DFT calculation–assisted stereo-structural assignment of arundifungin

Arundifungin ( 1 ) has been reported as a potent antifungal agent against Candida and Aspergillus spp; however, only its planar structure has been disclosed. This paper describes the assignment of the relative and absolute configuration of 1 , which includes (a) determination of the relative configuration of the ABCD polycyclic ring moiety on the basis of detailed nuclear magnetic resonance (NMR) analysis, followed by the confirmation with density functional theory (DFT)–based ¹³C NMR chemical shift calculations, (b) determination of the absolute configuration of the ABCD polycyclic ring moiety by observing a positive Cotton effect at 217 nm because of the C-8/C-9 tetrasubstituted double bond and its reproduction using DFT calculations, (c) determination of the configurational relationship between C-17 and C-20 by a combination of nuclear Overhauser effect (NOE) analysis and DFT-based conformational analysis, and (d) determination of the relative and absolute configuration of the C-24 and C-25 asymmetric centers on the acyclic side chain by a combination of chemical derivatization including modified Mosher's method and DFT-based conformational analysis, followed by electronic circular dichroism (ECD) spectral reproduction. Present study also discovered 26-deoxyarundifungin ( 2 ) of which relative structure was readily elucidated by ¹H and ¹³C spectral comparison with those of 1 . Since 2 exhibits slightly weaker antifungal activity against Cochliobolus miyabeanus than 1 , the hydroxy group at C-26 moderately contributes to the activity.     

Ultrasound-guided non-surgical embryo collection in the common marmoset

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.     

C lambda 2 and C lambda 4 immunoglobulin light chain genes in a wild-derived inbred mouse strain

A genomic clone containing the lambda constant (C) region genes (C lambda 2S and C lambda 4S) has been isolated from a genomic library from the mouse strain SPE. SPE is an inbred strain derived from progenitors trapped near Grenada in Spain and has been classified as mus 3 or mus spretus. The sequence of the C lambda 2S gene is virtually identical to that of BALB/c both in the coding region and in flanking sequences, suggesting that it is an expressed gene in the SPE strain. By contrast, the C lambda 4S gene on the same cluster has diverged in sequence from that of BALB/c and contains a large deletion that precludes its normal expression. Whereas BALB/c J lambda 4 region contains substitutions that probably preclude its usage, the SPE J lambda 4 gene includes all sequences required for a functional J gene. Comparison of the C lambda 2S and C lambda 4S gene sequences with those available for BALB/c C lambda 3 and C lambda 1 confirms the close relationship between the C lambda 1-C lambda 4 and C lambda 2-C lambda 3 gene pairs. The C lambda 3 gene of BALB/c is more closely related to C lambda 2S than is C lambda 1 of BALB/c to C lambda 4S. If it is assumed that C lambda 1 and C lambda 2 are respective duplicates of C lambda 4 and C lambda 3 and that these duplications occurred at the same time, then the C lambda 2 gene has been under stronger selective pressure than C lambda 4.     

Identification of the Region Required for Monomerization of the Rolling Circle Plasmid pKYM

Plasmid pKYM, isolated from the Gram-negative bacterium Shigellasonnei, is a small multicopy plasmid which replicates by a rollingcircle mechanism. The formation of multimers has been observedin a derivative of pKYM which lost a part of the origin region,and the loss of the monomerization mechanism would have ledto these multimers. By analyzing the constructs of several mutants,we discovered that a DNA region required for monomerizationwas present upstream of the RepK binding site in the replicationorigin. As either of the T-rich sequence or the inverted repeatsequences which were seen in that region have been lost in themultimer-forming plasmids, these sequences may be necessaryfor monomerization.     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

T-cell immune response to human herpesvirus-6 in healthy adults.

The proliferative response of peripheral blood mononuclear cells (PBMC) from healthy adults to human herpesvirus-6 (HHV-6) was examined. It was found that PBMC from 13 of 14 HHV-6-seropositive adults apparently proliferated in response to stimulation with HHV-6 antigen in contrast to the lack of response of cord blood mononuclear cells. In order to confirm the presence of HHV-6-specific memory T cells in the peripheral blood of healthy adults, we established HHV-6-specific T-cell clones from an HHV-6-seropositive individual. CD4+ T-cell clones generated from HHV-6-stimulated PBMC were found to proliferate upon stimulation with HHV-6 in the presence of autologous antigen-presenting cells, but not in response to herpes simplex virus type 1 antigen or mock-infected control antigen. These results indicate that a T-cell immune response against HHV-6 infection is generally present in healthy adult populations.