Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites.

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.

Cough and sputum in long COVID are associated with severe acute COVID-19: a Japanese cohort study.

BACKGROUND: Multiple prolonged symptoms are observed in patients who recover from acute coronavirus disease 2019 (COVID-19), defined as long COVID. Cough and sputum are presented by patients with long COVID during the acute and post-acute phases. This study aimed to identify specific risk factors for cough and sputum in patients with long COVID. METHODS: Hospitalized patients with COVID-19 aged 18 years were enrolled in a multicenter cohort study at 26 medical institutions. Clinical data during hospitalization and patient-reported outcomes after discharge were collected from medical records, paper-based questionnaires, and smartphone apps. RESULTS: At the 3-, 6-, and 12-month follow-ups, there were no differences in the incidence rates of wet and dry coughs. In contrast, the proportion of patients presenting sputum without coughing increased over time compared to those with sputum and coughing. Univariate analyses of cough and sputum at all follow-up visits identified intermittent mandatory ventilation (IMV), smoking, and older age as risk factors for prolonged symptoms. At the 12-month follow-up, persistent cough and sputum were associated with the characteristics of severe COVID-19 based on imaging findings, renal and liver dysfunction, pulmonary thromboembolism, and higher serum levels of LDH, KL-6, and HbA1C. The Kaplan-Meier curves showed that the severity of acute COVID-19 infection was correlated with prolonged cough and sputum production. Multivariable logistic regression analysis showed that IMV ventilator management were independent risk factors for prolonged cough and sputum at 12 months. CONCLUSIONS: In a Japanese population with long COVID, prolonged cough and sputum production were closely associated with severe COVID-19. These findings emphasize that a preventive approach including appropriate vaccination and contact precaution and further development of therapeutic drugs for COVID-19 are highly recommended for patients with risk factors for severe infection to avoid persistent respiratory symptoms.

Glycogen debranching pathway deduced from substrate specificity of glycogen debranching enzyme.

Glycogen debranching enzyme (GDE) is bifunctional in that it exhibits both 4-α-glucanotransferase and amylo-α-1,6-glucosidase activity at two distinct catalytic sites. GDE converts the phosphorylase-limit biantennary branch [G-G-G-G-(G-G-G-G↔)G-G- residue, where G = D-glucose, hyphens represent α-1,4-glycosidic bonds, and the double-headed arrow represents an α-1,6-glycosidic bond] into a linear maltooligosyl residue, which is then subjected to phosphorylase, and glycogen degradation continues. The prevailing hypothesis regarding the glycogen debranching pathway was that 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch into the G-G-G-G-G-G-G-(G↔)G-G- residue and amylo-α-1,6-glucosidase cleaves the remaining α-1,6-linked G residue. In the present study, we analyzed the substrate specificities of 4-α-glucanotransferase and amylo-α-1,6-glucosidase using fluorogenic biantennary dextrins such as G-G-G-G-(G-G-G-G↔)G-G-GPA (F4/4/2; where GPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol), G-(G-G-G-G↔)G-G-GPA (F1/4/2), and G-G-G-G-G-G-G-(G↔)G-G-GPA (F7/1/2). Contrary to the prevailing hypothesis, the main branch of F4/4/2 was an important donor substrate component of 4-α-glucanotransferase and did not serve as an acceptor substrate. However, when G-G-G-G-G-GPA was added to the mixture, it successfully accepted a maltotriosyl (G3-) residue from F4/4/2. In addition, amylo-α-1,6-glucosidase exhibited strong activity towards G-G-G-G-(G↔)G-G-GPA but weak activity towards F7/1/2. Furthermore, the debranching activity of GDE towards phosphorylase-limit glycogen substantially increased when methyl α-maltooligosides with lengths equal to or greater than that of methyl α-maltopentaoside (G5-OCH3) were added to the enzyme reaction mixture. Based on these results, we propose the following macroscopic debranching pathway: Via 4-α-glucanotransferase, the G3- residue of the donor branch is transferred to a long (n ≥ 5) linear Gn- residue linked to a different branching G residue.

A frameshift variant in the EXT1 gene in a feline leukemia virus-negative cat with osteochondromatosis.

