RESUMEN
d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.
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Bacillus subtilis , Racemasas y Epimerasas , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Fructosa/metabolismo , CetosasRESUMEN
PURPOSE: In our previous study, we constructed a one-pot multi-enzyme system for rare ketoses synthesis based on L-rhamnulose-1-phosphate aldolase (RhaD) from accessible glycerol in vitro. To eliminate tedious purification of enzymes, a facile Escherichia coli whole-cell cascade platform was established in this study. METHODS: To enhance the conversion rate, the reaction conditions, substrate concentrations and expressions of related enzymes were extensively optimized. RESULTS: The biosynthetic route for the cascade synthesis of rare ketoses in whole cells was successfully constructed and three rare ketoses including D-allulose, D-sorbose and L-fructose were produced using glycerol and D/L-glyceraldehyde (GA). Under optimized conditions, the conversion rates of rare ketoses were 85.0% and 93.0% using D-GA and L-GA as the receptor, respectively. Furthermore, alditol oxidase (AldO) was introduced to the whole-cell system to generate D-GA from glycerol, and the total production yield of D-sorbose and D-allulose was 8.2 g l-1 only from the sole carbon source glycerol. CONCLUSION: This study demonstrates a feasible and cost-efficient method for rare sugars synthesis and can also be applied to the green synthesis of other value-added chemicals from glycerol.
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Cetosas , Sorbosa , Sorbosa/química , Glicerol/metabolismo , Gliceraldehído/química , Gliceraldehído/metabolismoRESUMEN
Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: ⢠Transketolase catalyzes the formation of a 7-carbon sugar phosphate ⢠Structural and biochemical characterizations of thermophilic transketolase were done ⢠The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.
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Thermus thermophilus , Transcetolasa , Transcetolasa/química , Transcetolasa/metabolismo , Ribosa , Monosacáridos , Fosfatos , Cetosas , CarbonoRESUMEN
It is vitally important to characterize polysaccharides by monosaccharide composition method. In this study, a direct acetylation strategy combined with reversed-phase liquid chromatography electrospray tandem multiple reaction monitoring mass spectrometry (RPLC-ESI-MRM-MS) was developed for simultaneous determination of 8 aldoses (Glc, Gal, Man, Ara, Xyl, Rib, Rha and Fuc), a ketose (Fru), 2 alditols (Glc-ol and Man-ol) and 2 uronic acids (GlcA and GalA) on a high-pressure resistant reversed-phase column. Employing 1-MeIm as catalyst for direct acetylation, even though no DMSO was used to inhibit the transformation of configurations, each carbohydrate still produced a single chromatographic peak in RPLC conditions due to the É- and ß- isomers merged together. Except for Fru and Man, all the other 11 carbohydrates were base-line separated in a 1.7 µm CYANO column. Therefore, correction factor method is further proposed to perfectly solve co-elution problem of Fru and Man because of occurrence of a specific Q3 ion for aldoses rather than ketose. The result was verified on a 1.7 µm Fluoro-Phenyl column with a full separation of Fru and Man. Herein, the established direct acetylation as followed RPLC-ESI-MRM-MS method was successfully applied for compositional analysis of complex polysaccharides from edible plants and fungi.
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Cromatografía de Fase Inversa , Plantas Comestibles , Humanos , Acetilación , Polisacáridos/química , Monosacáridos/análisis , Espectrometría de Masas en Tándem/métodos , Carbohidratos , Hongos , Cetosas , Cromatografía Líquida de Alta PresiónRESUMEN
UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD+ and rotation of a decarboxylation-prone ß-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions.
