Nomogram to predict survival of patients with advanced and metastatic pancreatic Cancer.

BACKGROUND: Nomograms are rarely employed to estimate the survival of patients with advanced and metastatic pancreatic cancer (PC). Herein, we developed a comprehensive approach to using a nomogram to predict survival probability in patients with advanced and metastatic PC. METHODS: A total of 323 patients with advanced and metastatic PC were identified from the Chinese People's Liberation Army (PLA) General Hospital. A baseline nomogram was constructed using baseline variables of 323 patients. Additionally, 233 patients, whose tumors showed initial responses to first-line chemotherapy, were enrolled in the chemotherapy response-based model. 128 patients and 108 patients with advanced and metastatic PC from January 2019 to April 2021 were selected for external validating baseline model and chemotherapy response-based model. The 1-year and 2-year survival probability was evaluated using multivariate COX regression models. The discrimination and calibration capacity of the nomograms were assessed using C-statistic and calibration plots. The predictive accuracy and net benefit of the nomograms were evaluated using ROC curve and DCA, respectively. RESULTS: In the baseline model, six variables (gender, KPS, baseline TB, baseline N, baseline WBC and baseline CA19-9) were used in the final model. In the chemotherapy response-based model, nine variables (KPS, gender, ascites, baseline N, baseline CA 19-9, baseline CEA, change in CA 19-9 level at week, change in CEA level at week and initial response to chemotherapy) were included in the final model. The C-statistics of the baseline nomogram and the chemotherapy response-based nomogram were 0.67 (95% CI, 0.62-0.71) and 0.74 (95% CI, 0.69-0.77), respectively. CONCLUSION: These nomograms were constructed to predict the survival probability of patients of advanced and metastatic PC. The baseline model and chemotherapy response-based model performed well in survival prediction.

[Investigation of the cognition and behavior on drug safety in Beijing middle school students].

OBJECTIVE: To understand the cognition and behavior of drug safety in Beijing middle school students and provide advice for relevant education. METHODS: A cross-sectional survey using paper questionnaires was carried out on the student body of nine Beijing middle schools. Multi-stage proportionate stratified cluster sampling was adopted to enroll participants. In addition to demographic questions, the questionnaire included 17 questions assessing the cognition and behavior of safe drug use, prioritizing questions that aligned with the health education guideline for primary and secondary school students from Chinese Ministry of Education. Descriptive statistical methods were applied using the SAS 9.2 software. RESULTS: Of the 4 220 students investigated, 2 097(49.7%) were males and 2 123(50.3%) were females. The average age was (14.3±1.7) years. 2 030(48.1%) students were from downtown areas, 1 511(35.8%) were from urban-rural linking areas and 679(16.1%) were from rural areas. Half (51.5%) of the respondents were junior high school students, and the others were from senior high schools (34.2%) and vocational high schools (14.3%). Most of the students (89.6%) lived off campus. The awareness rate of drug safety knowledge was 74.4%, the median score of drug safety behavior was 4 points (full score was 5 points) and there was a statistically positive correlation between the two (Spearman's correlation coefficient was 0.156, P<0.001). Both the awareness rates and the drug safety behavior scores were statistically different among the students in different regions, different school types and different residence types (P<0.001). Multiple factors analysis demonstrated the correlation between the cognition degrees of both drug safety knowledge, behavior and the above factors. Of all the students, 80.4% agreed that any drug could have adverse drug reactions; 40.5% were aware that antibiotics couldn't kill viruses; as many as 49.6% mistook aspirin as antibiotic; 97.4% would read drug instructions before taking them; Only 42.4% put expired drugs into special recycling bins; 49.8% would deviate from the suggested dosage and frequency of their medication when they were sick with common diseases. CONCLUSION: Overall, the cognition of drug safety in Beijing middle school students is good, but problems still exist in medication adherence, the management of expired drugs and the antibiotics cognition, which need to be fixed through specific, pointed way of education. And more efforts should be made to improve the cognition in rural regions, vocational high schools and on campus students.

Therapeutic effect of Pleurotus eryngii cellulose on experimental fatty liver in rats.