Osteochondromatosis is a benign proliferative disorder characterized by cartilage-capped bony protuberances. In humans and most mammals, variants in the EXT1 or EXT2 gene are strongly correlated with the etiology of osteochondromatosis. However, in cats, osteochondromatosis has only been associated with feline leukemia virus infection. In this study, to explore other factors involved in the etiology of feline osteochondromatosis, we examined the EXT1 and EXT2 genes in a feline leukemia virus-negative cat with osteochondromatosis. Genetic analysis revealed a heterozygous single base pair duplication in exon 6 of the EXT1 gene (XM_023248762.2:c.1468dupC), leading to a premature stop codon in the EXT1 protein. Notably, this frameshift variant is recognized as one of the most common pathogenic variants in human osteochondromatosis. Our data suggest for the first time that genetic variants can have etiologic roles in osteochondromatosis in cats, as in humans and other animals.

New approach to prepare fluorogenic branched dextrins for assaying glycogen debranching enzyme.

Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA (B4/84, where Glc = D-glucose and GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1-6Glcα1-4Glcα1-4GlcPA (B3/31) was prepared from commercially available Glcα1-6Glcα1-4Glcα1-4Glc. Using α-cyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase.

A new interpretation of sulfate activation of rabbit muscle glycogen phosphorylase.

It is widely known that sulfate ion at high concentration serves like an allosteric activator of glycogen phosphorylase (GP). Based on the crystallographic studies on GP, it has been assumed that the sulfate ion is bound close to the phosphorylatable Ser14 site of nonactivated GP, causing a conformational change to catalytically-active GP. However, there are also reports that sulfate ion inhibits allosterically-activated GP by preventing the phosphate substrate from attaching to the catalytic site. In the present study, using a high concentration of sulfate ion, significant enhancement of GP activity was observed when macromolecular glycogen was used as substrate but not when smaller maltohexaose was used. In glycogen solution, nonreducing-end glucose residues are localized on the surface of glycogen and are not distributed homogenously in the solution. Using cyclodextrin-immobilized column chromatography, we found that sulfate at high concentration promoted GP-dextrin binding through the dextrin-binding site (DBS) located away from the catalytic site. This result is consistent with the properties of the DBSs found in glycogen-debranching enzyme and ß-amylase. Therefore, we propose a new interpretation of the sulfate activation of GP, wherein sulfate ions at high concentration promote glycogen-binding to the DBS directly, and glycogen-binding to the catalytic site indirectly. Our findings were successfully applied to the affinity purification of porcine brain GP.

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives.

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

Premature diagnosis of calciphylaxis without pathological indications finally diagnosed as cutaneous small-vessel vasculitis: a post-mortem case report.

A man in his fifties with diabetes had a past history of myocardial infarction and ventricular septal perforation. He underwent hemodialysis about a year ago and was taking amiodarone. He presented with sores and purpura on the lower limbs.-Skin biopsy showed immunofluorescence-negative leukocytoclastic vasculitis. Skin lesions were treated with ointments, which ameliorated the symptoms to some extent, but ulceration relapsed and deteriorated in both number and size. Calciphylaxis was suspected, and a second skin biopsy was performed. No calcium detection,on the arteries was observed, but leukocytoclastic vasculitis was seen. Antineutrophil cytoplasmic antibody-related vasculitis, cryoglobulin vasculitis, or anti-phospholipid syndrome were ruled out by negative findings for autoantibodies. Although he was treated with 30 mg prednisolone, his systemic condition deteriorated, and he died of disseminated intravascular coagulation. Autopsy findings showed no vasculitis in the lung, kidney or intestine, and perimyocardial patch infection was observed.Although calciphylaxis was clinically suspected, his condition was diagnosed finally as cutaneous small-vessel vasculitis.

Essential dextrin structure as donor substrate for 4-α-glucanotransferase in glycogen debranching enzyme.

Glycogen debranching enzyme is a single polypeptide with distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. To allow phosphorylase to degrade the inner tiers of highly branched glycogen, 4-α-glucanotransferase converts the phosphorylase-limit biantennary branch G-G-G-G-(G-G-G-G↔)G-G- (G: d-glucose, hyphens: α-1,4-linkages; double-headed arrow: α-1,6-linkage) into the G-G-G-G-(G↔)G-G- residue, which is then subjected to amylo-α-1,6-glucosidase to release the remaining G↔ residue. However, while the essential side-chain structure of the 4-α-glucanotransferase donor substrate has been determined to be the G-G-G-G↔ residue (Watanabe, Y., et al. (2008) J. Biochem.143, 435-440), its essential main-chain structure remains to be investigated. In this study, we probed the 4-α-glucanotransferase donor-binding region using novel fluorogenic dextrins Gm-(G4↔)G-Gn-F (F: 1-deoxy-1-[(2-pyridyl)amino]-d-glucitol) and maltohexaose (G6) as the donor and acceptor substrates, respectively. 4-α-Glucanotransferase exhibited maximum activity towards G4-(G4↔)G-F and G4-(G4↔)G-G-F, indicating that recognition of the G4-(G4↔)G-moiety was essential for full enzyme function. Notably, when the 4-α-glucanotransferase activity towards G4-(G4↔)G-G-F was taken as unity, those towards nonbranching dextrins were < 0.001. This indicated that the disproportionation activities towards maltooligosaccharides (Gm) are abnormal behaviours of 4-α-glucanotransferase. Notably, however, these activities have been traditionally measured to identify the 4-α-glucanotransferase mutations causing glycogen storage disease type III. This study provides a basis for more accurate identification.