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Racemasas y Epimerasas , Uridina Difosfato Ácido Glucurónico , Uridina Difosfato Ácido Glucurónico/metabolismo , Descarboxilación , Rotación , Ácido Glucurónico , Racemasas y Epimerasas/metabolismo , Cetosas , NAD/químicaRESUMEN
Non-ketotic hyperglycemia (NKH) is associated with a spectrum of symptoms and radiographic findings due to poorly-controlled diabetes mellitus. These lesions, which predominantly affect the parieto-occipital cortex, are commonly missed by neurologists and neuroradiologists due to their subtle hypointense appearance on T2-based imaging. We report four atypical cases of this syndrome to highlight its subtle, protean presentation in order to aid timely diagnosis. Based on our institutional case series, we describe four cases of NKH with atypical presentation and lesion burden affecting the anterior cortex. We review the clinical presentations, laboratory abnormalities, neuroimaging, and corresponding electroencephalography. Four patients with atypical NKH were characterized in our series. Presenting symptoms ranged from rhythmic hand-tapping to generalized tonic-clonic status epilepticus. Laboratory values were notable for marked hyperglycemia (range: 447 - 627 mg/dL), mild pseudo-hyponatremia (range: 127 - 136 mmol/L), and elevated hemoglobin A1C levels (range: 10.9 - 16.1%). All patients were found to have the classically described pattern of T2-based hypointensity; three with atypical distributions involving the "anterior" cortex. These lesions corresponded to the electrographic nidus of seizure burden. During follow-up, both seizures and T2-based hypointensity resolved within weeks of serum glucose normalization. Our series of four NKH patients with atypical findings of T2-based signal abnormalities expands the clinico-radiographic phenotype revealing a more protean distribution than previously described. Knowledge of these atypical imaging features will aid both the neurologist and radiologist in timely diagnosis and care of these patients.
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Epilepsia , Hiperglucemia , Electroencefalografía , Epilepsia/complicaciones , Glucosa , Hemoglobina Glucada , Humanos , Hiperglucemia/complicaciones , Cetosas , Fenotipo , Convulsiones/diagnósticoRESUMEN
Introduction: Epileptic seizures during non-ketotic hyperglycemia (NKH) represent a rare complication of uncontrolled diabetes mellitus. The definition associates a blood sugar level > 200mg/dL (11mmol/L), hyperosmolality, absence of ketosis, dehydration and seizure control after normalization of blood sugar levels. Material and methods: This retrospective observational study included patients hospitalized for epileptic seizures and NKH in the Cayenne Hospital Center between January 2010 and June 2020. The clinical, biological, and radiological results were collected. Results: 18 out of 228 (7.9%) patients with both diabetes and epileptic seizures had NKH. The mean age of the 12 women and 6 men was 64.8 years. In 8 patients, brain imaging did not show acute lesions and the seizures disappeared with control of hyperglycemia by hydration and insulin. In 6 patients, the seizures revealed a stroke, hemorrhagic in 4 cases, ischemic in 2 cases. 4 patients had a seizure in a context of known vascular epilepsy. The epileptic seizures were mainly focal seizures with motor symptoms that could be repeated, focal to bilateral tonic-clonic or focal status. Conclusion: Seizures in NKH are symptomatic of an acute brain lesion or vascular epilepsy more than 1 in 2 times. However, isolated NKH can cause seizures with a suggestive brain MRI.
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Epilepsia , Hiperglucemia , Cetosis , Glucemia , Electroencefalografía/efectos adversos , Femenino , Guyana Francesa , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/diagnóstico , Cetosas , Cetosis/complicaciones , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Convulsiones/complicacionesRESUMEN
Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of ß-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 µM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 µM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.
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Cetosis , Enfermedades de los Roedores , Femenino , Bovinos , Animales , Ratones , Ácido 3-Hidroxibutírico , Diacilglicerol O-Acetiltransferasa/metabolismo , Estrés del Retículo Endoplásmico , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Cetosas/metabolismo , Cetosas/farmacología , ARN Interferente Pequeño/metabolismo , Interleucina-6/metabolismo , Cetosis/veterinaria , Tejido Adiposo/metabolismo , Citocinas/metabolismo , Inflamación/veterinaria , Inflamación/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Choque Térmico/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Epinefrina/farmacología , ARN Mensajero/metabolismo , Inositol/metabolismo , Inositol/farmacología , Enfermedades de los Roedores/metabolismoRESUMEN
The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2-C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C-C bond-forming enzymes.
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Cetosas , Xilulosa , Antibacterianos , Bacterias/metabolismo , Gliceraldehído 3-Fosfato/metabolismo , Fosfatos , Tiamina Pirofosfato/metabolismo , Transferasas/metabolismoRESUMEN
l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production.
