The spectrum of mutations in the <Emphasis Type="Italic">PAH</Emphasis> gene in patients with hyperphenylalaninemia from the Karachay-Cherkess Republic

According to the neonatal screening conducted during the last nine years in Karachay-Cherkessia, the frequency of hyperphenylalaninemia (including PKU) was 1: 850 newborns, which significantly exceeded the average frequency of 1: 7000 in Russia. Analysis of DNA obtained from 25 patients with a diagnosis of “hyperphenylalaninemia” (HPA) from the Karachay-Cherkess Republic was performed to search for mutations in the PAH gene. Mutations were identified on 90% of the studied chromosomes, while at least one mutation in the PAH gene was observed in all patients. The allele frequency of a major mutation R261X was 32.5%. A correlation between genotype and phenotype was confirmed in patients with HPA.     

A method for the detection and characterization of GABA(A) receptors by calcium-sensitive fluorescent probes

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.     

Interactions between cognitive and sensory load while planning and controlling complex gait adaptations in Parkinson’s disease

Background

Recent research has argued that removal of relevant sensory information during the planning and control of simple, self-paced walking can result in increased demand on central processing resources in Parkinson’s disease (PD). However, little is known about more complex gait tasks that require planning of gait adaptations to cross over an obstacle in PD.

Methods

In order to understand the interaction between availability of visual information relevant for self-motion and cognitive load, the current study evaluated PD participants and healthy controls while walking toward and stepping over an obstacle in three visual feedback conditions: (i) no visual restrictions; (ii) vision of the obstacle and their lower limbs while in complete darkness; (iii) vision of the obstacle only while in complete darkness; as well as two conditions including a cognitive load (with a dual task versus without a dual task). Each walk trial was divided into an early and late phase to examine changes associated with planning of step adjustments when approaching the obstacle.

Results

Interactions between visual feedback and dual task conditions during the obstacle approach were not significant. Patients with PD had greater deceleration and step time variability in the late phase of the obstacle approach phase while walking in both dark conditions compared to control participants. Additionally, participants with PD had a greater number of obstacle contacts when vision of their lower limbs was not available specifically during the dual task condition. Dual task performance was worse in PD compared to healthy control participants, but notably only while walking in the dark regardless of visual feedback.

Conclusions

These results suggest that reducing visual feedback while approaching an obstacle shifts processing to somatosensory feedback to guide movement which imposes a greater demand on planning resources. These results are key to fully understanding why trips and falls occur in those with PD.

     

Results of the population and genetic study in the Republic of Tatarstan

Using data on the distribution of frequent surnames, the Crow index and its components, and nine polymorphic DNA loci, it was shown that the Tatar population of the Republic of Tatarstan is divided into subethnic groups (Mishars, Teptyars, and Kazan Tatars). No subdivision within each of these groups was found.     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.