Crystal structures of a marginally active thymidylate synthase mutant, Arg 126-->Glu

Thymidylate synthase (TS) is a long-standing target for anticancer drugs and is of interest for its rich mechanistic features. The enzyme catalyzes the conversion of dUMP to dTMP using the co-enzyme methylenetetrahydrofolate, and is perhaps the best studied of enzymes that catalyze carbon-carbon bond formation. Arg 126 is found in all TSs but forms only 1 of 13 hydrogen bonds to dUMP during catalysis, and just one of seven to the phosphate group alone. Despite this, when Arg 126 of TS from Escherichia coli was changed to glutamate (R126E), the resulting protein had kcat reduced 2000-fold and Km reduced 600-fold. The crystal structure of R126E was determined under two conditions--in the absence of bound ligand (2.4 A resolution), and with dUMP and the antifolate CB3717 (2.2 A resolution). The first crystals, which did not contain dUMP despite its presence in the crystallization drop, displayed Glu 126 in a position to sterically and electrostatically interfere with binding of the dUMP phosphate. The second crystals contained both dUMP and CB3717 in the active site, but Glu 126 formed three hydrogen bonds to nearby residues (two through water) and was in a position that partially overlapped with the normal phosphate binding site, resulting in a approximately 1 A shift in the phosphate group. Interestingly, the protein displayed the typical ligand-induced conformational change, and the covalent bond to Cys 146 was present in one of the protein's two active sites.     

Fatty Acid Binding Protein 6 Inhibition Decreases Cell Cycle Progression,Migration and Autophagy in Bladder Cancers

Bladder cancer (BC) has a high recurrence rate worldwide. The aim of this study was to evaluate the role of fatty acid binding protein 6 (FABP6) in proliferation and migration in human bladder cancer cells. Cell growth was confirmed by MTT and colony formation assay. Western blotting was used to explore protein expressions. Wound healing and Transwell assays were performed to evaluate the migration ability. A xenograft animal model with subcutaneous implantation of BC cells was generated to confirm the tumor progression. Knockdown of FABP6 reduced cell growth in low-grade TSGH-8301 and high-grade T24 cells. Cell cycle blockade was observed with the decrease of CDK2, CDK4, and Ki67 levels in FABP6-knockdown BC cells. Interestingly, knockdown of FBAP6 led to downregulation of autophagic markers and activation of AKT-mTOR signaling. The application of PI3K/AKT inhibitor decreased cell viability mediated by FABP6-knockdown additionally. Moreover, FABP6-knockdown reduced peroxisome proliferator-activated receptor γ and retinoid X receptor α levels but increased p-p65 expression. Knockdown of FABP6 also inhibited BC cell motility with focal adhesive complex reduction. Finally, shFABP6 combined with cisplatin suppressed tumor growth in vivo. These results provide evidence that FABP6 may be a potential target in BC cells progression.     

Prenatal diagnosis of de novo interstitial deletion of proximal 4q by maternal serum screening for Down syndrome

Deletion of the proximal portion of chromosome 4q is apparently rare. To our knowledge, prenatal diagnosis of the interstitial deletion of 4q12-21.1 has never been reported. We present a prenatal case of 4q deletion in association with a positive Down syndrome screening test of an elevated maternal serum free beta human chorionic gonadotrophin (beta-hCG) level. The prenatal sonogram revealed intra-uterine growth retardation (IUGR) and shortening of the femur. Facial dysmorphism included micrognathia, depressed nasal bridge and low-set ears, these anomalies were evident at the postnatal examination. All of the anomalies were consistent with those described in proximal 4q deletion syndrome. Our case suggests that chromosome studies may be indicated for patients with high maternal serum free beta-hCG and IUGR in the early second trimester.     

Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase

5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.     

Sequential invasions of pancreatic pseudocysts in pancreatic tail, hepatic left lobe, caudate lobe, and spleen

A 66-year-old male patient without a history of risk factors for pancreatitis suffered from pancreatitis and developed pseudocyst. During the course of treatment and follow-up, the pseudocyst was found to have migrated through the pancreatic tail, left hepatic lobe, caudate lobe, and spleen on abdominal sonography and computed tomography scan. Finally, emergent laparotomy was done for splenic abscess and removal of infected pseudocyst in the spleen and lesser sac of the abdomen. The patient made a full recovery after operation.     

Primary diffusely infiltrative adenocarcinoma of the colon and rectum

In this study, patient opinion on oral rehabilitation by means of Br?nemark implants was investigated. All patients were referred to a periodontal clinic for implant installation and treated by one and the same operator. Prosthetic restorations were performed by dentists, who had no previous experience with prostheses on implants, but had completed a postgraduate training course. Patient opinion was obtained through questionnaires, pertaining to satisfaction and oral function. A comparison was made between pre-implant situation, short-term (< 4 months) and long-term functioning (3 years) with the implant-restorative rehabilitation. In total, 61 patients participated in the study; 23 received a full lower arch bridge and 18 a full upper arch bridge, while 20 patients got partial bridges. Of 298 installed implants, 7 failed at abutment connection (2.3%) and 1 during the 3-year follow-up interval (0.3%). The study results indicated that a great majority of patients were very satisfied with the treatment. Comfort with eating, aesthetics, phonetics and overall satisfaction improved significantly and nearly all patients said that they would undergo the treatment again or recommend it to others. Patients experienced their implants as "natural" teeth. The conclusion is that rehabilitation ad modum Br?nemark, even in the hands of non-specialized dentists, can be of high quality, improving oral function and satisfying the needs and demands of patients.     