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Isomerasas Aldosa-Cetosa , Thermoanaerobacterium , Isomerasas Aldosa-Cetosa/metabolismo , Aminoácidos , Fructosa/metabolismo , Glucosa/metabolismo , Cetosas , Racemasas y Epimerasas/metabolismo , Ramnosa/metabolismo , Especificidad por Sustrato , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismoRESUMEN
Spatially-resolved profiling of tissue monosaccharides not only gives an insight into the spatial heterogeneity of monosaccharides, but also helps to decipher the possible roles of monosaccharides in biological processes. Here, we develop an on-tissue derivatization method, coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to image and quantify the aldose and ketose isomers of monosaccharide in biological tissues. A new derivatization reagent, 1-naphthaleneacethydrazide (NAH) was synthesized for the on-tissue derivatization of monosaccharides, and it significantly enhanced the imaging sensitivity of monosaccharides. Moreover, the NAH-derivatized aldose and ketose can generate isomer-specific diagnostic ions during MALDI-MS/MS analysis, and thus paves way for the isomer-specific MS imaging of aldose and ketose monosaccharides. On this basis, we further constructed a quantitative MALDI-MS imaging model based on isomer-specific diagnostic ions, and calculated the expression contents of aldose and ketose monosaccharide isomers in different tissue regions of carrot section. We expect that the development of this method should provide more precise view on the spatial distributions and contents of different monosaccharides in heterogeneous biological tissues.
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Monosacáridos , Espectrometría de Masas en Tándem , Isomerismo , Cetosas , Monosacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Diabetic striatopathy is a rare neurological manifestation of nonketotic hyperglycemia that presents with contralateral hemichorea-hemiballismus. Presentation with concurrent seizures is rarely reported. CLINICAL PRESENTATION: We report a case of diabetic striatopathy presenting with focal and generalized tonic-clonic seizures (GTCS) with right hemichorea-hemiballismus induced by a ketotic hyperglycemic state. Head MRI showed high T1-weighted signal intensity in the left lentiform nucleus with no significant diffusion restriction or postcontrast enhancement. The patient's condition gradually improved, with seizure control on AEDs. Hemichorea-hemiballismus significantly improved with adequate blood sugar control and resolved with low-dose haloperidol. CONCLUSIONS: Diabetic striatopathy presenting with hemichorea-hemiballismus and concurrent GTCS has been reported previously in two cases; however, it has never been reported in ketotic hyperglycemia. To the best of our knowledge, we herein report the first case report of focal and generalized seizures in a ketotic hyperglycemic state and mesial temporal sclerosis.
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Corea , Diabetes Mellitus , Discinesias , Hiperglucemia , Corea/diagnóstico por imagen , Corea/tratamiento farmacológico , Corea/etiología , Discinesias/etiología , Humanos , Hiperglucemia/complicaciones , Cetosas , Convulsiones/complicacionesRESUMEN
Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of ß-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)-a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction-was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.
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Enfermedades de los Bovinos , Cetosis , Ácido 3-Hidroxibutírico , Animales , Miosinas Cardíacas , Bovinos , Femenino , Cetosas , Cetosis/veterinaria , Lactancia , Cadenas Ligeras de Miosina , Neutrófilos , Proteína Quinasa C , Transducción de SeñalRESUMEN
Background: Ketosis is one of the most critical metabolic disorders that occur in dairy cows after parturition due to negative energy balance around calving. Aim: The study evaluated a specific therapeutic regimen of ketosis in Holstein dairy cattle by using the combination therapy including hormones, corticosteroids, propylene glycol, and vitamin B12 as well as the use of milk yield rates, insulin, insulin sensitivity, and abomasal functions monitors as diagnostic biomarkers for the recovery of ketotic cows either pre-therapy (0 days) or post-therapy (7 and 14 days). Methods: This study was conducted on ketotic cattle (n = 20) belonged to different dairy farms in Cairo and Giza governorates, Egypt. The diseased cows were undergoing clinical and biochemical investigations for the estimation of serum insulin. Quantitative Insulin Sensitivity Check Index (RQUICKI) and abomasal functions monitor mainly serum levels of gastrin, pepsinogen, and chloride. Results: The milk production rates, cost: benefit analysis ratio, and benefit of the dairy farm in ketotic animals were significantly increased post-treatment. An improvement of insulin sensitivity was stated as serum insulin, and RQUICKI were remarkably increased in post-therapeutic ketotic cows. Monitors of the abomasal function revealed abomasal functions improvement through the significant elevation of blood gastrin and a substantial reduction in serum pepsinogen due to treatment. Conclusion: The study revealed high efficacy of the applied therapeutic strategy regime. It led to a high recovery rate and a very low relapse rate for ketosis. An improvement in milk yield rates, insulin sensitivity, and abomasal function monitors was reported. Hypoinsulinaemia was still reported, however, serum insulin was improved.
