ia-excitatory input onto the SOL motoneurone pool needs to be reduced during the performance of shortening contractions.

Application of biological activated carbon anaerobic reactor for treatment of hazardous chemicals.

An expanded-bed anaerobic reactor with granular activated carbon (GAC) medium has been developed to treat wastewaters that contain a high concentration of inhibitory and/or refractory organic compounds as well as readily degradable organic compounds. The process is characterised by a combination of two removal mechanisms; adsorption on GAC and biological degradation by microorganisms grown on GAC. Applicability of the reactor to treatment of phenol, chloroacetaldehyde (CAA), pentachlorophenol (PCP) and tetrachloroethylene (PCE) was discussed based on experimental data. All chemicals focused on here were removed well and stably at a removal efficiency of more than 98% even during starting operation and shock load operation. Chemicals in influent that exceeded biological degradation capacity was initially adsorbed on GAC and then gradually degraded, and hence the adsorptive capacity of GAC was regenerated biologically. These results proved that a biological activated carbon anaerobic reactor was effective for treatment of wastewater containing hazardous chemicals, especially for strongly absorbable chemicals, as well as readily degradable organic compounds at high concentration.

[Mediastinal lymph node metastasis of thyroid cancer occurring 15 years after the initial operation; report of a case].

A 71-year-old woman was found to have an abnormal shadow on a chest X-ray. Fifteen years earlier she had undergone a subtotal thyroidectomy for thyroid cancer without any lymph node metastasis. Chest computed tomography (CT) revealed a mediastinal tumor with full of blood stream. Since the positron emission tomography (PET) disclosed an increased uptake of fluoro-2-deoxy-D-glucose (FDG) in the tumor, a malignant lymphatic tumor was therefore suspected. An immunohistological examination of biopsy specimens taken by thoracoscopic procedure demonstrated tumor to be lymph node metastasis of the previous thyroid cancer. After a tumor resection by means of a thoracotomy and total thyroidectomy, the patient was scheduled to receive radioiodine therapy. The previously reported cases are also herein reviewed.

[The ischemic mitral valve regurgitation due to acute coronary thromboembolism; report of a case].

A 71-year-old man with congestive heart failure due to acute myocardial infarction was referred to our hospital. He was under the support of mechanical ventilation and the intraaortic balloon pumping (IABP) and coronary angiogram revealed the thromboembolism of the obtuse marginal artery. We completed the revascularization by the direct percutaneous coronary intervention. However, grade II mitral valve regurgitation and heart failure were worsening. Mitral valvuloplasty and the modified maze procedure through the partial lower sternotomy were performed. He is still in good condition 4 years later. Ischemic mitral valve regurgitation due to the coronary thromboembolism is very rare. Careful follow-up on the grade of ischemic mitral valve regurgitation is necessary even after the early coronary recanalization. The surgical approach of the partial sternotomy should be used in such a case of acute mitral valve regurgitation.

Monoclinic superstructure of SrMgF4 with perovskite-type slabs.

Crystals of Ce-doped SrMgF4, strontium magnesium tetrafluoride, have been found to have a monoclinic P2(1) structure with doubled a and tripled c cell lengths compared with the orthorhombic Cmcm structure previously reported in the literature. The perovskite-type slabs, composed of corner-sharing MgF6 octahedra and Sr atoms, are stacked along the b axis. The six crystallographically independent MgF6 octahedra are rotated so as to provide long periodicities along a and c. The coordination numbers and bond distances around the six crystallographically independent Sr atoms are slightly different in each case. In the superstructure, the Sr atoms lie on local mirror planes which are thought to originate at the high-temperature phase transition.

DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein.

The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs. It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases. In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule. A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75. AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity. The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity. Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA. In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA. These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription. The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.

Frustrations in polymer conformation in gels and their minimization through molecular imprinting.

We report an experimental realization of a gel system in which frustrations exist and can be minimized, thus meeting two crucial criteria predicted to enable memory of conformations in polymers. The gels consist of a thermosensitive major monomer component and two minor components. One minor component is positively charged and will form complexes around negatively charged target molecules placed in solution. The complexes can be imprinted into the gel by then cross-linking the second minor component, which will form cross-links additional to those in the major polymer matrix. The complexes are destroyed and reformed upon swelling and reshrinking of the gels, showing that memorization has been achieved.

Gel catalysts that switch on and off.