The aim of this study was to explore the therapeutic effect of Pleurotus eryngii cellulose on experimental fatty liver in rats. Rats were fed high-fat fodder to establish a rat fatty liver model, and were then fed different concentrations of Pleurotus eryngii cellulose for six weeks. Lipitor was used as a positive control. Measured levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and total triglyceride (TG); the activity of malondialdehyde (MDA), superoxide dismutase (SOD), hepatic lipase (HL), and lipoprotein lipase; and liver histopathological changes. Successfully established rat fatty liver model after feeding high-fat fodder for one week. A diet of P. eryngii cellulose for six weeks significantly reduced ALT, AST, TC, and TG levels in rat serum (P < 0.01); TC and AST levels in P. eryngii cellulose high-dose group and Lipitor group were not significantly different from those of the control (P > 0.05). SOD activity increased significantly, while MDA and HL activity decreased (P < 0.05); fatty degeneration and fat accumulation both decreased in hepatic tissue. Hepatic protection of P. eryngii cellulose showed dose-related effect. P. eryngii cellulose can affect lipid metabolism, having therapeutic effects on fatty liver in rats.

Screening of differentially expressed genes between multiple trauma patients with and without sepsis.

The purpose of this study was to identify critical genes associated with septic multiple trauma by comparing peripheral whole blood samples from multiple trauma patients with and without sepsis. A microarray data set was downloaded from the Gene Expression Omnibus (GEO) database. This data set included 70 samples, 36 from multiple trauma patients with sepsis and 34 from multiple trauma patients without sepsis (as a control set). The data were preprocessed, and differentially expressed genes (DEGs) were then screened for using packages of the R language. Functional analysis of DEGs was performed with DAVID. Interaction networks were then established for the most up- and down-regulated genes using HitPredict. Pathway-enrichment analysis was conducted for genes in the networks using WebGestalt. Fifty-eight DEGs were identified. The expression levels of PLAU (down-regulated) and MMP8 (up-regulated) presented the largest fold-changes, and interaction networks were established for these genes. Further analysis revealed that PLAT (plasminogen activator, tissue) and SERPINF2 (serpin peptidase inhibitor, clade F, member 2), which interact with PLAU, play important roles in the pathway of the component and coagulation cascade. We hypothesize that PLAU is a major regulator of the component and coagulation cascade, and down-regulation of PLAU results in dysfunction of the pathway, causing sepsis.

[A 5-year retrospective study of computer aided design and computer aided manufacturing ceramic endocrowns in endodontically treated posterior teeth].

Objective: To evaluate the clinical outcomes of computer aided design and computer aided manufacturing (CAD/CAM) ceramic endocrowns in endodontically treated posterior teeth after five years by a retrospective study. Methods: Patients who received CAD/CAM ceramic endocrowns after endodontically treatment in Department of Endodontics, School of Stomatology, The Fourth Military Medical University between January 2016 and June 2017 were invited for this clinical study. Clinical performance was evaluated in the aspect of color match, anatomic form,marginal adaptation, restoration integrity and secondary caries. Survival rate of the restorations was calculated by the use of Kaplan-Meier method. Log-rank test was applied as well for the sake of analyzing the effect of tooth position, sex and materials to the survival rate of the restorations. Results: Seventy-four patients, 25 men and 49 women with age of (38.8±10.2) years, participated in this study for a total of 101 CAD/CAM ceramic endocrowns after observation period of (62.8±12.0) months. There were 8 failed cases among 101 restorations, 5 were loss of retention, 2 were ceramic fracture and 1 was secondary caries respectively. In particular, 93% (89/96) restorations got score A on anatomic form and 95% (91/96) restorations got score A on marginal adaptation, while 38% (36/96) restorations showed the good color match compared with the abutment teeth. The estimated cumulative survival rate of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after 5 years was 93.0% (95%CI: 87.9%-98.1%). The single-factor Log-rank analysis demonstrated that there was no statistically significant difference in the survival rate of CAD/CAM ceramic endocrowns among men and women, premolars and molars, position in the dental arch, or different materials (χ²<0.01, P=0.957; χ²=0.64, P=0.422; χ²=0.69, P=0.407; χ²=0.88, P=0.349). Conclusions: Based on this clinical study, the clinical performance of CAD/CAM ceramic endocrowns in endodontically treated posterior teeth after five years is reliable, which could be a general option to restore nonvital teeth.

