Predictive value of immature granulocyte and delta neutrophil index in the diagnosis of complicated acute cholecystitis.

OBJECTIVE: The complicated gallbladder disorders are associated with increased mortality and morbidity. Thus, this study was aimed at evaluating the predictive value of immature granulocyte count and delta neutrophil index in the prediction of complicated cholecystitis. PATIENTS AND METHODS: We retrospectively reviewed patients who underwent surgery for acute cholecystitis between January 2018 and April 2022. Overall, 351 patients fulfilling the inclusion criteria were included in the study. In all patients, demographic data, immature granulocyte count (IGC), delta neutrophil index (DNI), white blood cell (WBC) count, C-reactive protein (CRP), and albumin levels were recorded. Based on operative findings and histopathological examination, the patients were classified into 2 groups uncomplicated (group I) and complicated (e.g., perforation, gangrenous and emphysematous cholecystitis; group II) groups. The IBM SPSS version 26.0 (SPSS Corp, Armonk, NY, USA) was used to assess differences in blood parameters between groups. The predictive values of the parameters evaluated were estimated using ROC analysis. A p-value<0.05 was considered statistically significant. RESULTS: Acute complicated cholecystitis was found in 138 of 351 patients. No significant difference was detected in age and gender distribution between groups (p=0.352 and p=0.214, respectively). When blood parameters were assessed, it was found that IGC, DNI, WBC, and CAR values were significantly higher in group II (p<0.001; p<0.001, p<0.001, and p=0.036, respectively), while there was no significant difference in CRP and albumin between groups (p=0.099 and p=0.53, respectively). In the ROC analysis, the highest AUC value was found for IG count and DNI (0.784 and 0.775, respectively). The sensitivity and specificity were found as 68.8% and 86.9% for IG count and 49.3% and 96.2% for DNI, respectively. CONCLUSIONS: The IG count and DNI are two novel parameters with strong predictive value in the early diagnosis of acute complicated cholecystitis, which may support clinical findings, imaging studies, and other laboratory parameters.

Can serum basal cortisol values predict successful operations for bariatric surgery patients?

OBJECTIVE: Obesity is a chronic metabolic disease declared as the 21st century pandemic by the World Health Organization. Obesity has become an alarming situation for society, and it has to be treated. If the appropriate criteria are met by patients, bariatric surgery is an effective treatment method that provides weight loss in a short time. There are no definitive criteria regarding which groups of patients and characteristics may benefit most from bariatric surgery. In this study, we evaluate whether serum basal cortisol levels can predict successful outcomes of bariatric surgery and whether there is any difference in outcome between diabetic and non-diabetic patients. PATIENTS AND METHODS: This observational study included 244 obese patients who had undergone laparoscopic sleeve gastrectomy (LSG). Preoperative and postoperative 6-month weight, body mass index (BMI), serum basal cortisol, fasting plasma glucose, 1-mg dexamethasone suppression test (DST) results, and Type 2 Diabetes Mellitus (T2D) history were recorded. We analyzed the patients first by dividing them into two groups as excessive weight loss (%EWL) ≥50% and %EWL< 50%, and then into two groups as diabetic and nondiabetic patients. RESULTS: The mean age of patients with %EWL≥ 50% was found to be statistically significantly lower than that of patients with %EWL< 50% [39 (19-60) vs. 47 (36-61) years; p=0.046]. While there was no significant difference in basal cortisol values (p=0.513), DST results were statistically significantly lower in patients with %EWL≥ 50% than patients with %EWL< 50% [0.6 (0.1-2.1) vs. 0.8 (0.7-1.1); p=0.040]. CONCLUSIONS: In obese patients undergoing LSG, serum basal cortisol level may not predict the success of operation. However, the result of 1 mg DST may predict the operation success.

Antimicrobial Resistance of E. coli and Salmonella Isolated from Wild Birds in a Rehabilitation Center in Turkey.