Hilar lymphadenopathy, development of tubulointerstitial nephritis, and dense deposit disease following Pfizer-BioNTech COVID-19 vaccination.

Despite the reports on glomerulonephritis associated with COVID-19 mRNA vaccines, no study has reported about the dense deposit disease (DDD). Here, we present a case of hilar lymphadenopathy after the COVID-19 mRNA vaccination, following which the patient developed tubulointerstitial nephritis (TIN) and DDD. A 74-year-old man received his second dose of mRNA vaccine, and on the next day, he developed fever, urticaria, and dyspnea. On further examination, he had pleural effusion and right hilar lymphadenopathies, which were improved with conservative therapy. After 48 days of the second vaccination, he developed renal dysfunction and new-onset hematuria. Light microscopy findings by renal biopsy revealed apparent mesangial cell proliferation, increased mesangial matrix in the glomeruli, and diffuse inflammatory cell infiltration in the interstitium. Immunofluorescence analysis revealed 1 + positive results for IgG and IgM, negative results for IgA, and 2 + positive results for C3 with a garland pattern on the capillary walls. Electron microscopy revealed that severe cell proliferation in the capillary rumen, and continuous, thickened, and highly dark-stained spotty dense deposits in the glomerular basement membrane; and noncontinuous spotty dense deposits in the tubular basement membrane. Based on the decrease in C3 and pathological findings, TIN accompanied with DDD was diagnosed. The mRNA vaccine might have contributed to the development of lymphadenopathies, TIN, and DDD in this case. Moreover, TIN and DDD might be associated with the activated alternative pathway induced by the mRNA vaccine.

Short-term effects of roxadustat on serum copper and iron changes in a peritoneal dialysis patient.

Dysregulation in total body copper causes severe complications and excess copper can be toxic. Divalent metal transporter 1, duodenal cytochrome B, and copper transporter ATPase7A are included in the many intestinal genes transactivated by HlF-α. On July X, 2022 an 80-year-old female patient on peritoneal dialysis was prescribed roxadustat 100 mg, because darbepoetin was unable to increase hemoglobin level effectively. On the same day, icodextrin 1 L was initiated to mitigate edema. Laboratory data showed hemoglobin 9.1 g/dL, transferrin saturation 77%, copper 123 µg/dL, and iron 170 µg/dL before changing to roxadustat. The patient visited us 6 days after the change because of the appetite loss. Transferrin saturation and serum copper and iron levels increased to 90%, 170 and 203 µg/dL, respectively, which were decreased or normalized after discontinuing roxadustat and icodextrin, suggesting that even short-term roxadustat administration can influence copper levels as well as iron levels. Excess copper and iron levels during roxadustat treatment do not immediately equate with toxicity, but indicate a physiological compensation or transient imbalance of metabolism especially in patients treated with ferric citrate. Further investigation for the hypoxia-inducible factor-prolyl hydroxylase inhibitors effects on iron and copper metabolisms is needed. Determining the short-term effect of roxadustat on serum copper and iron in only this case is impossible. Therefore, further accumulation of similar cases is necessary to clarify the short-term effects of roxadustat on serum copper and iron.

Role of sphingosine-1-phosphate inß-adrenoceptor desensitization via Ca(2+) sensitization in airway smooth muscle.

