Sedation for Colonoscopy Procedures Using Dexmedetomidine Versus Propofol-Fentanyl Infusions: A Prospective Randomized Controlled Trial.

Objective: Different anaesthetists for sedation or monitored anaesthesia care have been used for colonoscopy. The target of this research was the ability to perform colonoscopy under a painless degree of sedation and the prevalence of undesired proceedings. Methods: A total of 60 patients were randomly divided into two groups: Group D received dexmedetomidine and Group PF received propofol-fentanyl. Patients in both groups received the same infusion ratio. The minimum infusion amount of dexmetatomidine is (0.1 to 0.4 µg kg-1 h-1) in Group D, whereas fentanyl is administered at a rate of 0.01 to 0.05 µg kg-1 min-1 in the PF group during the approximately 45-min colonoscopy. Results: Group D exhibited significantly lower modified Observer's Assessment of Alertness/Sedation (OAA/S) scores at intraoperative time points T1-T12. Group D also exhibited significantly lower visual analog scale scores for pain at intraoperative time points T4 and T7. The mean arterial pressure was significantly lower in Group D at intraoperative times T6-T8 and T11-T12, as well as upon admission to the post-anaesthesia care unit (PACU) and 30 min after admission to the PACU. The results of the ANOVA tests revealed a significantly lower heart rate in Group D. The respiratory rate exhibited a notable decrease during time intervals T8 and T10 in the PF group. Conclusion: The administration of dexmetatomidine and propofol-fentanyl during colonoscopy was found to be safe. In addition, dexmetatomidine may present significant benefits in this context because of its lower occurrence of adverse respiratory events.

Therapeutic potentials of mesenchymal stem cells in the treatment of inflammatory bowel disease in rats.

Interleukin-1beta (IL-1ß) and interleukin-17A (IL-17A) have strong pro-inflammatory activities that are involved in inflammatory bowel diseases (IBDs). Mesenchymal stem cell (MSC) therapy is considered a promising treatment for IBD. This study was performed to understand the role of rat Nlrp3 inflammasome, Hmgb1, and pro-inflammatory cytokines (IL-1ß and IL-17a) in the pathogenesis of IBD. Also, to evaluate the role of human umbilical cord blood-MSCs (hUCB-MSCs) in the management of IBD. The rats were in four groups: normal controls, indomethacin-induced IBD group, indomethacin-induced IBD rats that received phosphate-buffered saline (PBS), and the IBD group that received hUCB-MSCs as a treatment. The messenger RNA (mRNA) expression levels of rat Nlrp3, Hmgb1, IL-1ß, and IL-17a were evaluated by quantitative real-time polymerase chain reaction. Histopathological examination of the small intestinal tissues of the studied rats was performed. There was a significant upregulation of the rat Nlrp3, IL-1ß, IL-17a mRNA expression (p < 0.001 for the three parameters), and Hmgb1 (p < 0.05) in the untreated IBD group compared to the normal control group. In the MSC-treated group, IL-1ß, IL-17a, and rat Nlrp3 mRNA expression significantly decreased compared to both the untreated IBD group and PBS group (p < 0.05 for all). hUCB-MSCs ameliorated IBD in rats by downregulating the pro-inflammatory cytokines (IL-1ß and IL-17a) and other inflammatory mediators such as Hmgb1 and rat Nlrp3.

Leveraging cross-view geo-localization with ensemble learning and temporal awareness.

The Global Navigation Satellite System (GNSS) is unreliable in some situations. To mend the poor GNSS signal, an autonomous vehicle can self-localize by matching a ground image against a database of geotagged aerial images. However, this approach has challenges because of the dramatic differences in the viewpoint between aerial and ground views, harsh weather and lighting conditions, and the lack of orientation information in training and deployment environments. In this paper, it is shown that previous models in this area are complementary, not competitive, and that each model solves a different aspect of the problem. There was a need for a holistic approach. An ensemble model is proposed to aggregate the predictions of multiple independently trained state-of-the-art models. Previous state-of-the-art (SOTA) temporal-aware models used heavy-weight network to fuse the temporal information into the query process. The effect of making the query process temporal-aware is explored and exploited by an efficient meta block: naive history. But none of the existing benchmark datasets was suitable for extensive temporal awareness experiments, a new derivative dataset based on the BDD100K dataset is generated. The proposed ensemble model achieves a recall accuracy R@1 (Recall@1: the top most prediction) of 97.74% on the CVUSA dataset and 91.43% on the CVACT dataset (surpassing the current SOTA). The temporal awareness algorithm converges to R@1 of 100% by looking at a few steps back in the trip history.

Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity.

Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines "MDA-MB, HepG-2, and PC-3" was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.

In Vitro and In Silico Pharmacological and Cosmeceutical Potential of Ten Essential Oils from Aromatic Medicinal Plants from the Mascarene Islands.

In this study, 10 essential oils (EOs), from nine plants (Cinnamomum camphora, Curcuma longa, Citrus aurantium, Morinda citrifolia, Petroselinum crispum, Plectranthus amboinicus, Pittosporum senacia, Syzygium coriaceum, and Syzygium samarangense) were assessed for their antimicrobial, antiaging and antiproliferative properties. While only S. coriaceum, P. amboinicus (MIC: 0.50 mg/mL) and M. citrifolia (MIC: 2 mg/mL) EOs showed activity against Cutibacterium acnes, all EOs except S. samarangense EO demonstrated activity against Mycobacterium smegmatis (MIC: 0.125-0.50 mg/mL). The EOs were either fungistatic or fungicidal against one or both tested Candida species with minimum inhibitory/fungicidal concentrations of 0.016-32 mg/mL. The EOs also inhibited one or both key enzymes involved in skin aging, elastase and collagenase (IC50: 89.22-459.2 µg/mL; 0.17-0.18 mg/mL, respectively). Turmerone, previously identified in the C. longa EO, showed the highest binding affinity with the enzymes (binding energy: -5.11 and -6.64 kcal/mol). Only C. aurantium leaf, C. longa, P. amboinicus, P. senacia, S. coriaceum, and S. samarangense EOs were cytotoxic to the human malignant melanoma cells, UCT-MEL1 (IC50: 88.91-277.25 µg/mL). All the EOs, except M. citrifolia EO, were also cytotoxic to the human keratinocytes non-tumorigenic cells, HaCat (IC50: 33.73-250.90 µg/mL). Altogether, some interesting therapeutic properties of the EOs of pharmacological/cosmeceutical interests were observed, which warrants further investigations.

Hybrid pointer networks for traveling salesman problems optimization.

In this work, we proposed a hybrid pointer network (HPN), an end-to-end deep reinforcement learning architecture is provided to tackle the travelling salesman problem (TSP). HPN builds upon graph pointer networks, an extension of pointer networks with an additional graph embedding layer. HPN combines the graph embedding layer with the transformer's encoder to produce multiple embeddings for the feature context. We conducted extensive experimental work to compare HPN and Graph pointer network (GPN). For the sack of fairness, we used the same setting as proposed in GPN paper. The experimental results show that our network significantly outperforms the original graph pointer network for small and large-scale problems. For example, it reduced the cost for travelling salesman problems with 50 cities/nodes (TSP50) from 5.959 to 5.706 without utilizing 2opt. Moreover, we solved benchmark instances of variable sizes using HPN and GPN. The cost of the solutions and the testing times are compared using Linear mixed effect models. We found that our model yields statistically significant better solutions in terms of the total trip cost. We make our data, models, and code publicly available https://github.com/AhmedStohy/Hybrid-Pointer-Networks.

Magnetic Resonance Imaging Features in Different Types of Invasive Breast Cancer: A Systematic Review of the Literature.

Breast cancer is the most common malignancy affecting women worldwide, and early diagnosis of breast cancer is the key to its successful and effective treatment. Traditional imaging techniques such as mammography and ultrasound are used to detect and configure breast abnormalities; unfortunately, these modalities have low sensitivity and specificity, particularly in young patients with dense breast tissue, breast implants, or post-surgical scar/architecture distortions. Therefore, breast magnetic resonance imaging (MRI) has been superior in the characterization and detection of breast cancer, especially that with invasive features. This review article explores the importance of breast MRI in the early detection of invasive breast cancer versus traditional tools, including mammography and ultrasound, while also analyzing the use of MRI as a screening tool for high-risk women. We will also discuss the different MRI features for invasive ductal carcinoma and lobular carcinoma and the role of breast MRI in the detection of ductal carcinoma in situ with a focus on the utilization of new techniques, including MR spectroscopy and diffusion-weighted imaging.