Wildlife plays a critical role as a reservoir for zoonosis especially pathogenic enteric bacteria. In this study we evaluated the presence of E. coli and Salmonella isolates from wild birds and determined their antimicrobial resistance. Intestine and fecal samples from 82 dead wild birds obtained from rehabilitation centre, were examined by microbiological analysis, antibiotic susceptibilities against of 18 antimicrobials and presence of tetracycline resistance genes by multiplex and singleplex PCR were investigated. A total of 51 E. coli were identified as well as Salmonella Kentucky and Salmonella Bisberg. A majority of the E. coli isolates were resistant to lincomysin (100%), penicilline (96.1%), kanamycin (80.4%), tetracycline (68.6%), and oxytetracycline (64.7%). All Salmonella serotypes were resistant to lincomycin, nalidixic acid and penicilline.In addition, 58.82% of E. coli isolates had phenotypic resistance to at least three or more antimicrobials. Our results indicated that the high frequency of tetracycline resistance (68.62%) due to the tet (A), tet (B), and tet (D) genes. This is the first report isolating S. Bisberg and determining antibiotic susceptibility of E.coli and Salmonella isolates from wild birds in Turkey. These results will help providing better understand of the dissemination of antibiotic resistancy in the environment, which can be used to potentially decrease spread through bird migration. Moreover, these results help assess the risk of spread of resistance from wild birds to humans.

Characterization of class 1, 2, 3 integrons, ESBL genes and antibiotic susceptibility of Salmonella serotypes from broiler and cattles in Turkey.

Antimicrobial resistance in Salmonella has been associated with the presence of integrons and many other resistance mechanisms contributing to the spread of antimicrobial-resistant genes within and between livestock and human populations. In this study, the presence of Salmonella serovars from broiler and cattle samples and their antimicrobial resistance, integrons, tet resistance, ESBL and resistance genes carriage were investigated. Total of 209 litter (broiler farms) and fecal samples (cattle farms) were examined by bacteriological procedures, susceptibilities against 18 antimicrobials and genes carriages were detected by singleplex and multiplex PCR. A total of 46/209 (22 %) Salmonella strains were isolated. Six different Salmonella serotypes from 46 Salmonella isolates were identified and the most common serotype was S. Infantis 38 (82.6%) from broiler litter; followed by S. Kitenge 3 (6.5 %) from fecal sample. The highest occurrence of resistance observed for penicilline (46/46, %100), lincomycin (43/46, 93.5%) and 42 isolates (43/46, 93.5%) exhibited MDR. The overall occurrence of class 1, 2 and 3 integrons carrying Salmonella in tested samples were 63.04% (29/46), 43.5% (20/46) and 84.8% (39/46) respectively. Out of the 27 isolates produced an ESBL, mostly CTX and TEM. On 46 Salmonella isolates, in 16 (34.8%) Tcr' genes were determined. Genotypic and phenotipic detection of ESBL genes found within integrons from Salmonella isolates from different sources (broiler and cattle) can provide powerful information about health and economic risk associated with transferable multidrug resistance.

Precision medicine in human heart modeling : Perspectives, challenges, and opportunities.

Precision medicine is a new frontier in healthcare that uses scientific methods to customize medical treatment to the individual genes, anatomy, physiology, and lifestyle of each person. In cardiovascular health, precision medicine has emerged as a promising paradigm to enable cost-effective solutions that improve quality of life and reduce mortality rates. However, the exact role in precision medicine for human heart modeling has not yet been fully explored. Here, we discuss the challenges and opportunities for personalized human heart simulations, from diagnosis to device design, treatment planning, and prognosis. With a view toward personalization, we map out the history of anatomic, physical, and constitutive human heart models throughout the past three decades. We illustrate recent human heart modeling in electrophysiology, cardiac mechanics, and fluid dynamics and highlight clinically relevant applications of these models for drug development, pacing lead failure, heart failure, ventricular assist devices, edge-to-edge repair, and annuloplasty. With a view toward translational medicine, we provide a clinical perspective on virtual imaging trials and a regulatory perspective on medical device innovation. We show that precision medicine in human heart modeling does not necessarily require a fully personalized, high-resolution whole heart model with an entire personalized medical history. Instead, we advocate for creating personalized models out of population-based libraries with geometric, biological, physical, and clinical information by morphing between clinical data and medical histories from cohorts of patients using machine learning. We anticipate that this perspective will shape the path toward introducing human heart simulations into precision medicine with the ultimate goals to facilitate clinical decision making, guide treatment planning, and accelerate device design.

