D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.     

Optical measurement of spindle radial motion by moiré technique of concentric-circle gratings

This paper describes an optical moiré method intended for the testing of the radial motion of a rotating spindle. Two concentric-circle gratings of fine pitches were configured for the radial motion to be directly detected by analyzing the interferometric fringes generated by the gratings. This method was immune to mechanical and electrical disturbances since neither master cylinder nor electrical gap gage was needed. Test results demonstrated that a measuring accuracy of less than 0.01 μm can practically be achieved using the method.     

Cross-specialty linkage and extrapolation of resource-based relative value scales

We used the fluorescent labelled dopamine D1-receptor antagonist Bodipy-SCH 23390 for the cellular localization of D1-ligand binding sites in the retinae of different vertebrates (teleosts, Xenopus, turtle, rat and rabbit). Competition experiments with unfixed cryosections of fish retina were performed to characterize the binding conditions of Bodipy-labelled SCH 23390. Tissue bound [3H]SCH 23390 was displaceable with increased amounts of bodipy-SCH 23390. The pharmacological specificity of the D1 fluorescent antagonist was determined by competition experiments with an excess of unlabelled SCH 23390. This treatment significantly reduced the level of fluorescence of the retina confirming the specificity of the binding. We observed a homogeneously distributed fluorescence signal in both plexiform layers in unfixed cryosections of fish, frog, turtle, rat and rabbit. Similar staining intensities of both plexiform layers were found in frog, turtle, rat and rabbit retina. In teleosts, the label of the outer plexiform layer was markedly more intense. Non-specific label was associated with photoreceptor outer and inner segments. The specific labelling of both plexiform layers indicates a mismatch of dopamine releasing and D1-binding sites, and suggests a possible extrasynaptic localization of the D1-receptor. The physiological significance of the observed distribution of D1-ligand binding sites is discussed with respect to the role of dopamine in controlling adaptational processes in the retina.     

Parasitic Bipolar Junction Transistors in a Floating-Gate MOSFET for Fluorescence Detection

In this letter, we examined whether the parasitic bipolar junction transistors (BJTs) in the MOSFET fabricated by the standard CMOS process can play a role as a fluorescence detector. To suppress the action of two vertical parasitic BJTs, the gate and n-well were tied in the parasitic BJTs, and the body node was connected to the drain. The proposed device was compared with the inherent and the parasitic diodes in the MOSFET. It had 100 times higher photocurrents than the diodes in the MOSFET. In addition, it was applied for the detection of the fluorescent signal, and could detect near 10 nM of Alexa 546. Therefore, CMOS-process-compatible parasitic BJTs can be used as a photodetector in an integrated fluorescence detector.     

A novel bidirectional CMOS transceiver for chip-to-chip optical interconnects

A novel bidirectional complementary metal-oxide-semiconductor (CMOS) transceiver for chip-to-chip optical interconnects operating at 2.5 Gb/s is proposed, which shares the common block of a receiver and a transmitter on a single chip. The share of the common block of two circuits makes it possible to save 55% or 20% of power dissipation, depending on the operating mode. The chip in 0.18-/spl mu/m CMOS technology occupies an area of 0.82/spl times/0.82 mm/sup 2/, 70% of the total area of a typical unshared transceiver chip. The transmitting and receiving modes of operation show -3-dB bandwidths of 2.2 and 2.4 GHz and electrical isolations of -28 and -40 dB, respectively.     

Drying of persimmons (Diospyros kaki L.) and the following changes in the studied bioactive compounds and the total radical scavenging activities

Fresh persimmons were subjected to two different processes: sun-drying during 1 month and dehydration at 60 °C during 12 h. To assess the effect of this process on nutritional and health-related properties of persimmons dietary fibers, minerals, trace elements, polyphenols and the total radical scavenging activities (TRSAs) were determined before and after processing. It was found that the contents of dietary fibers, minerals and trace elements in fresh and dried persimmons fruits were comparable. Total polyphenols in fresh persimmons was higher than in dried fruits (1.3 vs. 0.9 and 0.8 mg/100 g FW, respectively) and percentage of inhibition was higher than in dried fruits (70% vs. 59% and 55% and 58% vs. 53% and 46% for 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) [ABTS] radicals, respectively (P>0.05 in all cases). In conclusion: (1) the differences in the contents of dietary fibers, minerals and trace elements in fresh and dried persimmons are not significant; (2) the contents of polyphenols and the level of the TRSA are higher in fresh persimmons than in dried fruits; however, both variables are also high in dried persimmons; (3) when fresh fruits are not available, proper dried persimmons could be used as a valuable substitute.     

Anatomical basis of a congenital hearing impairment: basilar papilla dysplasia in the Belgian Waterslager canary

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.     

A new method of preform design in hot forging by using electric field theory

The preform design in metal forging plays a key role in improving product quality, such as ensuring defect-free property and proper metal flow. In industry, preforms are generally designed by the iterative trial-and-error approach. This approach, however, leads not only to the increase of significant tool cost but also to the extended down-time of the production equipment. It is thus necessary to reduce time and man power through an effective method of preform design. In this paper, the equi-potential lines designed in the electric field are introduced to find an appropriate preform shape. The equi-potential lines generated between two conductors of different voltages show similar trends for the minimum work paths between the undeformed shape and the deformed shape. Based on this similarity, the equi-potential lines obtained by the arrangement of the initial and final shapes are utilized for the design of the preform, and then the artificial neural network is used to find the range of initial volume and potential value of the electric field.