Immunogenically optimized peptides derived from natural mutants of HIV CTL epitopes and peptide combinatorial libraries

Two strategies were aimed at identifying immunogenically optimized peptides for the potential use in the formulation of an effective prophylactic or therapeutic HIV-1 vaccine. Three CTL epitopes were investigated: Gag p24(19-27) TV9, Gag p17(77-85) SL9, and RT(309-317) IV9. The first strategy derives from the hypothesis that a number of rare mutant CTL epitopes of HIV-1 may be more immunogenic than the common ones. As such, these rare mutant sequences might be highly effective in generating cross reactive anti-HIV-1 CTL responses against a range of mutant sequences. As anticipated, several rare mutant peptide sequences were identified that generated strong CTL responses against both the consensus sequences and several naturally occurring mutants in human PBL cultures primed ex vivo and in HLA-A2 transgenic mice immunized in vivo. Finally, to reach beyond the sequence diversity of the "natural" library of mutated sequences, a synthetic combinatorial peptide library was screened with a TV9 specific T-cell line; this resulted in the identification of an immunogenically optimized mimic peptide sequence that provoked highly effective CTL immune responses against TV9 and mutants. Sequence homologies between the natural mutants and synthetic mimic may provide insight into key contact positions in the MHC/TCR/peptide complex.     

ATP depletion alters the mode of cell death induced by benzyl isothiocyanate

Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 microM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 microM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the rho degrees cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (rho(+)) cells, whereas the ROS production was significantly inhibited in the rho degrees cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the rho degrees cells than in the rho(+) cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.     

The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins

A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.     

Rat mutations cvd and hob with cerebellar malformations map to chromosome 2.

In this paper, we executed genome mapping and comparative mapping analyses for cvd and hob, autosomal recessive mutations with cerebellar vermis defect and cerebellar dysplasia in the rat. For the linkage analysis, we produced three sets of backcross progeny, (ACI x CVD)F(1) and (F344 x CVD)F(1) females crossed to a cvd homozygous male rat, and (HOB x WKY)F(1) males crossed to hob homozygous female rats. Analysis of the segregation patterns of simple sequence length polymorphism (SSLP) markers scanning the whole rat genome allowed the mapping of these autosomal recessive mutations to rat Chromosome (Chr) 2. The most likely gene order is D2Mgh12 - D2Rat86 - D2Mit15 - D2Rat185 - cvd - D2Rat66 - D2Mgh13, and D2Mit18 - Fga -D2Mit14 - D2Rat16 - hob - D2Mgh13. Crossing test between a proven cvd heterozygous and a hob heterozygous rats demonstrated their allelism. Furthermore, comparative mapping indicated the cvd locus corresponds to mouse chromosome 3 and a strong candidate gene Unc5h3, a causative gene for the rostral cerebellar malformation mouse, was implicated.     

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.     

Coaggregation among Nonflocculating Bacteria Isolated from Activated Sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Effect of various reagents on the susceptibility of target cells to the cytotoxicity of guinea pig lymphotoxin (GLT).

The cytotoxic activity of guinea pig lymphotoxin (GLT) was increased when the target cells were treated with the following metabolic inhibitors: puromycin, actinomycin D, NaN3, NaF, and 2,4-dinitrophenol, whereas calf serum, N-acetyl-D-galactosamine, colchicine, and vinblastin inhibited GLT-cytotoxicity. Puromycin, an inhibitor of protein synthesis, exhibited the most potent enhancing effect on GLT-cytotoxicity. It seems likely that there are certain cellular metabolic processes depending on cellular protein synthesis which antagonize GLT-cytotoxicity. Taking advantage of this effect of puromycin, a time-saving assay method for GLT-cytotoxicity was developed.