[Association between statins use and liver injury based on prescription sequence symmetry analysis].

OBJECTIVE: To study the association between statins use and liver-injury through prescription sequence symmetry analysis(PSSA)and evaluate the feasibility of the method to be used in Chinese Medical Insurance Database. METHODS: The data of the patients who prescribed both statins and liver-proactive drugs in Chinese Basic Medical Insurance Database in 2013 were selected as study subjects to calculate the adjusted sequence ratio(ASR)with signal detection methods to determine the study parameters and investigate the potential association between statins use and liver-injury. RESULTS: In 5 649 individuals which met the inclusion criteria, the washout period was set as one month and interval period was set as 60 days. The overall ASR of statins was 1.471(95%CI: 1.395-1.550), the ASR of atorvastatin was 1.419(95% CI: 1.335-1.508), the ASR of simvastatin was 1.307(95%CI: 1.164-1.467). The positive signal was strong in 30 days interval period. CONCLUSIONS: PSSA indicated that there might be potential association between statins use and liver-injury, especially the uses of atorvastatin and simvastatin. This signal detection method may be a fast and effective method in drug safety evaluation and can be used in Chinese Medical Insurance Database.

The complete nucleotide sequence of the mouse thyroid-specific enhancer-binding protein (T/EBP) gene: extensive identity of the deduced amino acid sequence with the human protein.

A mouse thyroid-specific enhancer-binding protein (T/EBP) gene and its flanking regions have been cloned and completely sequenced. The gene consists of 2 exons and exhibits high similarity (83-97%) to the rat sequence throughout the coding region and including an intron and up to 1.3 kbp upstream to the ATG initiation codon. A cDNA clone encoding human T/EBP has been also isolated and sequenced. Comparison of the deduced amino acid sequence of T/EBP revealed an extensive identity of 98% between mouse and the human protein.

Demonstration of a functionally active tPA-like plasminogen activator in human platelets.

The mechanism of platelet-enhanced fibrinolysis is unclear. We therefore investigated the fibrinolytic activity of human platelets and demonstrated that they contain a tissue plasminogen activator (tPA)-like plasminogen activator, abbreviated as tPA-like-PA. This activator was detected by ELISA in platelet incubation medium and in platelet Triton extracts. Plasminogen activation assays showed that this tPA-like-PA could induce plasminogen activation to form plasmin. Western blots of Triton extracts incubated with anti-tPA antibody demonstrated a major 64-kD protein band, compared to a 70-kD band for standard single chain tPA, plus a minor 118-kD band corresponding to a complex of tPA-like-PA and plasminogen activator inhibitor (PAI-1). Western blots of Triton extracts incubated with anti-PAI-1 antibody produced an approximately similar high-molecular-weight (118 kD) protein band. Fibrin zymographic analysis of affinity-purified tPA-like-PA demonstrated a major and a minor fibrin lysis zone, which approximately corresponded to the tPA-like-PA and its complex with PAI-1 observed by Western blots. Immunogold labelling and electron microscopy demonstrated that platelet activator, either as the free form or co-localized with PAI-1, was present in granules and in channels of the open canalicular system. We conclude that platelets contain a functionally active tPA-like-PA, whose low fibrinolytic activity might be due to its readily forming a complex with PAI-1. This functionally active tPA-like-PA might contribute to the enhanced fibrinolytic activity of platelets observed in platelet-rich thrombi.

Optical coherence tomography for noninvasive diagnosis of epithelial cancers.