We report development of a polymer gel with a catalytic activity that can be switched on and off when the solvent composition is changed. The gel consists of two species of monomers. The major component, N-isopropylacrylamide, makes the gel swell and shrink in response to a change in composition of ethanol/water mixtures. The minor component, vinylimidazole, which is capable of catalysis, is copolymerized into the gel network. The reaction rate for catalytic hydrolysis of p-nitrophenyl caprylate was small when the gel was swollen. In contrast, when the gel was shrunken, the reaction rate increased 5 times. The activity changes discontinuously as a function of solvent composition, thus the catalysis can be switched on and off by an infinitesimal change in solvent composition. The kinetics of catalysis by the gel in the shrunken state is well described by the Michaelis-Menten formula, indicating that the absorption of the substrate by the hydrophobic environment created by the N-isopropylacrylamide polymer in the shrunken gel is responsible for enhancement of catalytic activity. In the swollen state, the rate vs. active site concentration is linear, indicating that the substrate absorption is not a primary factor determining the kinetics. Catalytic activity of the gel is studied for substrates with various alkyl chain lengths; of those studied the switching effect is most pronounced for p-nitrophenyl caprylate.

Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.

A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels.

Methylglyoxal modification of protein. Chemical and immunochemical characterization of methylglyoxal-arginine adducts.

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction (glycation), reacts with proteins and forms advanced glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct. In addition, we describe the immunochemical study on the MG-arginine adducts using monoclonal antibody directed to MG-modified protein. Upon incubation of Nalpha-acetyl-L-arginine with MG at 37 degrees C, two nonfluorescent products and one fluorescent product were detected as the major products. The nonfluorescent products were identified as the Ndelta-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetrahydropyrimidine moiety (Ndelta-(4-carboxy-4,6-dimethyl-5, 6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2-yl)-L-ornithine). On the basis of the following chemical and spectroscopic evidence, the major fluorescent product, putatively identified as Ndelta-(5-methylimidazolon-2-yl)-L-ornithine (5-methylimidazolone), was found to be identical to Ndelta-(5-hydroxy-4, 6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and high resolution fast atom bombardment-mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and a molecular formula of C10H25O6N4, respectively, which coincided with argpyrimidine; (ii) the 1H NMR spectrum of this product in d6-Me2SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5-methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, as an immunochemical approach to the detection of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major fluorescent product, argpyrimidine, as the dominant epitope. The immunohistochemical analysis of the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.

A novel biomarker for hyperglycemia, MRX isolated from hydrolysate of glycated proteins.

Long-lived proteins can undergo non-enzymatic glycation to form highly crosslinked structures with characteristic fluorescence during aging and diabetes processes. In this paper, a typical fluorophore, named Maillard reaction product X (MRX), was isolated from the hydrolysate of glycated proteins. MRX could be formed by incubation of bovine serum albumin with glucose, followed by acid hydrolysis. The structure of MRX was determined to be 8-hydroxy-5-methyldihydrothiazolo[3,2-alpha] pyridinium-3-carboxylate. MRX was also found to be formed by the incubation of cysteine and arginine with glucose, followed by hydrolysis. We found the formation of MRX in the recently developed genetically diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and compared them with that in the control Long-Evans Tokushima Otsuka (LETO) rats. Significantly higher levels of MRX were observed from the serum (p < 0.005) and urinary protein (p < 0.001) of OLETF rats in comparison with those of LETO rats. MRX must be a potential candidate as a biomarker for hyperglycemia.

Protein modification by a Maillard reaction intermediate methylglyoxal. Immunochemical detection of fluorescent 5-methylimidazolone derivatives in vivo.

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes, is a common intermediate in nonenzymatic glycation (Maillard reaction) in vivo. Here we describe the immunochemical approach to the detection of MG adducts in proteins in vitro and in atherosclerotic lesions of human aorta in vivo. The reaction of protein (bovine serum albumin) with MG led to selective loss of arginine and lysine residues, accompanied by the formation of 5-methylimidazolone (N delta-(5-methylimidazolon-2-yl)ornithine) and imidazolysine (1,3-di-lysino-4-methylimidazole) derivatives, respectively. The anti-5-methylimidazolone antibody was prepared by immunizing rabbits with a MG-keyhole limpet hemocyanin conjugate and purifying the serum on an affinity gel prepared by covalent attachment of the 5-methylimidazolone derivative. The antibody cross-reacted with the proteins treated with not only MG but trioses, such as hydroxyacetone, dihydroxyacetone, and glyceraldehyde. The immunohistochemical analysis revealed that atherosclerotic lesions of human aorta contained 5-methylimidazolone derivatives whose distributions were identical to those of advanced glycation end products (AGEs) detected by the anti-AGE antibody.

Spice constituents scavenging free radicals and inhibiting pentosidine formation in a model system.