Patchy Vasoconstriction Versus Inflammation: A Debate in the Pathogenesis of High Altitude Pulmonary Edema.

High altitude pulmonary edema (HAPE) occurs in individuals rapidly ascending at altitudes greater than 2,500 m within one week of arrival. HAPE is characterized by orthopnea, breathlessness at rest, cough, and pink frothy sputum. Several mechanisms to describe the pathophysiology of HAPE have been proposed in different kinds of literature where most of the mechanisms are reported to be activated before a drop in oxygen saturation levels. The majority of the current studies favor diffuse hypoxic pulmonary vasoconstriction (HPV) as a pathophysiological basis for HAPE. However, some of the studies described inflammation in the lungs and genetic basis as the pathophysiology of HAPE. So, there is a major disagreement regarding the exact pathophysiology of HAPE in the current literature, which raises a question as to what is the exact pathophysiology of HAPE. So, we reviewed 23 different articles which include clinical trials, review articles, randomized controlled trials (RCTs), and original research published from 2010 to 2020 to find out widely accepted pathophysiology of HAPE. In our study, we found out sympathetic stimulation, reduced nitric oxide (NO) bioavailability, increased endothelin, increased pulmonary artery systolic pressure (PASP) resulting in diffuse HPV, and reduced reabsorption of interstitial fluid to be the most important determinants for the development of HAPE. Similarly, with the evaluation of the role of inflammatory mediators like C-reactive protein (CRP) and interleukin (IL-6), we found out that inflammation in the lungs seems to modulate but not cause the process of development of HAPE. Genetic basis as evidenced by increased transcription of certain gene products seems to be another promising hypoxic change leading to HAPE. However, comprehensive studies are still needed to decipher the pathophysiology of HAPE in greater detail.

Safety and Efficacy of Stem Cell Therapy in Patients With Ischemic Stroke.

Stem cell therapy is emerging as a promising treatment strategy to treat patients with stroke. While there are established modes of treatment for stroke patients such as thrombolysis and endovascular intervention, most of the stroke patients frequently end up with major residual deficits or even death. The use of stem cells to treat stroke has been found to be beneficial in the animal models but strict evidence for the same in humans is still lacking. We reviewed 13 clinical trials of stem cell therapy in stroke patients conducted between 2014 and 2020 based on the search using the database PubMed, and the clinical trial registry (www.clinicaltrials.gov). We aimed to assess the safety and efficacy of stem cell treatment in stroke patients who participated in the trials. Quality assessment of the clinical trials revealed a sub-optimal score. We found mixed results regarding the efficacy of stem cells in the treatment of ischemic stroke although we could not do a quantitative analysis of the effect outcomes. Assessment for safety revealed promising results as there were only minor side effects related to cell therapy. Although stem cell therapy seems to be a promising strategy to treat stroke patients in the future, we concluded that the field needs more evidence regarding the safety and efficacy of the use of stem cells in stroke patients before we use them in the clinic.

Diabetes Mellitus and Parkinson's Disease: Shared Pathophysiological Links and Possible Therapeutic Implications.

Diabetes mellitus (DM) is the most common chronic metabolic disease. Parkinson's disease (PD) is considered one of the most common neurodegenerative diseases. There are many similarities between both conditions. Both disorders are chronic diseases. Both diseases result from a decrease in a specific substance: dopamine in PD, and insulin in DM. Besides, both disorders arise due to the destruction of particular cells, dopaminergic cells in PD, and pancreatic beta-cell in DM. Recently, many epidemiological and experimental studies showed a connection between DM and PD. There are common underlying mechanisms in the pathophysiology of both diseases. These underlying mechanisms include mitochondrial dysfunction, oxidative stress, hyperglycemia, and inflammation. Insulin resistance is indeed the hallmark of DM, especially type 2 diabetes mellitus (T2DM), which plays a significant role in these pathophysiological and molecular mechanisms. Besides, many studies revealed that anti-diabetic drugs have a beneficial effect on PD. In this current literature review, we aim to explore the standard pathophysiological and molecular linkages between these two disorders as well as how DM could affect the incidence and progression of PD. We also review how anti-diabetic drugs impact PD. In the future, further experimental and expanded clinical studies are needed to fully understand the exact pathophysiological connections between the two disorders and the efficacy of insulin and other anti-diabetic drugs in the treatment of PD in diabetic patients. Fully understanding and targeting these pathophysiological and molecular links could result in de novo curative therapy for PD and DM.