Assessing users'' product-specific knowledge for personalization in electronic commerce

While many electronic commerce (EC) companies are adopting one-to-one marketing approaches using various personalization technologies to make their products and services unique for the purpose of attracting and retaining customers and improving their completion edges in the EC ecosystem, which, nevertheless, has low entrance barriers for new players to join and further intensify the competition, none or few of them consider a fundamental issue—the user's product-specific knowledge. Our research proposed to add this new domain of the customer's knowledge on appropriate target products into the personalization process as a part of the overall EC strategy for businesses. In this paper, we present our initial design for assessing the user's product-specific knowledge using the proposed innovative method for detecting it directly in a non-intrusive way without asking users to answer or fill out any types of questionnaires. Our method is based on customer's on-line navigation behaviors by analyzing their navigation patterns through pre-trained artificial neural networks. An empirical study designed for a case of EC store selling digital cameras was conducted in our research to prove the concept, and a good preliminary result was derived from the study.

For the purpose of comparing the performances between the conventional approach of using questionnaire and the proposed innovative approach of navigation pattern mining, a questionnaire based approach for evaluating the user's product-specific knowledge was designed and incorporated into our knowledge level assessment system (KLAS). Our study result shows that although the pure questionnaire-based KLAS is intrusive and may not be accepted by some users, for those users willing to complete the questionnaire, the proposed navigation pattern approach can be combined with the questionnaire-based approach to create a hybrid KLAS which has a significantly improved accuracy rate in detecting the customer's product knowledge level.     

Abdominal pregnancy--report of two cases and review of literature

Ectopic pregnancy is one of the major complications of pregnancy. The abdominal pregnancy is a comparatively rare type of ectopic gestation with a high fetal and maternal morbidity and mortality. Although abdominal pregnancy had been well documented, it still remains a serious dilemma for most clinicians in the contemporary obstetric practice because of the difficulties in early diagnosis and proper management. We reported two cases of early abdominal pregnancy with accurate preoperative diagnosis followed by immediate surgical intervention and total removal of the placental tissue. The important concepts of management with this uncommon but ominous condition are discussed, including the predisposing risk factors, clinical features, diagnostic difficulties, appropriate surgery and management of the placenta.     

Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3. Molecular cloning, primary sequence, and enzyme expression

The genes for Flavobacterium meningosepticum Endo (endoglycosidase) F2 and Endo F3 were cloned, and their nucleotide sequences were determined. The deduced amino acid sequences were verified independently to a large extent by direct peptide microsequencing of 66 and 84% of native Endo F2 and Endo F3, respectively. Structurally, the Endo F2 and Endo F3 genes code for a typically long leader sequence of 45 and 39 amino acids, respectively, and, in both cases, a mature protein of 290 amino acids. Comparative structural analysis demonstrated minimum overall homology (15-30%) between Endo F1, Endo F2, and Endo F3, but revealed distinct clusters of identical residues distributed throughout the entire sequence, which represent motifs for binding and hydrolysis of beta 1,4-di-N-acetylchitobiosyl linkages in complex carbohydrates. The mobility of native Endo F2 and Endo F3 on SDS-polyacrylamide gel electrophoresis, unlike Endo F1, did not correlate with the molecular weights determined from the coding region of the corresponding genes. Mass spectrometry confirmed that Endo F2 and Endo F3 were heterogeneous and contained approximately 4000 and 1200 daltons of mass not accounted for in the gene structure. We presume that Endo F2 and Endo F3 are variably post-translationally modified during secretion by possible linkage to the hydroxyl of serine.     

Mining association rules procedure to support on-line recommendation by customers and products fragmentation

Electronic Commerce (EC) has offered a new channel for instant on-line shopping. However, there are too many various products available from a great number of virtual stores on the Internet for Internet shoppers to select. On-line one-to-one marketing therefore becomes a great assistance to Internet shoppers. One of the most important marketing resources is the prior daily transaction records in the database. The great amount of data not only gives the statistics, but also offers the resource of experiences and knowledge. It is quite natural that marketing managers can perform data mining on the daily transactions and treat the shoppers the way they prefer. However, the data mining on a significant amount of transaction records requires efficient tools. Data mining from automatic or semi-automatic exploration and analysis on a large amount of data items set in a database can discover significant patterns and rules underlying the database. The knowledge can be equipped in the on-line marketing system to promote Internet sales.

The purpose of this paper is to develop a mining association rules procedure from a database to support on-line recommendation. By customers and products fragmentation, product recommendation based on the hidden habits of customers in the database is therefore very meaningful. The proposed data mining procedure consists of two essential modules. One is a clustering module based on a neural network, Self-Organization Map (SOM), which performs affinity grouping tasks on a large amount of database records. The other rule is extraction module employing rough set theory that can extract association rules for each homogeneous cluster of data records and the relationships between different clusters. The implemented system was applied to a sample of sales records from a database for illustration.