We summarize our recent progress in the development of the optical coherence tomography (OCT) systems suitable for clinical diagnosis and the preliminary results for in vivo diagnosis of epithelial cancers (e.g., bladder cancers). The endoscopic spectral-domain OCT system allows simultaneous, real-time, cross-sectional OCT images of tissue structure and functions (i.e., local Doppler blood flow) of biological tissue for enhanced diagnosis. A new approach to use spectral demodulation of elastic scattering is discussed for potential cancer grading. The transverse and axial resolutions of the OCT scopes are 12 microm and 10 microm, respectively. Results of the preliminary clinical studies show that unlike animal carcinogenesis models, bladder cancers in humans are more complicated in terms of epithelial backscattering changes: some lesions exhibit enhanced backscattering; some show reduced scattering owing to complex surface condition changes such as asperities or invaginations induced by tumorigenesis (e.g., papillary transitional cell cancers). Nevertheless, promising results can be provided by incorporating other diagnostic parameters such as changes in local vasculature and urothelial heterogeneity.

Fluorescence guided optical coherence tomography for the diagnosis of early bladder cancer in a rat model.

PURPOSE: We describe the technique of fluorescence image guided optical coherence tomography (FG-OCT). We examined its ability to enhance specificity and sensitivity for the noninvasive diagnosis of early bladder cancer. MATERIALS AND METHODS: Transitional cell carcinoma was developed in 54 Fisher 344 female rats by intravesical methyl-nitroso-urea instillations. Two or three rats were diagnosed sequentially by 5-ALA (5-aminolevulinic acid hydrochloride) induced fluorescence imaging, cross-sectional OCT and histological microscopy weekly during weeks 11 to 33 following initial methyl-nitroso-urea instillation to track the course of carcinogenesis. RESULTS: The specificity of fluorescence detection was significantly enhanced by FG-OCT (53% and 93%, respectively, p <0.0001). The sensitivity of fluorescence detection and FG-OCT was 79% and 100%, respectively. CONCLUSIONS: FG-OCT cystoscopy has the potential to diagnose early bladder cancer with high sensitivity and specificity with drastically decreased imaging time compared to that of white light guided OCT cystoscopy.

High-resolution imaging characterization of bladder dynamic morphophysiology by time-lapse optical coherence tomography.

We report an experimental study of the possibility of high-speed optical coherence tomography (OCT) for high-resolution imaging characterization of detrusor dynamic morphophysiology and analysis of the mechanisms that lead to geriatric incontinence (GI). The spontaneous contractility of intact fresh rabbit bladders was imaged with two-dimensional (2D) OCT ex vivo at up to 8 frames/s. The time-lapse 2D OCT images were postprocessed by image segmentation and fast-Fourier-transform analysis to characterize the dynamic morphological changes of the bladder contractility. In addition, we studied young and aging rat bladders to analyze the differences in dynamics. Preliminary results of our ex vivo study reveal that time-lapse OCT can track the contractile waves of bladders at high spatial resolution and characterize their dynamic morphophysiology in terms of amplitude, phase, and frequency. The results suggest that time-lapse OCT has the potential to act as a detrusor optical biopsy to enhance the diagnosis of detrusor dysfunction and thus of the mechanisms that lead to GI.

Inhibition of the trehalose-P synthase of mycobacteria by various antibiotics.

A number of antibiotics were tested as potential inhibitors of the purified trehalose-P synthase of Mycobacterium smegmatis. Of about 30 compounds tested, 4 (cathomycin, circulin, diumycin, and moenomycin) were active against this enzyme. Thus each of these compounds inhibited the formation of trehalose-P by the purified trehalose-P synthase when either UDP-glucose or GDP-glucose was used as the glucosyl donor. However, preincubation of the synthase with heparin, a polyanion activator of the enzyme when UDP-glucose is used as the substrate, prevented the inhibition by these various antibiotics. Fifty percent inhibition by diumycin and moenomycin occurred at a concentration of about 50 microg/ml (Ki of about 1 x 10(-5) M), but 50% inhibition by cathomycin and circulin required substantially higher concentrations (about 50 to 200 microg/ml). The inhibition by cathomycin, diumycin, and moenomycin was of the competitive type, whereas that by circulin was noncompetitive in nature. However, the inhibition was of a complex nature and the data suggest two different binding sites for these inhibitors. Photoaffinity labeling of the synthase with an azido-UDP-[32P]glucose probe was effectively blocked by diumycin, moenomycin, or cathomycin indicating that these inhibitors do interact at the substrate binding site. These antibiotics also inhibited the growth of M. smegmatis when added to cells innoculated into trypticase soy broth. The inhibition of growth was concentration-dependent and directly proportional to the size of the bacterial innoculum. These antibiotics, however, did not inhibit protein synthesis nor did they inhibit the incorporation of mannose into lipid-linked saccharides.

The effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells.

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with [2-3H]mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Methylation analysis also indicated that this Man5GlcNAc2 contained 1----3 linked mannose residues. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps by inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with [3H]mannose or [3H]galactose. In contrast, control cells produced complex and high-mannose structures but no hybrid oligosaccharides were detected. The inhibition by mannosamine could be overcome by adding high concentrations of glucose to the medium.

The formation of lipid-linked oligosaccharides in Madin-Darby canine kidney cells. Changes in oligosaccharide profiles induced by glucosamine.

Glucosamine inhibits the incorporation of [2-3H]mannose into lipid-linked oligosaccharides and into glycoproteins in influenza virus-infected MDCK cells. Fifty per cent inhibition of these components requires about 2 mM glucosamine. The oligosaccharide portions of the lipid-linked oligosaccharides in cells inhibited with glucosamine were compared to that of normal cells by chromatography on Bio-Gel P-4 columns. In uninhibited cells, the major oligosaccharide formed from [2-3H]mannose was the Glc3Man9GlcNAc2 species as demonstrated by the products of endoglucosaminidase H and alpha-mannosidase digestion. At low concentrations of glucosamine (approximately 2 mM) or in short term incubations (1 to 2 h), the large oligosaccharide disappeared and was replaced by a Man7GlcNAc2 species. This was also characterized by various enzymatic treatments as well as its migration rate on Bio-Gel P-4 as compared to known oligosaccharides. At still higher glucosamine concentrations or longer incubation times, the Man7GlcNAc2 species also disappeared and was replaced by a Man3GlcNAc2 species. The effect of glucosamine was reversible such that when the cells were washed free of this inhibitor, they resumed the synthesis of the Glc3Man9GlcNAc2 species and the other two oligosaccharides disappeared. These smaller oligosaccharides were not observed when glucosamine was replaced by either 5 mM galactosamine or 5 mM N-acetylglucosamine.

Control of N-linked oligosaccharide synthesis: cellular levels of dolichyl phosphate are not the only regulatory factor.

When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.

The effects of castanospermine and swainsonine on the activity and synthesis of intestinal sucrase.

Castanospermine is an indolizidine alkaloid that is found in the seeds of the Australian tree Castanospermum australe. These seeds have been reported to be toxic to animals and to cause severe gastrointestinal upset. In order to determine whether castanospermine is responsible for this toxicity, the alkaloid was injected into young mice or rats, and its effects on various intestinal disaccharidases were determined. Another indolizidine alkaloid, the alpha-mannosidase inhibitor swainsonine, was also tested to compare its effects to those of castanospermine. Castanospermine strongly and rapidly inhibited the activity of the disaccharidases, sucrase, maltase, and trehalase, with sucrase being the most sensitive to inhibition. The loss of activity of these enzymes, especially sucrase, in injected animals appeared to be due to a direct inhibition of enzyme activity, rather than to a change in the structure of the glycan chains of the enzyme, since only minor alterations in carbohydrates were observed. On the other hand, swainsonine, when injected into animals, also profoundly decreased the activity of the sucrase, but this alkaloid had no direct effect on sucrase activity although it did markedly alter the carbohydrate nature of this glycoprotein. This change in oligosaccharide structure may affect protein conformation, stability, or targeting, any or all of which may in turn affect activity. In in vitro studies with the purified enzyme, castanospermine was found to be a competitive inhibitor of intestinal sucrase, but it was a noncompetitive inhibitor of intestinal maltase. A number of other glucosidase inhibitors that inhibit sucrase activity in vitro are also described.

Trehalose-P synthase of mycobacteria: its substrate specificity is affected by polyanions.