The harm of anticoagulation in patients with low-risk by CHADS2 and reclassified as high-risk by CHA2DS2VASc: inferences from TRAF cohort.

OBJECTIVE: There is a gap in the knowledge concerning oral anticoagulation (OAC) in atrial fibrillation (AF) patients with a non-high risk of stroke. CHA2DS2VASc and CHADS2 scores generated imprecise risk estimates for low risk patients. We aimed to assess OAC in patients with low risk by CHADS2 and reclassified as high-risk by CHA2DS2VASc. PATIENTS AND METHODS: In this study, retrospective nationwide population-based study, data were obtained from the Turkish claims and utilization management system. Patients with non-valvular AF (n=451,113) between 2007 and 2012 sub-divided into those with a CHA2DS2VASc≥1 and CHADS2=0 (n=41,273) who were off-warfarin (n=29,448) and on-warfarin (n=11,825). Stroke and systemic embolism, major bleeding, all-cause mortality, net clinical benefit (NCB) and ultimate NCB (UNCB) were assessed. RESULTS: Of the total cohort (mean age 66.1 ± 14.1 years, 56.1% female), CHA2DS2VASc improved the net reclassification index of observed 5-year composite thromboembolic endpoint by 6.9% (p<0.05). CHA2DS2VASc reclassified 9.7% low risk patients as high risk. Among reclassified-high-risk category (patients with a CHA2DS2VASc score of ≥1 and CHADS2 score of 0), major bleeding for that prescribed warfarin was 3% and higher than the rate of thromboembolism among those off-warfarin. NCB (-0.035) and UNCB (-0.021) were negative. Death and hospitalization at 1 year were significantly higher for on-warfarin group. CONCLUSIONS: Clinical outcomes, net clinical benefit indices are negative; rates of death and hospitalization were significantly higher for OAC in reclassified category. This emphasizes the importance of greater attention to balancing the risks and benefits of OAC in patients with low risk by CHADS2 and reclassified as high-risk by CHA2DS2VASc.

Comparison of incisional hernia models in rats: an experimental study.

PURPOSE: Incisional hernia repair is a frequently performed operation worldwide. In this experimental study, our aim is to present the incisional hernia model after creating midline laparotomy and several type of defects on abdominal wall of the rats. Thereby, the method determined here may be used in future experimental incisional hernia repair studies. METHODS: After approval, 32 male rats were randomly seperated into 4 groups of 8 animals each, and were operated to form an incisional hernia; Sham group, 5 cm incision group, 5 cm incision plus capitonnage group, and 5 cm incision plus 2 × 4 cm muscle excision group, respectively. On the 28th postoperative day after killing, the abdominal anterior wall of rats were removed for histopathological and biomechanic examination. RESULTS: The incisional hernia size was found to be statistically different in at least one group (p = 0.001). The incisional hernia size in Group 4 was found to be significantly higher than Group 2 (p = 0.001). When the tension and elongation values were examined, there was a difference in at least one group (p < 0.001 and p = 0.029, respectively). Histopathological examination shows that the degree of inflammation and fibrosis varies significantly (p = 0.001 and p = 0.002, respectively). CONCLUSION: This study has lead us to believe that the rat model created by applying muscle excision from the midline of the abdomen is the ideal incisional hernia model that can be used in future experimental incisional hernia studies.

Effects of atorvastatin on talinolol absorption: A potential drug-drug interaction.

In this study, we aimed to determine the drug-drug interaction potential between atorvastatin (ATOR), and talinolol (TAL). Concentration-dependent effects of ATOR on the intestinal permeability of TAL were investigated by an in situ intestinal perfusion method. Dose-dependent effects of ATOR on TAL exposure were evaluated by measuring plasma concentrations after oral administration in rats. ATOR slightly changed the intestinal secretion of TAL in jejunum but not in colon. Plasma AUC levels of TAL were elevated by co-administration of ATOR at low and high doses whereas medium doses of ATOR resulted in a decrease in TAL bioavailability. However, these changes were not statistically significant. In our study, the pharmacokinetics of TAL were not affected by the concurrent use of ATOR in rats. In conclusion, it should be considered that complex interplay between the efflux and uptake transporters in the tissues and inhibition of these transporters by modulating agents may overshadow individual effects of each other.