Many antioxidants have been found in spices and herbs, and some of them are well known as strong scavengers of active oxygen radicals. We have isolated active products, which markedly inhibited the formation of malondialdehyde (MDA from 2-deoxyribose and the hydroxylation of benzoate with the hydroxyl radical, from methanol extracts of allspice and clove. Pimentol from allspice, and biflorin and its isomer, abbreviated as clove3, from clove were identified as the active principles. These revealed strong activity as hydroxyl radical scavengers at a concentration of 2.0 microM. The antioxidative activities in an in vitro model system involving the rabbit erythrocyte membrane ghost were as strong as those of alpha-tocopherol at 200 microM. Such advanced glycation end products (AGE) as pentosidine are biomarkers of diabetes mellitus, and active oxygens have been suggested to be involved in the formation of AGE. The above-mentioned free radical scavengers effectively inhibited the formation of pentosidine in a model system of N alpha-t-butoxycarbonyl-fructoselysine and N alpha-t-butoxycarbonyl-arginine.

[Hypothermic management of brain dead dogs].

For preventing graft failure, the effects of hypothermic management of brain dead dogs was investigated. Forty-three brain dead dogs were divided into two groups according to the degree of esophageal temperature; a normothermic group (37.2 +/- 0.3 degree C, mean +/- SEM, n = 22), and a hypothermic group (31.8 +/- 0.3 degree C, n = 21) which was obtained by introducing ice slush in the peritoneal cavity. During the management of brain dead dogs, 1) heart rate, pressure product, and a total amount of catecholamine were significantly lower (p < 0.05) in the hypothermic group than in the normothermic group, 2) mean blood pressure, the maximum rate of the rise of left ventricle (LVdp/dt) and cardiac output were not different between both groups, 3) lactate content in the coronary sinus, and O2-extraction rate of the heart tended to be lower in the hypothermic group than in the normothermic group. After transplantation, the recovery of cardiac function was better in the hypothermic group than in the normothermic group. Hypothermic management of brain dead dogs may safely decrease cardiac stress, and keep cardiac aerobic circumstances.

[Superselective local infusion therapy with tissue-plasminogen activator for acute massive pulmonary thromboembolism: preliminary clinical experience].

Three cases of acute massive pulmonary thromboembolism were treated with the superselective infusion of tissue-plasminogen activator. Superselective pulmonary angiography immediately after administration of tissue-plasminogen activator demonstrated angiographic improvement in all patients. No complications were encountered during or after the procedure. It is considered that superselective infusion of tissue-plasminogen activator can be an effective therapy for massive pulmonary thromboembolism.

[Quantitative analyses of glycosaminoglycans in tear fluids in normal human eyes and eyes with corneal epithelial disorders].

The existence and pathophysiological role of glycosaminoglycans in the tear fluid in humans was investigated using quantitative analyses of hyaluronic acid and chondroitin sulfate in the tear fluid. The subjects were 42 eyes of 31 normal controls, 9 eyes of 9 patients with superficial punctate keratitis (SPK), and 13 eyes of 13 patients with epithelial defect. After an instillation of 100 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-de-sac. The glycosaminoglycans in the tears were then treated with chondroitinase ABC to make fractions of unsaturated disaccharides. The quantities of disaccharides were determined by high-performance liquid chromatography. Concentrations were expressed as nanomoles of unsaturated disaccharides per protein in the tears. The concentrations of hyaluronic acid and chondroitin sulfate in the normal controls were 0.07 +/- 0.12(n mol/mg protein) and 6.91 +/- 3.63 (n mol/mg protein), respectively. The mean concentration of hyaluronic acid was significantly higher in patients with epithelial erosion than in normal controls, whereas the mean concentration of chondroitin sulfate was significantly lower in patients with epithelial erosion than in normal controls. There was no significant difference in the concentration of glycosaminoglycans between the patients with SPK and normal controls. The results of our study suggest that glycosaminoglycans are synthesized and endogenously secreted into the tear fluids and, especially in the case of hyaluronic acid, may play an important role in corneal epithelial wound healing in patients with epithelial erosion.

[Quantitative analysis of glycosaminoglycans in tear fluids during corneal epithelial wound healing in rabbits].

We performed quantitative analyses of glycosaminoglycans in the tear fluids in a rabbit wound healing model. We ablated rabbit corneal epithelium with trephine and spatula, and sampled tear fluids before the epithelial ablation, and at 3, 24, 48, and 72 hours after it. After an instillation of 200 microliters saline solution in the conjunctival sac, as much tear fluid as possible was collected from the lower cul-del-sac. The glycosaminoglycans in the tears were then treated with chondoroitinase ABC to make fractions of disaccharides. The quantities of disaccharides were determined with high-performance liquid chromatography as weight per unit protein in the tears. The concentrations of delta Di-HA in the tear fluids at 3 and 24 hours were significantly higher than those before the treatment and returned to the initial value at 72 hours after making the epithelial wound. Among the disaccharides of chondroitin sulfate, delta Di-0S and delta Di-6S showed a significant increase at 3 hours after the treatment but delta Di-4S did not show any significant variation. The results suggest that the glycosaminoglycans in the rabbit tear fluids may play an important role in the corneal epithelial wound healing process.