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Enfermedades de los Bovinos , Resistencia a la Insulina , Cetosis , Ácido 3-Hidroxibutírico , Animales , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Femenino , Insulina , Cetosas , Cetosis/tratamiento farmacológico , Cetosis/veterinaria , Lactancia , LecheRESUMEN
Disruption of endoplasmic reticulum (ER) homeostasis, a condition termed "ER stress," contributes to the development of liver injury in nonruminants. Because liver injury is a prominent pathological feature associated with overproduction of ketone bodies in dairy cows with ketosis, understanding the ER stress state and its functional consequences on liver injury is of particular interest. Here, 30 multiparous cows (within 3 wk postpartum) classified based on blood ß-hydroxybutyrate (BHB) as healthy (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Compared with healthy cows, ketotic cows had greater levels of serum fatty acids and activities of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and glutamate dehydrogenase but lower serum glucose. Furthermore, dairy cows with ketosis had greater protein abundance of ER stress markers in liver tissue, including protein kinase RNA-like ER kinase (PERK), inositol-requiring protein-1α (IRE1α), and cleaved activating transcription factor-6 (ATF6). Cows with ketosis also had higher mRNA levels of hepatic 78-kDa glucose-regulated protein (GRP78) and spliced X-box binding protein 1 (sXBP1). These data confirmed an enhanced ER stress state in clinically ketotic cows. To explore whether enhanced hepatic ER stress was induced by elevated ketone bodies and the possible contribution of ER stress to liver injury, in vitro experiments were then performed using isolated primary calf hepatocytes treated with incremental concentrations of BHB (0, 0.6, 1.2, 3.0, and 4.8 mM) for 12 h with or without overexpression of GRP78 (the master regulator of unfolded protein response). Phosphorylation levels of PERK and IRE1α proteins, level of cleaved ATF6 protein, and mRNA abundance of GRP78 and sXBP1 in hepatocytes increased after treatment with high (3.0 and 4.8 mM) BHB, indicating a mechanistic link between excessive BHB and enhanced hepatic ER stress. Furthermore, treatment with 3.0 and 4.8 mM BHB markedly elevated activities of aspartate aminotransferase and alanine aminotransferase in cell supernatant, indicating exacerbated hepatocyte damage after ER stress was enhanced. Overexpression of GRP78 attenuated both BHB-induced ER stress and the ensuing cellular damage, suggesting that hepatocyte damage caused by excessive BHB can be mediated via enhanced ER stress. Overall, the present study revealed that ER stress may exacerbate liver injury development in clinically ketotic cows, underscoring the biological relevance of this pathway in the context of liver injury.
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Enfermedades de los Bovinos , Cetosis , Ácido 3-Hidroxibutírico , Animales , Bovinos , Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Endorribonucleasas , Femenino , Homeostasis , Cetosas , Cetosis/veterinaria , Hígado , Proteínas Serina-Treonina QuinasasAsunto(s)
Diabetes Mellitus , Hiperglucemia , Humanos , Hiperglucemia/diagnóstico , Cetosas , Imagen por Resonancia MagnéticaRESUMEN
BACKGROUND: In acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases. METHODS: Membrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor. RESULTS: In this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose. CONCLUSIONS: This reaction may represent an exception to Bertrand Hudson's rule. GENERAL SIGNIFICANCE: Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.