Is Low-level laser therapy and gaseous ozone application effective on osseointegration of immediately loaded implants?

PURPOSE: The purpose of this study was to investigate the effects of biostimulation lasers and ozone therapy on osseointegration of immediately loaded implants. MATERIALS AND METHODS: A total number of 100 implants (DTI Implant Systems) were applied to 25 patients evenly. Temporary crowns were applied to each patient on the same session as the surgery. Implants were divided into four treatment groups (Group 1: low-level laser therapy (LLLT) group, Group 2: ozone therapy group, Group 3: different protocol of ozone therapy group, and Group 4: control group) each with 25 implants. The irradiations were performed with a gallium-aluminum-arsenide diode low-level laser (Laser BTL-4000) to Group 1. Ozone therapy was performed using an ozone generator (OzoneDTA) with an intraoral probe to Group 2 and Group 3. RESULTS: In this study, the overall implant survival rate was 92% after a 6-month observation period. The implant stability quotient values were found significantly higher in Group 1 (LLLT group) and Group 3 (different protocol of ozone therapy group) than the other groups (P < 0.05). There was no significant difference in Group 2 (ozone therapy group) and the control group (P > 0.05). CONCLUSIONS: Our results suggest that both LLLT and ozone therapy with prolonged application time are promising methods to enhance bone healing around immediately loaded implants and increase implant stability; however, there is a need for more studies on this subject for these methods to become routine applications.

Pharmacokinetics of talinolol is modified by barnidipine: implication of P-glycoprotein modulation.

Concomitant administration of P-glycoprotein substrates and inhibitors may cause pharmacokinetic drug interactions leading to increased concentrations associated with serious side effects and toxicities. Barnidipine is a longacting calcium-channel blocker and potent inhibitor of P-glycoprotein in vitro, and talinolol is a beta-blocker and probe substrate of P-glycoprotein. This study was designed to investigate the effects of single and repeated oral doses of barnidipine on talinolol pharmacokinetics in rats. In the single-dose study, talinolol (20 mg/kg) alone and with barnidipine at low (1 mg/kg) and high doses (10 mg/kg) were orally administered to rats. In the repeated-dose study, rats were treated with barnidipine (1 mg/kg/day) or vehicle only for four days, then with talinolol (20 mg/kg, on day 5). Blood samples were collected at 0.5, 1, 2, 4, 6 h following last dose and plasma talinolol levels were determined by HPLC. Compared to the control, Cmax of talinolol elevated 10% (p=0.79) and 110% (p<0.05); plasma AUC0-6h increased 33% (p=0.41) and 46% (p<0.05) following low and high single doses of barnidipine co-administration, respectively. In the repeated-dose study, Cmax and AUC0-6h of talinolol increased 131% (p<0.05) and 130% (p<0.05) respectively, following co-administration of a low barnidipine dose. Double-peaks were observed when single or repeated low doses of barnidipine were co-administered. There may be coupling between occurrence of double-peak phenomenon and P-glycoprotein inhibition. Increment of talinolol bioavailability upon low and high doses of barnidipine co-administration may be due to P-glycoprotein inhibition. The higher increase of talinolol plasma AUC0-6h due to the repeated doses of barnidipine may be explained by downregulation of P-glycoprotein.

Transuterine migration as a complication of intrauterine contraceptive devices: six case reports.