BACKGROUND: The correlation between inflammatory cells and airway smooth muscle plays fundamental roles in the pathophysiology of asthma. This study was designed to determine whether pre-exposure of airway smooth muscle to sphingosine-1-phosphate (S1P), which is released from mast cells by allergic reactions, causes a deterioration of ß-adrenoceptor function. METHODS: Isometric tension and the ratio of fluorescence intensities at 340 and 380 nm (F(340)/F(380)), an indicator of intracellular Ca2+ levels, were simultaneously measured using fura-2 loaded guinea-pig tracheal tissues. Intracellular cAMP levels were also measured. RESULTS: Pre-exposure to S1P caused a reduction in the inhibitory effects of 0.3µM isoprenaline, a ß-adrenoceptor agonist, and 10µM forskolin, a direct activator of adenylyl cyclase, against 1µM methacholine-induced contraction in concentration- and time- dependent manners. In contrast, the values of F(340)/F(380) were not augmented under this experimental condition. After incubation with S1P in the presence of 0.001-1µM Y-27632, a Rho-kinase inhibitor, the reduced responsiveness to forskolin induced by S1P was reversed in a concentration-dependent manner. Moreover, pre-treatment with pertussis toxin (PTX), an inhibitor of G(i), suppressed the loss of forskolin-induced relaxation induced by S1P. Pre-exposure to S1P markedly inhibited the augmentation of cAMP accumulation induced by forskolin. However, addition of Y-27632 and pre-exposure to PTX returned forsokin-induced cAMP accumulation to the control level. CONCLUSIONS: Pre-exposure to S1P causes heterologus desensitization of ß-adrenoceptors by increasing the sensitivity of airway smooth muscle to intracellular Ca2+. Ca2+ sensitization regulated by G(i) and Rho-kinase is involved in this phenomenon.

Four Cases of Serum Copper Excess in Patients with Renal Anemia Receiving a Hypoxia-Inducible Factor-Prolyl Hydroxylase Inhibitor: A Possible Safety Concern.

Copper is an indispensable trace metal element and is mainly absorbed in the stomach and small intestine and excreted into the bile. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have emerged as a novel approach for renal anemia management. Many intestinal genes, including divalent metal transporter 1, duodenal cytochrome B, and copper transporter ATPase7A, related to iron absorption are transactivated by HlF-α, during iron deficiency. We first report 4 cases of patients with renal anemia who showed excess in serum copper level during roxadustat or daprodustat treatment, which were decreased to the normal level after discontinuing HIF-PHIs and changing the drug to darbepoetin alfa, suggesting that HIF-PHI is associated with serum copper excess. HIF-PHI modulates iron metabolism, such as iron absorption, sequestration, and mobilization, and may increase serum copper levels by increasing copper absorption and/or redistribution of copper in tissues. Therefore, it is urgent to examine the correlation between HIF-PHI use and serum copper levels because copper excess might be involved in several acute or chronic adverse events.

A case of a hemodialysis patient with secondary hyperparathyroidism who was resistant to etelcalcetide treatment but not to cinacalcet hydrochloride.

Although both cinacalcet and etelcalcetide are calcimimetics that directly inhibit parathyroid hormone (PTH) secretion by activating the calcium (Ca)-sensing receptor (CaSR), their binding sites are different. We report a first case of a hemodialysis (HD) patient with secondary hyperparathyroidism (SHPT), in whom cinacalcet, but not etelcalcetide, could reduce serum intact PTH (i-PTH) levels. A HD patient received total parathyroidectomy (PTx) with auto-transplantation 16 years earlier. Due to SHPT relapse, cinacalcet was started at 7 years after PTx. His i-PTH levels had been controlled with both 75-100 mg of cinacalcet and 4.5 µg/week of calcitriol for a year before switching from cinacalcet to etelcalcetide. At 1 month following the switch, his serum i-PTH level increased to 716 pg/mL. The dose of etelcalcetide was gradually increased and finally reached the maximal dose of 45 mg/week. Because even the maximal dose of etelcalcetide for > 4 months did not reduce his serum i-PTH levels to < 700 pg/mL, etelcalcetide was switched to 50 mg/day of cinacalcet, which reduced the levels to 208 pg/mL at 2 months after the switch. Genomic sequencing test using whole blood revealed no mutation in the portion including Cys 482 of CaSR gene. The patient was resistant to etelcalcetide treatment but not to cinacalcet, suggesting the possibility that the enlarged parathyroid gland has some change in the portion including Cys 482 in the CaSR gene. Therefore, considering the possibility of etelcalcetide resistance during SHPT treatment should be kept in mind.

Epidermal growth factor receptor mutation-positive advanced lung adenocarcinoma presenting with acute respiratory failure diagnosed by thin bronchoscope through transnasal route under high-concentration oxygen mask.