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Ácido Acético/metabolismo , Galactitol/metabolismo , Gluconobacter/metabolismo , Hexosas/metabolismo , Cetosas/metabolismo , Proteínas Bacterianas/metabolismo , Gluconobacter/enzimología , Oxidación-Reducción , Oxidorreductasas/metabolismo , Cofactor PQQ/metabolismoRESUMEN
Biocatalysis using molecular oxygen as the electron acceptor has significant potential for selective oxidations at low cost. However, oxygen is poorly soluble in water, and its slow rate of mass transfer in the aqueous phase is a major obstacle, even for laboratory-scale syntheses. Oxygen transfer can be accelerated by vigorous mechanical methods, but these are often incompatible with biological catalysts. Gentler conditions can be achieved with shallow, high surface area bag reactors that are designed for single use and generally for specialized cell culture applications. As a less-expensive alternative to these high-end bioreactors, we describe repurposing inflatable shipping pillows with resealable valves to provide high surface area mixing under oxygen for preparative synthesis of glucosone (D-arabino-hexos-2-ulose) from D-glucose using non-growing Escherichia coli whole cells containing recombinant pyranose 2-oxidase (POX) as catalyst. Parallel reactions permitted systematic study of the effects of headspace composition (i.e., air vs 100% oxygen), cell density, exogenous catalase, and reaction volume in the oxidation of 10% glucose. Importantly, only a single charge of 100% oxygen is required for stoichiometric conversion on a multi-gram scale in 18 h with resting cells, and the conversion was successfully repeated with recycled cells.
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Reactores Biológicos , Escherichia coli/metabolismo , Cetosas/biosíntesis , Oxígeno/metabolismo , Embalaje de Productos , CatálisisRESUMEN
INTRODUCTION: Diabetic striatopathy is a rare condition characterized by unilateral hemichorea and/or hemiballismus in the settings of uncontrolled nonketotic diabetes mellitus. Imaging studies usually reveal striatal abnormality - subtle hyperdensity on CT and T1 hyperintensity on MRI. The resolution of clinical symptoms is prompt when optimal glycaemic control is achieved. CASE REPORT: We present the case of a 90-year-old male who came to our attention for acute involuntary choreiform movements of his left-sided extremities lasting two-weeks. Apart from that neurological examination was unremarkable. His medical history included hypertension, atrial fibrillation, previous stroke with no residual disability and poorly controlled type 2 diabetes mellitus on metformin treatment. There was no history of movement disorders or exposure to neuroleptics. His glucose level on admission was 512.6 mg/dL, glycated hemoglobin was 14%. CT scan of the head demonstrated an abnormally increased intensity within the right striatum. Treatment consisted of symptomatic treatment of chorea and improvement of blood glucose control. Tiapride was started with a dose of 100âmg 4 times a day. The patient was initiated on intensive insulin therapy which included insulin glargine 10 units every evening and 12 units of insulin glulisine 3 times a day with meals. Abnormal movements resolved after normoglycemia was achieved approximately 7 days after admission. Though striatal hyperdensity was still present at follow-up CT scan after 10 days, it was less pronounced. CONCLUSION: Diabetic striatopathy is a rare but treatable disorder and should be considered in patients with poorly controlled diabetes who present with hemichorea.
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Corea , Diabetes Mellitus Tipo 2 , Hiperglucemia , Accidente Cerebrovascular , Anciano de 80 o más Años , Corea/diagnóstico por imagen , Corea/tratamiento farmacológico , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/tratamiento farmacológico , Cetosas , Imagen por Resonancia Magnética , Masculino , Pirazoles , PiridonasRESUMEN
A facile approach is introduced here for the synthesis of rare ketoses from glycerol and d-/l-glyceraldehyde (d-/l-GA). The reactions were carried out in a one-pot multienzyme fashion in which the only carbon source is glycerol. In the enzymatic cascade, glycerol is phosphorylated and then oxidized at C2 to afford dihydroxyacetone phosphate (DHAP), the key donor for enzymatic aldol reaction. Meanwhile, the primary alcohol of glycerol is also oxidized to give the acceptor molecule GA in situ (d- or l-isomer could be formed stereospecifically with either alditol oxidase or horse liver alcohol dehydrogenase). Different DHAP-dependent aldolases were used to generate the aldol adducts (rare ketohexose phosphates) with various stereoconfigurations and diastereomeric ratios. It is worth noting that the enzyme that catalyzes the phosphorylation reaction in the first step could also help recycle the phosphate in the last step to provide free rare sugar molecules. This study provides a useful method for rare ketose synthesis on a 100 mg to g scale, starting from relatively inexpensive materials which solved the problem of supplying both glycerol 3-phosphate and GA in our previous work. It also demonstrates an example of green synthesis due to highly efficient carbon usage and recycling of cofactors.