OBJECTIVES: We retrospectively analyzed six cases between 1986 and 2002 that had been operated within our unit with the diagnosis of migration of an intrauterine contraceptive device (IUD). Although an IUD is an effective contraceptive method, the migration of one is a rare but serious complication. The aim of this report is to emphasize the management and therapy of this complication. CASES: Out of six patients, three patients with occurring pregnancy, two with pelvic pain and one with a missing IUD incidentally diagnosed during a routine follow-up gynecological examination, were admitted to our clinic. The diagnosis of perforation and transuterine migration of the IUD was confirmed with a plain abdominal X-ray with a hysterometer placed in the uterus, hysterosalpingography and ultrasound. One patient was diagnosed as having a perforated rectosigmoid bowel intraoperatively and one presented with perforation of the bladder. In the remaining four cases, the IUD only migrated into the abdominal cavity without any organ perforations. One IUD was extracted laparoscopically, one was removed through the vagina by colpotomy and, in the other four cases, a laparotomy had to be performed. Patients were discharged without any complications. CONCLUSION: The most serious potential complication of IUD use is uterine perforation and this can cause severe morbidity. When an IUD is located in the abdominal cavity, it should be carefully managed and removed, even in an asymptomatic patient.

A comparative study on detection of Ornithobacterium rhinotracheale antibodies in meat-type turkeys by dot immunobinding assay, rapid agglutination test and serum agglutination test.

The objectives of the present study were to develop a dot-immunobinding assay (DIA) and a serum agglutination test (SAT) for detection of Ornithobacterium rhinotracheale, to compare the rapid agglutination test (RAT) and the SAT, and to make a serosurvey of O. rhinotracheale exposure on turkey farms in Turkey. Antiserum against O. rhinotracheale bacterin was prepared in rabbits and 72 serum samples were collected from turkeys with respiratory signs on four farms. Comparison of the tests showed that 55.5, 48.6 and 40.3% of serum samples were positive by RAT, SAT and DIA, respectively. The sensitivity of the DIA appeared to be lower than that of the agglutination tests but the specificity is not known.

Sucralfate in the prevention of radiation-induced oral mucositis.

Radiotherapy-induced mucositis decreases the quality of life by impairing eating, swallowing, and talking and by disturbing sleep. Mucositis may also predispose to local and systemic infections and may cause interruption of radiotherapy course. We studied the efficacy of sucralfate suspension in the prevention and management of oral mucositis and pain during radiotherapy in a double-blind, placebo-controlled, randomized, prospective trial. Twenty-eight patients with head and neck cancer were included in the study. The patients were randomized to use either sucralfate mouth washing (n = 18) or placebo washing (n = 10) during irradiation. Oral mucositis and symptoms were assessed by the same physician using Radiation Therapy Oncology Group Acute Radiation Morbidity Scoring criteria. All patients developed varying degrees of radiation-induced mucositis. Grade 4 mucositis was not encountered in any patient. One patient had grade 1, seven patients grade 2, and two patients grade 3 mucositis in placebo group. In sucralfate group, nine patients each had grade 1 and grade 2 with no grade 3 mucositis. Patients in the sucralfate group experienced significantly lower degree of mucositis than placebo group (p < 0.05). Sucralfate mouth washing is beneficial in decreasing the intensity of radiation-induced mucositis and oral discomfort. It is cheap, easy to administer with no serious side effect, and may be routinely used in patients receiving head and neck radiotherapy.

Expression and purification of HIV-I p15NC protein in Escherichia coli.

An efficient method for the expression and purification of nucleocapsid precursor protein (p15NC) from HIV-I (BH 10 isolate) was developed and used to obtain large quantities of this viral protein for structural studies, protein biochemistry, and high-throughput screening efforts. We have engineered an existing p15NC clone into a new vector developed at the University of Heidelberg, Germany. Using PCR, we introduced new restriction sites and a strong ribosome-binding site in the p15NC gene and expressed authentic p15NC protein. Our protocol enabled us to rapidly obtain soluble and highly stable p15NC expressed in Escherichia coli and to purify several milligrams of p15NC to homogeneity. In the current purification scheme, lysis of cell paste followed by a simple three-step FPLC procedure yields about 0.4-0.5 mg of purified p15NC per gram of E. coli cell paste expressing the protein with an overall yield of 45%. The purified p15NC retained its ability to bind full-length HIV-I p15NC mRNA in solution- or solid-phase-based assays. A specific stem-loop forming RNA fragment (24-mer) and its antisense DNA oligomer (21-mer) derived from the full-length p15NC mRNA were also able to bind to p15NC. In addition, antisense DNA oligos with bulky 5-iodouracil and 5-iodocytidine substituents were able to bind to p15NC with little or no perturbations as assessed by their ability to compete with the full-length p15NC mRNA in filter-binding competition assays. In addition, RNA-dependent cleavage of the purified p15NC in vitro by HIV-I protease occurred at rates similar to those reported previously.

