Mobilization of the transposable element mdg4 by hybrid dysgenesis generates a family of unstable cut mutations in Drosophila melanogaster

Summary A family of unstable mutations at the cut locus in Drosophila melanogaster was obtained under the conditions of hybrid dysgenesis (Gerasimova 1981, 1982). The in situ hybridization experiments have shown that, in the original unstable ct ^MR2 mutation, the 7B region of the X chromosome (where cut is located) contains a mobile dispersed genetic element, mdg4. All other unstable ct mutations derived from ct ^MR2 including visible and lethal alleles and unstable ct ⁺ reversions, also contain mdg4 in the 7B region. The X chromosomes of the parent strain (wild type) do not contain mdg4 at all. All stable revertants derived from ct ^MR2, from other unstable ct mutations, or from ct lethals lost mdg4 from the 7B region. The ct ^MR2 X chromosome does not contain P-elements, although a few copies are present in the autosomes. The instability of the ct ^MR2./ct ^MR2 strain remained at a high level for 50 generations (1.5 years) and then rapidly decreased. A new cross with an MRh12/Cy strain (originally used for dysgenesis induction and containing a number of P-elements) increased the instability to a level exceeding the original one. The data strongly suggest that unstable ct mutations in our system are induced by transpositions of mdg4, possibly activated by P-elements.     

Electrically operated sodium channels in the somatic membrane of sympathetic neurons

Electrically operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion were investigated using an intracellular dialysis technique and voltage clamping. It was found that sodium currents can be conveyed along two independent systems of sodium channels in these neurons. A mathematical analysis was made of voltage-dependent tetrodotoxin-sensitive fast sodium currents within the framework of the Hodgkin-Huxley model and their kinetic properties were compared with those described in other subjects. It was also shown that the tetrodotoxin-sensitive sodium channels in the somatic membrane of sympathetic neurons have a high affinity for sodium ions. The kinetic and voltage-dependent characteristics of slow tetrodotoxin-sensitive inward sodium current are described. It is also noted that this component of the sodium current was observed in only a limited number of neurons (not more than 2%).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 108–117, January–February, 1986.     

Application of cellulose-based self-assembled tri-enzyme system in a pseudo-reagent-less biosensor for biogenic catecholamine detection

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.     

Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 degrees C) and high (18 degrees C) temperatures leads to differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5'-UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3'-UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH-A(alpha) and LDH-A(beta) subunits. It is expected that the beta-subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH-A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm-acclimated fish the relative expression between alpha and beta isoforms of the LDH-A mRNA is around 5 : 1, whereas in cold-acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH-A is more homogeneous in terms of alpha/beta-subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine-tuning of the properties of skeletal LDH-A, which is in line with previously observed kinetic and thermodynamic differences between 'cold' and 'warm' LDH-A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.     

Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel.We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from approximately 4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm(3), i.e. about 40% of the channel's volume in the high conductance state.     

Ovariectomy is protective against renal injury in the high-salt-fed older mRen2. Lewis rat

Studies in experimental animals and younger women suggest a protective role for estrogen; however, clinical trials may not substantiate this effect in older females. Therefore, the present study assessed the outcome of ovariectomy in older mRen2. Lewis rats subjected to a high-salt diet for 4 wk. Intact or ovariectomized (OVX, 15 wk of age) mRen2. Lewis rats were aged to 60 wk and then placed on a high-salt (HS, 8% sodium chloride) diet for 4 wk. Systolic blood pressures were similar between groups [OVX 169 +/- 6 vs. Intact 182 +/- 7 mmHg; P = 0.22] after the 4-wk diet; however, proteinuria [OVX 0.8 +/- 0.2 vs. Intact 11.5 +/- 2.6 mg/mg creatinine; P < 0.002, n = 6], renal interstitial fibrosis, glomerular sclerosis, and tubular casts were lower in OVX vs. Intact rats. Kidney injury molecule-1 mRNA, a marker of tubular damage, was 53% lower in the OVX HS group. Independent from blood pressure, OVX HS rats exhibited significantly lower cardiac (24%) and renal (32%) hypertrophy as well as lower C-reactive protein (28%). Circulating insulin-like growth factor-I (IGF-I) levels were not different between the Intact and OVX groups; however, renal cortical IGF-I mRNA and protein were attenuated in OVX rats [P < 0.05, n = 6]. We conclude that ovariectomy in the older female mRen2. Lewis rat conveys protection against salt-dependent increase in renal injury.     

Polysaccharides isolated from Açaí fruit induce innate immune responses

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease.     

Brain-computer interface based on generation of visual images

This paper examines the task of recognizing EEG patterns that correspond to performing three mental tasks: relaxation and imagining of two types of pictures: faces and houses. The experiments were performed using two EEG headsets: BrainProducts ActiCap and Emotiv EPOC. The Emotiv headset becomes widely used in consumer BCI application allowing for conducting large-scale EEG experiments in the future. Since classification accuracy significantly exceeded the level of random classification during the first three days of the experiment with EPOC headset, a control experiment was performed on the fourth day using ActiCap. The control experiment has shown that utilization of high-quality research equipment can enhance classification accuracy (up to 68% in some subjects) and that the accuracy is independent of the presence of EEG artifacts related to blinking and eye movement. This study also shows that computationally-inexpensive bayesian classifier based on covariance matrix analysis yields similar classification accuracy in this problem as a more sophisticated Multi-class Common Spatial Patterns (MCSP) classifier.