Vascular endothelial cells have impaired capacity to present immunodominant, antigenic peptides: a mechanism of cell type-specific immune escape

Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides.     

Transactivation of erbB2 by short and long isoforms of leptin receptors

We generated kinase-positive and kinase-negative erbB2 tagged with YFP and the long form of leptin receptor (LEPRb) tagged with CFP. Both were as active as their untagged analogs. Both short and long isoforms of leptin receptor phosphorylated and thereby activated erbB2 upon leptin binding and enhanced MAPK activity. Our results unveil a novel route by which leptin may provoke erbB2's phosphorylation and thus enhance its oncogenic potential independently of HER family ligands or its overexpression. Using FRET technology in living cells, we found no evidence of complex formation between erbB2 and prolactin or leptin receptors, indicating that the transactivation occurs through an indirect interaction.     

Reproductive strategy of a small endemic cyprinid, the Yarqon bleak (Acanthobrama telavivensis), in a mediterranean-type stream

A mediterranean-type climate exists in five widely separated regions; the Mediterranean basin, parts of western North America, parts of western and southern Australia, southwestern South Africa and parts of central Chile. Streams in these regions feature seasonal disturbances of contrasting hydrology with high predictability of the timing of flooding and drying but low constancy. We would expect fish living in these streams to avoid scouring flow and breed after cessation of the flood period. The aim of the present study was to examine the adaptation of the Yarqon bleak, Acanthobrama telavivensis, an endemic cyprinid in the coastal streams of Israel, to mediterranean-type stream (mediterranean—written with a small m, is used in connection with climate or ecological region and is distinguished from Mediterranean that is used in a geographical context, referring to the Mediterranean basin.) conditions. For that we studied its reproductive strategy (age at maturity and life span, gonad activity, oocyte maturation, spawning activity and habitats, appearance of juveniles), in a major costal stream (Yarqon). Our findings show that the Yarqon bleak exhibits life history traits attuned with a mediterranean-climate hydrologic regime. It breeds in late winter and early spring, a window of opportunity between flash floods and habitat desiccation. Being a multiple spawner allows the fish to compensate for the potential loss of part of its reproductive output due to scouring flows of late floods. The ability of the Yarqon bleak to spawn on different substrate-types enables it to take advantage of different stream conditions that pertain in different years. The fish attains pre-adult size (ca. 33–42 mm) within the first year, prior to drying out of most stream reaches, and matures by the beginning of the second year (males >41; females >42 mm). The cost of these tactics is a short life span (4–5 age groups). The reproductive strategy of the Yarqon bleak falls into the category of in-channel breeding but, unlike the case suggested by a low flow recruitment model, the fish breed during the period of flood cessation, a transitional time between high and low flows, rather than at the time of low flow. Breeding at this time of the year in mediterranean-type streams puts early stages somewhat at risk of being washed away by late floods, but gains them a longer period of growth under favorable conditions. We suggest an additional positive tradeoff that should be investigated: the reduced competition with age 0 of other fish that breed later in the season. This suggested model of recruitment during the period of flood cessation seems appropriate for fish in streams with seasonal contrasting flows of high predictability but low constancy.     

New measures of insecticidal efficacy and safety obtained with the 39K promoter of a recombinant baculovirus

Baculoviruses are orally infectious to insects and considered to be natural insecticides. To enhance their speed-of-kill these viruses were engineered to express arthropod neurotoxins under the control of various strong promoters. Although this strategy proved to be efficient, it raised recently concerns about safety. We analyzed the speed-of-kill and safety of Autographa californica multiple nucleopolyhedrovirus expressing the insecticidal scorpion neurotoxin AaIT and found that the mortality of Helicoverpa armigera larvae was enhanced significantly when the expression was controlled by the baculovirus delayed-early promoter 39K rather than the very late promoter p10. This improvement was also reflected in better protection of cotton leaves on which these insects were fed. Using lacZ as a sensitive reporter we also found that expression driven by the 39K promoter was detected in insect but not in mammalian cells. These results imply that by selection of an appropriate viral promoter, engineered baculoviruses may comply with the high standard biosafety requirements from a genetically modified organism (GMO). Our results provide further support for the potential use of engineered baculoviruses in insect pest control in a safely manner.     

Comparative testing of various pancreatic cancer stem cells results in a novel class of pancreatic-cancer-initiating cells

No systemic therapy is effective against pancreatic cancer (PC). Pancreatic cancer stem cells (PCSC) are hypothesized to account for therapeutic resistance. Several PCSC subpopulations were reported, each characterized by different markers. To be able to target PCSC, we sought to better define this putative heterogeneity. Therefore, we tested most of the known putative PCSC markers in established and fresh tumor cell lines. CD20, CD24, CD44, CD133, CD184 (CXCR4), CD326 (EpCam, ESA), Sox-2, OCT 3/4, and the side-population (SP) were tested in five PC cell lines, and the effects of confluency, hypoxia, radiation, and gemcitabine on the SP. The testing phase suggested several putative PCSC populations that were further tested and validated for their tumor-initiating capacity against known PCSC in 3 established and 1 fresh PC cell lines. Cell surface and intracellular markers showed significant variability among cell lines. SP was the only common marker in all cell lines and consistently less than 1%. SP response to confluence, hypoxia, radiation, and gemcitabine was inconsistent between cell lines. The initial testing phase suggested that SP/CD44-CD24-CD326+ cells might be a novel PCSC subpopulation. Tumor initiation capacity tests in nude mice confirmed their increased tumorigenicity over previously reported PCSC. Our data better define the heterogeneity of reported PCSC in cell lines tested in this study. We propose that prior to targeting PC via PCSC, one will need to gain more insight into this heterogeneity. Finally, we show that SP/CD44-CD24-CD326+ cells are a novel subpopulation of pancreatic cancer tumor initiating cells. Further mechanistic studies may lead to better targeting of PC via targeting this novel PCSC.     

Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.     

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.     

A new UV-B absorbing mycosporine with photo protective activity from the lichenized ascomycete Collema cristatum.

A novel photo protective mycosporine was isolated from the lichenized ascomycete Collema cristatum. Biological activity was measured in terms of protection against UV-B induced membrane destruction and pyrimidine dimer formation in cultured human keratinocytes, and prevention of UV-B induced erythema. It was found that the pure isolated compound prevented UV-B induced cell destruction in a dose-dependent manner, that the compound partially prevented pyrimidine dimer formation and completely prevented UV-B induced erythema when applied to the skin prior to irradiation.