Response of murine gamma delta T cells to the synthetic polypeptide poly-Glu50Tyr50

Random heterocopolymers of glutamic acid and tyrosine (pEY) evoke strong, genetically controlled immune responses in certain mouse strains. We found that pE50Y50 also stimulated polyclonal proliferation of normal gamma delta, but not alpha beta, T cells. Proliferation of gamma delta T cells did not require prior immunization with this Ag nor the presence of alpha beta T cells, but was enhanced by IL-2. The gamma delta T cell response proceeded in the absence of accessory cells, MHC class II, beta 2-microglobulin, or TAP-1, suggesting that Ag presentation by MHC class I/II molecules and peptide processing are not required. Among normal splenocytes, as with gamma delta T cell hybridomas, the response was strongest with V gamma 1+ gamma delta T cells, and in comparison with related polypeptides, pE50Y50 provided the strongest stimulus for these cells. TCR gene transfer into a TCR-deficient alpha beta T cell showed that besides the TCR, no other components unique to gamma delta T cells are needed. Furthermore, interactions between only the T cells and pE50Y50 were sufficient to bring about the response. Thus, pE50Y50 elicited a response distinct from those of T cells to processed/presented peptides or superantigens, consistent with a mechanism of Ig-like ligand recognition of gamma delta T cells. Direct stimulation by ligands resembling pE50Y50 may thus selectively evoke contributions of gamma delta T cells to the host response.     

Histochemical and Ultrastructural Analyses of the Epithelial Cells of the Body Surface Skin from the Terrestrial Slug, Incilaria fruhstorferi

Dorsal and ventral epithelium of the terrestrial slug, Incilaria fruhstorferi, is simple and consists of five cell types: microvillous, ciliated, round mucous, tubular mucous and channel. Microvillous cells were similar to human intestinal epithelial cells morphologically and functionally. At the base of microvilli, pinocytic vesicles which ultimately fused to form larger vacuoles, or multivesicular bodies were present. At the edge of tail or mouth, ciliated epithelial cells possessed the typical axonemes (9 plus 2 arrangement of microtubles). Mucous secretory cells were either tubular or round and their granules were membrane-bound and secreted by exocytosis. Granules of round mucous cells were proteinaceous but those of tubular cells were acidic mucopolysaccharides. Channel cells were elongate U-shaped and the central lumen was filled with a large amount of fluid (hemolymph). The function of channel cells is thought to remove hemolymph accumulated during hyperhydration. Our experiments of some markers-injection revealed that the fluid containing large molecules passed transcellularly from the hemolymph, across the basal or side region of the cell and into the central lumen. These results suggest that channel cell of the slug skin and vertebrate nephron showed some parallels in structure and function.     

Synthesis and biological activity of 1beta-methyl-2-[5'-isoxazoloethenylpyrrolidin-3'-ylthio]carbapenems

A new series of 1beta-methylcarbapenems 1a-i bearing isoxazoloethenyl groups on the pyrrolidine ring has been prepared and evaluated for in vitro antibacterial activity and stability to DHP-I. Most compounds showed excellent antibacterial activity and high stability to DHP-I superior to that of meropenem. Of these new carbapenems, 1a,b,h exhibited the best combination of antibacterial activity and DHP-I stability.     

Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties

Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin.     

p97 ATPase, an ATPase involved in membrane fusion, interacts with DNA unwinding factor (DUF) that functions in DNA replication

DNA unwinding factor (DUF) unwinds duplex DNA and is supposed to function in DNA replication in Xenopus egg extracts. Here we report the isolation and analysis of a DUF-interacting factor. By immunoprecipitation, we found that p97 ATPase (p97) interacts with DUF in Xenopus egg extracts. This interaction was confirmed by the in vitro binding of purified p97 with DUF. When sperm chromatin was added to Xenopus egg extracts to construct nuclei active in DNA replication, p97 was incorporated into the nuclei. These data suggest that the complex of DUF and p97 may function in DNA replication.     

New bioactive aromatic compounds from Vismia guianensis

Five benzophenones, vismiaguianones A-E, and two benzocoumarins, vismiaguianins A and B were isolated from the CHCl3 extract of the roots of Vismia guianensis by bioassay-directed fractionation using the DNA strand-scission assay and KB cell line. Of the isolates obtained, vismiaguianone B exhibited DNA strand-scission activity, whereas vismiaguianones D and E and vismiaguianin A were found to be significantly cytotoxic.     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.