Alvogyl and absorbable gelatin sponge as palatal wound dressings following epithelialized free gingival graft harvest: a randomized clinical trial.

OBJECTIVES: This randomized controlled trial compares for the first time effects of Alvogyl versus absorbable gelatin sponge as palatal wound dressings on postoperative pain, amount of analgesic consumption, post-surgical bleeding, and wound re-epithelization. MATERIALS AND METHODS: Following sample size calculation, 36 systemically healthy patients requiring palatal mucosal graft harvesting were randomized to receive Alvogyl (intervention group, 18 patients) or absorbable gelatin sponge (control group, 18 patients) palatal dressings. Patient-reported VAS pain scores over 2 weeks were defined as primary outcome. Post-surgical bleeding, number of analgesics consumed, and complete re-epithelialization of the palatal wound for up to 5 weeks were defined as secondary outcomes. RESULTS: Although significantly higher VAS pain scores were reported in the control as compared with the intervention group up to 12 days post-surgically (from (median [range]) 8.5 [2-10] to 1 [0-2] and from 6 [0-10] to 0 [0-2] respectively), with higher analgesics consumption (from 2 [1-3] to 1 [0-3] and from 1 [0-3] to 0 [0-2] tablets respectively), a multivariate regression analysis considering age, gender, graft width/length, tissue thickness, analgesics intake, and dressing type demonstrated no statistically significant effect of any factor, including dressing type on VAS pain scores. At 4 weeks, 22.2% of patients in the intervention group versus 11.1% in the control group demonstrated complete re-epithelization of their palatal engraftment site, before complete re-epithelization in both groups at 5 weeks. No post-surgical bleeding was reported with both dressings. CONCLUSIONS: Within the study's limitations, results suggest Alvogyl as a practical palatal surgical dressing, comparable with absorbable gelatin sponge in cost, pain reduction, hemostasis, and re-epithelization properties. TRIAL REGISTRATION: www.ClinicalTrials.gov Identifier: NCT03402321 CLINICAL RELEVANCE: Alvogyl could present a novel palatal wound dressing material, comparable with gelatin sponge.

Evaluating the safety of Liptruzet (ezetimibe and atorvastatin): what are the potential benefits beyond low-density lipoprotein cholesterol-lowering effect?

INTRODUCTION: The combination of ezetimibe and atorvastatin (Liptruzet - referred to in this article as eze/ator), has recently been approved by the FDA for reducing low-density lipoprotein cholesterol (LDL-c) in patients with primary or mixed hyperlipidemia as in case of homozygous familial hypercholesterolemia. It helps block intestinal absorption of cholesterol and it inhibits the production of cholesterol in the liver. AREAS COVERED: The safety and effectiveness of the eze/ator combination as treatment of hyperlipidemia. Medline was searched for atorvastatin and/or ezetimibe. EXPERT OPINION: The combination of (eze/ator) is proven to be effective in lowering LDL-c. It is not only a safe and effective treatment of hyperlipidemia, but it also reduces inflammatory markers and atherosclerosis. It is not yet clear, however, whether the combination therapy can decrease the risk of diabetes associated with statin administration. Insulin sensitivity is improved by the single administration of ezetimibe, a finding that is documented by several clinical and animal studies. More specifically, ezetimibe has been shown to decrease insulin resistance associated with nonalcoholic fatty liver disease (NAFLD). The effects of combination therapy that have to be explored in future research and clinical trials include whether this combination can be used in the treatment of NAFLD, cholesterol gallstones and portal hypertension.

Potential Benefit of Metformin as Treatment for Colon Cancer: the Evidence so Far.

Metformin is known as a hypoglycaemic agent that regulates glucose homeostasis by inhibiting liver glucose production and increasing muscle glucose uptake. Colorectal cancer (CRC) is one of the most common cancers worldwide, with about a million new cases diagnosed each year. The risk factors for CRC include advanced age, smoking, black race, obesity, low fibre diet, insulin resistance, and the metabolic syndrome. We have searched Medline for the metabolic syndrome and its relation to CRC, and metformin as a potential treatment of colorectal cancer. Administration of metformin alone or in combination with chemotherapy has been shown to suppress CRC. The mechanism that explains how insulin resistance is associated with CRC is complex and not fully understood. In this review we have summarised studies which showed an association with the metabolic syndrome as well as studies which tackled metformin as a potential treatment of CRC. In addition, we have also provided a summary of how metformin at the cellular level can induce changes that suppress the activity of cancer cells.