The trehalose-P synthase was purified to near homogeneity from the cytoplasmic fraction of Mycobacterium smegmatis. At the final stage of purification, the enzyme preparation showed one major band of 59 kDa on SDS gels. The 59 kDa band became labeled with N3-UDP[32P]-glucose, and this labeling was inhibited in a concentration-dependent manner by either unlabeled UDP-glucose or GDP-glucose. The native enzyme also had a molecular weight of about 60 kDa by gel filtration, indicating that the active enzyme is a monomer. The 59 kDa protein was subjected to endoproteinase Lys-C digestion, and three peptides isolated by HPLC were sequenced. The sequences of 56 amino acids in these three peptides showed 60% identity to the trehalose-P synthases of Saccharomyces cerevesiae and Schizosaccharomyces pombe. The purified mycobacterial enzyme catalyzed the synthesis of trehalose-P from glucose-6-P and a variety of nucleoside diphosphate glucose derivatives, depending on whether a polyanion was absent or present. Thus, UDP-glucose and GDP-glucose were the best glucosyl donors, but maximum activity with UDP-glucose required the presence of a polyanion such as heparin, whereas activity with GDP-glucose was relatively independent of polyanion. The presence of heparin in the incubation mixture increased the affinity of the enzyme for UDP-glucose by a factor of 100, or more. However, the affinity for GDP-glucose was only twofold better in the presence of heparin. The purified synthase also utilized ADP-glucose and CDP-glucose, but the K(m) for these glucosyl donors was quite high even in the presence of polyanion. The effect of heparin on UDP-glucose activity was dose-dependent and maximum at about 1-2 micrograms of heparin/incubation. However, the size of the heparin molecule (i.e., the number of monosaccharide residues) was critical for activation, and only those heparins with 18 or more monosaccharide units were effective in stimulating activity.

Trehalose-phosphate synthase of Mycobacterium tuberculosis. Cloning, expression and properties of the recombinant enzyme.

The trehalose-phosphate synthase (TPS) of Mycobacterium smegmatis was previously purified to apparent homogeneity and several peptides from the 58 kDa protein were sequenced. Based on that sequence information, the gene for TPS was identified in the Mycobacterium tuberculosis genome, and the gene was cloned and expressed in Escherichia coli with a (His)6 tag at the amino terminus. The TPS was expressed in good yield and as active enzyme, and was purified on a metal ion column to give a single band of approximately 58 kDa on SDS/PAGE. Approximately 1.3 mg of purified TPS were obtained from a 1-L culture of E. coli ( approximately 2.3 g cell paste). The purified recombinant enzyme showed a single band of approximately 58 kDa on SDS/PAGE, but a molecular mass of approximately 220 kDa by gel filtration, indicating that the active TPS is probably a tetrameric protein. Like the enzyme originally purified from M. smegmatis, the recombinant enzyme is an unusual glycosyltransferase as it can utilize any of the nucleoside diphosphate glucose derivatives as glucosyl donors, i.e. ADP-glucose, CDP-glucose, GDP-glucose, TDP-glucose and UDP-glucose, with ADP-glucose, GDP-glucose and UDP-glucose being the preferred substrates. These studies prove conclusively that the mycobacterial TPS is indeed responsible for catalyzing the synthesis of trehalose-P from any of the nucleoside diphosphate glucose derivatives. Although the original enzyme from M. smegmatis was greatly stimulated in its utilization of UDP-glucose by polyanions such as heparin, the recombinant enzyme was stimulated only modestly by heparin. The Km for UDP-glucose as the glucosyl donor was approximately 18 mm, and that for GDP-glucose was approximately 16 mm. The enzyme was specific for glucose-6-P as the glucosyl acceptor, and the Km for this substrate was approximately 7 mm when UDP-glucose was the glucosyl donor and approximately 4 mm with GDP-glucose. TPS did not show an absolute requirement for divalent cations, but activity was increased about twofold by 10 mm Mn2+. This recombinant system will be useful for obtaining sufficient amounts of protein for structural studies. TPS should be a valuable target site for chemotherapeutic intervention in tuberculosis.