A DNA immunization model study with constructs expressing the tick-borne encephalitis virus envelope protein E in different physical forms

We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the envelope glycoprotein E of the flavivirus tick-borne encephalitis virus. These included a secreted recombinant subviral particle, a secreted carboxyl-terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-gamma, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.     

Placebo medication use in patient care: a survey of medical interns

The use of placebo medication, long recognized by clinicians, often has serious practical implications, such as patient deception. Past evidence has suggested that resident physicians tend to misuse placebo medication. Interns from two consecutive years of a residency program were surveyed anonymously to assess their knowledge and use of placebos. Of the 74 interns surveyed, 44 (59%) were familiar with placebo use in patient care. Fifty percent of these interns familiar with placebo use had learned about placebos from another physician. All interns who had learned about placebos during their internships had learned from another physician, whereas interns who had gained their knowledge of placebos as medical students were as likely to have learned from the medical literature as they were to have learned from a physician (P = 0.027). Interns aware of placebo use were more likely to consider placebo administration for suspected, factitious pain (P = 0.022). The present study uncovered no relationship between interns' estimations of placebo efficacy and the utility they attributed to placebos in assessing a complaint of pain. This suggests that conceptual inconsistencies underlie their use of placebos. Interns often learn of placebos as medical students and are influenced by physician-mentors. Placebo use in patient care is an area of attention for medical educators.     

Degradation of alpha-pinene oxide and [2H7]-2,5,6-trimethyl-hept-(2E)-enoic acid by Pseudomonas fluorescens NCIMB 11761

When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.     

The influence of microgravity on memorized arm movements

To investigate sensory and motor functions in microgravity, goal-oriented arm movements were performed by 9 cosmonauts in weightlessness. The ability to reproduce predefined motor patterns was examined pre-, in-, and post-flight under two different paradigms: In a first test, the cosmonaut had to reproduce passively learned movements with eyes closed, while in the second test, the cosmonaut learned the pattern with eyes open. The different learning paradigms effected the metric parameters of the memorized stimulus pattern while the influence of the different gravity levels resulted in significant offsets and torsions of the reproduced figures. In comparing the inflight condition with preflight, intact proprioceptive afference seemed to play an important role for reproducing movements from motor short-time memory correctly.     

Salinity Adaptations and Bathymetric Distribution of Bivalve Mollusks Portlandia arcticaand Nuculana pernula in the White Sea

We examined changes in the oxygen consumption rate, the number of active animals, rate of losses of salts, and the level of cell hydration under conditions of decreased salinity in the mollusks Portlandia arctica(Gray) and Nuculana pernula(O.F. Müller), inhabiting the White Sea in the deepest depths under high salinity (about 30) and negative temperatures of –1.3 to –1.4°C throughout the year. It has been shown that the mollusks demonstrate a relatively great tolerance of desalination at the organism level and a capability to regulate cell volume under decreased salinity of the environment. We concluded that the depletion of fauna of the White Sea in the deepest depths is not connected with the tolerance level of deep-sea animals to salinity fluctuations, but rather is due to environmental conditions (liquid silts and negative temperatures).     

TRAIL-induced apoptosis is independent of the mitochondrial apoptosis mediator DAP3

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2) and the adapter protein Fas-associated death domain protein (FADD). Recently, an adapter function for death-associated protein 3 (DAP3) between DR4/DR5 and FADD has been proposed. However, DAP3 has been reported to be a ribosomal protein localized to the mitochondrial matrix. To address these discrepancies, the intracellular localization of DAP3 after apoptosis induction in human T-lymphocytes with recombinant TRAIL was analyzed. DAP3, in contrast to cytochrome c, remained intra-mitochondrial during apoptosis. No interaction between FADD and DAP3 after cell fractionation could be detected as long as subcellular compartments remained intact. Only whole cell lysate co-immunoprecipitation revealed an ex vivo interaction between DAP3 and FADD. Therefore, DAP3 and FADD interact only in vitro after disruption of the cellular compartments. TRAIL-induced and DR4-mediated apoptosis in Jurkat cells is independent of DAP3.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila

The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes. We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila. TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13(suc1) protein. In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity. Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T. thermophila. A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.     

Arabidopsis haiku mutants reveal new controls of seed size by endosperm

In flowering plants, maternal seed integument encloses the embryo and the endosperm, which are both derived from double fertilization. Although the development of these three components must be coordinated, we have limited knowledge of mechanisms involved in such coordination. The endosperm may play a central role in these mechanisms as epigenetic modifications of endosperm development, via imbalance of dosage between maternal and paternal genomes, affecting both the embryo and the integument. To identify targets of such epigenetic controls, we designed a genetic screen in Arabidopsis for mutants that phenocopy the effects of dosage imbalance in the endosperm. The two mutants haiku 1 and haiku 2 produce seed of reduced size that resemble seed with maternal excess in the maternal/paternal dosage. Homozygous haiku seed develop into plants indistinguishable from wild type. Each mutation is sporophytic recessive, and double-mutant analysis suggests that both mutations affect the same genetic pathway. The endosperm of haiku mutants shows a premature arrest of increase in size that causes precocious cellularization of the syncytial endosperm. Reduction of seed size in haiku results from coordinated reduction of endosperm size, embryo proliferation, and cell elongation of the maternally derived integument. We present further evidence for a control of integument development mediated by endosperm-derived signals.