5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

Patient safety attitude among health care providers in family health care centers in Cairo governorate.

Two hundred and eighty Health care; physicians, nurses and technicians were included in the study, 133 (47.5) from 6th discrete HCF, 54 (19.3%) from Duwaiqa and 93 (33.2%) from Al Haggana. Ages ranged between 22 and 59 years (35.14 ± 10.13), years of experience in the study group ranged between 1 and 35 years (8.72 ± 8.18) and years of work in group ranged between 1 & 40 years (10.43 ± 8.33). Doctors and nurses were males (72.55%) & females (86%) respectively while technicians were mostly males (60%). The teamwork climate score was 3.98 ± 0.87, 64.0% answered high or very high, 16.4% answered inadequate and 18% had answered few or very few. Safety climate score was 3.61 ± 0.63, 49.28% had high or very high score, 17.14% answered inadequate and 32.15% had answered few or very few. Job satisfaction score was 3.91 ± 0.80, 32.15% had answered few or very few, 17.14% answered inadequate and 49.28% answered high or very high. Stress recognition score was 3.61 ± 0.79, 25% had answered few or very few, 28.6% answered inadequate and 45.7% answered high or very high. Perception of management score was 3.48 ± 0.80, 23.2% had answered few or very few, 17.8% answered inadequate and 57.6% answered high or very high. Working condition score was 3.51 ± 0.84, 46.8% had answered few or very few, 17.1% answered inadequate and 35.7% answered high or very high. A significant difference regarding team work score, safety climate score, perception of management score, working condition score with highest value in doctors and lowest in technicians. On the other hand no significant difference was detected regarding job satisfaction score and stress recognition score. A significant difference regarding team work score, safety climate score and perception of management score with high values among older groups. No significant difference was detected regarding job satisfaction score, stress recognition score and working condition score. Also, a significant difference regarding team work score and safety climate score with higher value in males. Significant difference regarding job satisfaction score and working condition score was detected with higher value in females. No significant difference was detected regarding stress recognition score and perception of management scores.

Medicinal plants and their uses by the people in the Region of Randa, Djibouti.

ETHNOPHARMACOLOGICAL RELEVANCE: The article presents the local knowledge on medicinal plants and their relevance in managing health problems. Important ethnobotanical leads are given with priority species and disease categories, casting insight on future phytochemical and pharmacological studies. AIM OF THE STUDY: The use of traditional medicinal plants has been an integral part of the traditional healthcare systems in Djibouti. However, scientific studies on the traditional herbal healing systems of the various cultural groups have never been undertaken. This study has, therefore, aimed at assessing plant-related ethnomedicinal knowledge of the people in Randa Region; prioritising the plants with respect to common disease categories and inferring about prospects of new pharmacological products. MATERIALS AND METHODS: Interview-based ethnobotanical field study was carried out to document the plant-based ethnomedicinal knowledge handed down to the present by the oral tradition of people living in 24 villages in Tadjourah District of Randa Region (north Djibouti). Informant Consensus Factors (ICF) and Fidelity Level (FL) values of the medicinal plants were calculated to check the level of informant agreement and the healing potentials of the species. RESULTS: A total of 91 plant species that belong to 72 genera and 40 families were documented. Most of these species (92%) were collected from non-cultivated areas. Their local names and traditional uses in medicine were also studied. The plant family Fabaceae was represented by the highest number of taxa (17 species). Strong informant agreements hinted at good healing potentials of some species as shown by high values of consensus factors for eye diseases (0.98), mouth diseases (0.93), kidney problems (0.89) and microbial infections (0.84). Dodonea angustifolia, Solanum cordatum, Grewia erythraea, Acalypha indica, Acacia etbaica, Fagonia schweinfurthii, Solanum coagulans, Senna alexandrina and Grewia tembensis scored high FL values emerging as promising priority species for future pharmacological screening against microbial infections. CONCLUSION: The results of this study may inspire further ethnobotanical and ethnopharmacological research and investigations toward drug discovery in Djibouti and beyond.

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations.

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis.

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.