A 59-year-old woman complained of continuous dyspnea. Computed tomography revealed multiple pulmonary nodules, mildly small enlarged mediastinal lymph nodes and a nodule in the liver segment 8. Her dyspnea worsened with respiratory failure 4 days after presentation. Liver biopsy was not possible as she could not hold her breath; thus, we performed bronchoscopy. For biopsy, the pulmonary nodules with a positive bronchus sign were preferred over the mildly small enlarged mediastinal lymph nodes. Bronchoscopy under non-invasive positive pressure ventilation (NPPV) or high-flow nasal cannula (HFNC) was impossible because of the lack of equipment. Therefore, we biopsied via thin bronchoscope through nasal cavity under a high-concentration oxygen mask. Pathological findings revealed epidermal growth factor receptor mutation-positive lung adenocarcinoma. For patients with respiratory failure who cannot undergo bronchoscopy under NPPV or HFNC, thin bronchoscopy through the nasal cavity under a high-concentration oxygen mask may be clinically useful to prevent hypoxaemia during the procedure.

Biochemical evaluation of processed ascites in patients undergoing cell-free and concentrated ascites reinfusion therapy.

The biochemical composition of processed ascites is not well researched and may differ among institutions. This prospective study was conducted to evaluate the biochemical characteristics of processed ascites of 11 patients with liver cirrhosis and carcinoma who underwent cell-free and concentrated ascites reinfusion therapy. The ascites due to carcinoma were more acidic and had higher lactate dehydrogenase activity than those due to liver cirrhosis. The ascites due to liver cirrhosis contained a higher amount of immunoglobulin than those due to carcinoma. Immunoglobulin preparations were approximately 2.95% IgG in liver cirrhosis ascites and 2.25% IgG in carcinoma ascites. Moreover, the concern about IgA infusion in the patient with IgA deficiency made it important to identify the source of the ascites. The present study provided fundamental information regarding the safety of cell-free and concentrated ascites reinfusion therapy.

Donor substrate specificity of 4-alpha-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching.

Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained.

Role of Ca(2+) mobilization in desensitization of beta-adrenoceptors by platelet-derived growth factor in airway smooth muscle.

Platelet-derived growth factor (PDGF), which is released from eosinophils and fibroblasts, may be implicated in the pathophysiology of bronchial asthma. To examine the involvement of airway inflammation in beta-adrenergic desensitization, the present study was designed to determine whether pre-exposure to PDGF deteriorates beta-adrenoceptor function in airway smooth muscle. We focused on Ca(2+) signaling as an intracellular mechanism involved in this phenomenon. Isometric tension and F(340)/F(380) (an indicator of intracellular Ca(2+) concentration) induced by isoprenaline and other cAMP-related agents were simultaneously measured before and after exposure to PDGF in fura-2-loaded guinea-pig tracheal smooth muscle. Indomethacin was applied throughout the experiments to abolish prostaglandin synthesis by PDGF. After exposure of the tissues to 10 ng/ml PDGF for 15 min, the effects of isoprenaline, a beta-adrenoceptor agonist, and forskolin, a direct inhibitor of adenylyl cyclase, against methacholine-induced contraction were markedly reduced with increasing F(340)/F(380). However, in the presence of verapamil, an inhibitor of voltage-dependent Ca(2+) channels, the reduced responsiveness to isoprenaline and forskolin induced by pre-exposure to PDGF was reversed with reducing F(340)/F(380). Reduced responsiveness to isoprenaline by PDGF was also not observed in the presence of Ca(2+)-free solution. The inhibitory effects of db-cAMP, an analogue of cAMP, and theophylline, a nonselective inhibitor of phosphodiesterase, were not attenuated by PDGF. In conclusion, pre-exposure to PDGF causes impairment of the beta-adrenoceptors/adenylyl cyclase processes in airway smooth muscle that is independent of cyclooxygenase synthesis by PDGF. Ca(2+) mobilization by Ca(2+) influx through voltage-dependent Ca(2+) channels is involved in this heterologous desensitization of beta-adrenoceptors.

Atypical Hemolytic Uremic Syndrome Associated with Complement Factor H Mutation and IgA Nephropathy: A Case Report Successfully Treated with Eculizumab.

We present a rare case of IgA nephropathy in a patient who developed atypical hemolytic uremic syndrome (aHUS) associated with a complement factor H (CFH) gene mutation, and who was successfully treated with eculizmab. A 76-year-old man was admitted as the patients had thrombotic microangiopathies findings. The patient was treated with plasma exchange, hemodialysis and methylprednisolone. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 level was not decreased. Light microscopy findings were consistent with hemolytic uremic syndrome and immunofluorescence analysis revealed IgA and C3 were detected. Genetic analysis revealed that mutation of p.Arg1215Gln in CFH was identified. The diagnosis of aHUS was confirmed and eculizmab therapy was currently effective for 5 months.

Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides.

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.