Determinants of the human immunodeficiency virus type 1 p15NC-RNA interaction that affect enhanced cleavage by the viral protease.

During human immunodeficiency virus type 1 (HIV-1) virion assembly, cleavage of the Gag precursor by the viral protease results in the transient appearance of a nucleocapsid-p1-p6 intermediate product designated p15NC. Utilizing the p15NC precursor protein produced with an in vitro transcription-translation system or purified after expression in Escherichia coli, we have demonstrated that RNA is required for efficient cleavage of HIV p15NC. Gel mobility shift and nitrocellulose filter binding experiments indicate that purified p15NC protein specifically binds its corresponding mRNA with an estimated Kd of 1.5 nM. Binding was not affected by the presence or absence of zinc or EDTA. Moreover, mutagenesis of the cysteine residues within either of the two Cys-His arrays had no effect on RNA binding or on RNA-dependent cleavage by the viral protease. In contrast, decreased binding of RNA and diminished susceptibility to cleavage in vitro were observed with p15NC-containing mutations in one or more residues within the triplet of basic amino acids present in the region between the two zinc fingers. In addition, we found that 21- to 24-base DNA and RNA oligonucleotides of a particular sequence and secondary structure could substitute for p15 RNA in the enhancement of p15NC cleavage. Virus particles carrying a mutation in the triplet of NC basic residues (P3BE) show delayed cleavage of p15NC and a defect in core formation despite the eventual appearance of fully processed virion protein. These results define determinants of the p15NC-RNA interaction that lead to enhanced protease-mediated cleavage and demonstrate the importance of the triplet of basic residues in formation of the virus core.

A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase.

BACKGROUND: Quinolinic acid (QA) is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS and meningitis. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for QA metabolism and is also an essential step in de novo NAD biosynthesis. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide (NAMN) from QA and 5-phosphoribosyl-1-pyrophosphate (PRPP). The structures of several phosphoribosyltransferases (PRTases) have been reported, and all have shown a similar fold of a five-strandard beta sheet surrounded by four alpha helices. A conserved sequence motif of 13 residues is common to these 'type I' PRTases but is not observed in the QAPRTase sequence, suggestive of a different fold for this enzyme. RESULTS: The crystal structure of QAPRTase from Salmonella typhimurium has been determined with bound QA to 2.8 A resolution, and with bound NAMN to 3.0 A resolution. Most significantly, the enzyme shows a completely novel fold for a PRTase enzyme comprising a two-domain structure: a mixed alpha/beta N-terminal domain and an alpha/beta barrel-like domain containing seven beta strands. The active site is located at the C-terminal ends of the beta strands of the alpha/beta barrel, and is bordered by the N-terminal domain of the second subunit of the dimer. The active site is largely composed of a number of conserved charged residues that appear to be important for substrate binding and catalysis. CONCLUSIONS: The seven-stranded alpha/beta-barrel domain of QAPRTase is very similar in structure to the eight-stranded alpha/beta-barrel enzymes. The structure shows a phosphate-binding site that appears to be conserved among many alpha/beta-barrel enzymes including indole-3-glycerol phosphate synthase and flavocytochrome b2. The new fold observed here demonstrates that the PRTase enzymes have evolved their similar chemistry from at least two completely different protein architectures.

The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate.

The three-dimensional structure of Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5--phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base orotic acid has been solved and refined with X-ray diffraction data extending to 2.3 A resolution to a crystallographic R-factor of 18.7%. The complex was generated by carrying out catalysis in the crystal. Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex [Scapin, G., Grubmeyer, C., and Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] revealed that the enzyme backbone undergoes only small movements. The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5'-phosphate binding loop, and a long, solvent-exposed loop at the dimer interface. The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP.orotate complexes, with its anomeric carbon moving approximately 7 A across the binding cavity. In the PRPP.orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate with the substrate in the OMP complex.

Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase.

Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)