Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual,SPR and NMR screening

NDM-1 can hydrolyze nearly all available β-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of ¹⁵N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.     

Cord-factor analogs: synthesis of 6,6'-di-O-mycoloyl- and -corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside)

Appropriate solvolysis of 2,3,2',3'-tetra-O-benzyl-4,6,4', 6'-tetra-O-mesyl-alpha,alpha-trehalose gave 2,3,2',3' -tetra-O-benzyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) (2). Selective tosylation or mesylation of 2 respectively gave the 6, 6'-ditosylate (3) and 6,6'-dimesylate (4), the structures of which were confirmed by the 1H-n.m.r. spectra of the corresponding 4,4'-di-O-acetyl derivatives. Treatment of 3 with potassium mycolate in toluene, and subsequent hydrogenolysis, gave the 6'-mycolate 6-tosylate derivative. Treatment of 3 with potassium mycolate or potassium corynomycolate in hexamethylphosphoric triamide, followed by catalytic hydrogenolysis, yielded the respective cord-factor analogs 6,6'-di-O-mycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside) and 6,6'-di-O-corynomycoloyl-(alpha-D-galactopyranosyl alpha-D-galactopyranoside). The same 6,6'-diesters were obtained from the 6,6'-dimesylate 4. Putative 4,6-anhydro-6'-monomycolates are also described.     

The structure of 180° supernumerary limbs and a hypothesis of their formation

The results of a detailed analysis of 100 supernumerary limbs generated by 180° ipsilateral rotation (on the same limb stump) of regeneration blastemas is presented. The limbs were analyzed in terms of their position of origin, frequency, cartilage structure by Victoria blue staining, and muscle structure by serial sections. Single, double, or triple supernumeraries can be produced at no unique position of origin, although the posterodorsal quadrant was preferred. Four classes of supernumerary limbs were generated by such operations—normal; double dorsal or double ventral; part normal/part mirror imaged; part normal/part inverted in approximately equal frequencies. After amputation of these supernumeraries the same muscle patterns are faithfully regenerated. A hypothesis to explain the production of these abnormal limbs is proposed based on the observed phenomenon of fusion of supernumerary blastemata, but their regenerative behaviour presents problems for current models of pattern formation. Similar results have been obtained with developing limb buds and the relation between development and regeneration is discussed.     

In vitro growth of murine T cells. IV. Use of T-cell growth factor to clone lymphoid cells

Murine bone marrow cells can suppress the in vitro primary antibody response of normal spleen cells without apparent cytotoxicity. The bone marrow cells suppress the response to both T-dependent (SRBC) and T-independent (DNP-Ficoll) antigens. When bone marrow cells are fractionated on a sucrose density gradient, the suppressive activity is found in the residue rather than the lymphocyte fraction. The suppressive activity is either unaffected or enhanced by treatment with anti-T- and anti-B-cell serums. Pretreatment of mice with phenylhydrazine which reduces the number of pre-B cells did not reduce the suppressive activity of their bone marrow cells. Suppressive activity is abolished by irradiation of the marrow cells in vitro with 1000 R prior to assay. The activity is present in the marrow of thymus deficient (nude) mice, infant mice, and mice which have been made polycythemic by transfusion. Furthermore, the suppressor cell can phagocytize iron carbonyl particles, is slightly adherent to plastic and Sephadex G-10, and can bind to EA monolayers. We conclude that the suppressor cell is not a mature lymphocyte or granulocyte nor a member of the erythrocytic series, but is likely to be an immature cell possibly of the myeloid series. We speculate on the physiologic role of this cell.     

Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at ?30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.