Lake Baikal amphipods and their genomes, great and small.

Endemic amphipods (Crustacea: Amphipoda) of Lake Baikal represent an outstanding example of large species flocks occupying a wide range of ecological niches and originating from a handful of ancestor species. Their development took place at a restricted territory and is thus open for comprehensive research. Such examples provide unique opportunities for studying behavioral, anatomic, or physiological adaptations in multiple combinations of environmental conditions and thus attract considerable attention. The existing taxonomies of this group list over 350 species and subspecies, which, according to the molecular phylogenetic studies of marker genes, full transcriptomes and mitochondrial genomes, originated from at least two introductions into the lake. The studies of allozymes and marker genes have revealed a significant cryptic diversity in Baikal amphipods, as well as a large variance in genetic diversity within some morphological species. Crossing experiments conducted so far for two morphological species suggest that the differences in the mitochondrial marker (cytochrome c oxidase subunit I gene) can potentially be applied for making predictions about reproductive isolation. For about one-tenth of the Baikal amphipod species, nuclear genome sizes and chromosome numbers are known. While genome sizes vary within one order of magnitude, the karyotypes are relatively stable (2n = 52 for most species studied). Moreover, analysis of the diversity of repeated sequences in nuclear genomes showed significant between-species differences. Studies of mitochondrial genomes revealed some unusual features, such as variation in length and gene order, as well as duplications of tRNA genes, some of which also underwent remolding (change in anticodon specificity due to point mutations). The next important steps should be (i) the assembly of whole genomes for different species of Baikal amphipods, which is at the moment hampered by complicated genome structures with high repeat content, and (ii) updating species taxonomy taking into account all the data.

Comparison of the evolutionary patterns of DNA repeats in ancient and young invertebrate species flocks of Lake Baikal.

DNA repeat composition of low coverage (0.1-0.5) genomic libraries of four amphipods species endemic to Lake Baikal (East Siberia) and four endemic gastropod species of the fam. Baicaliidae have been compared to each other. In order to do so, a neighbor joining tree was inferred for each quartet of species (amphipods and mollusks) based on the ratio of repeat classes shared in each pair of species. The topology of this tree was compared to the phylogenies inferred for the same species from the concatenated protein-coding mitochondrial nucleotide sequences. In all species analyzed, the fraction of DNA repeats involved circa half of the genome. In relatively more ancient amphipods (most recent common ancestor, MRCA, existed approximately sixty millions years ago), the most abundant were species-specific repeats, while in much younger Baicaliidae (MRCA equal to ca. three millions years) most of the DNA repeats were shared among all four species. If the presence/absence of a repeat is regarded as a separate independent trait, and the ratio of shared to total numbers of repeats in a species pair is used as the measure of distance, the topology of the NJ tree is the same as the quartet phylogeny inferred for the mitogenomes protein coding nucleotide sequences. Meanwhile, in each group of species, a substantial number of repeats were detected pointing to the possibility of non-neutral evolution or a horizontal transfer between species occupying the same biotope. These repeats were shared by non-sister groups while being absent in the sister genomes. On the other hand, in such cases some traits of ecological significance were also shared.

Structural uterine changes in postpartum endometritis in cows.

AIM: The purpose of this work was to study the dynamics of structural manifestations of acute cases of postpartum endometritis in cows. MATERIALS AND METHODS: The light and electron microscopy methods were used when studying structural changes in the endometrium in case of postpartum endometritis in seven cows. Sections of endometrial specimens for light microscopy, 5-7 µm thick, were stained with hematoxylin and eosin and also by Van Gieson's. For electron microscopy, semi-thin sections were stained with Azur-2 in combination with basic fuchsin, as well as contrasting by lead citrate and uranyl acetate. RESULTS: As a result of the study, we have established the following: Necrobiosis of the epithelial layer of the mucosa, cellular infiltration with shaped elements of blood in the functional layer, swelling of the cells of the uterine gland, and single microbial cells on the surface of the mucosa. We have noted edema of the stroma of the functional layer of the endometrium, swelling of the epithelial layer of the endometrial mucosa, and swelling of fibroblastic and lymphoid cells. Ultrastructural changes in endometrial cells in case of acute postpartum endometritis in cows are accompanied by the destruction of microvilli on the apical surface of the epithelium, an abundance of coccal microflora on the surface of the epithelium, necrobiosis of epithelial cells, and partial edema of the nucleus, and cytoplasm of the histiocyte. CONCLUSION: We had established that acute purulent-catarrhal dystrophic processes were observed in the structural organization of the endometrium. In the depth of catarrhal mucus on the surface of the endometrium, there was an abundance of bacterial flora, with diplococci being prevalent. In ultrastructural organization of the endometrium, we observed deep dystrophic and necrobiotic processes in the parenchyma and endometrial stroma, as well as exudative processes with a change in the integrity of the microcirculatory bed. Thus, to prevent an inflammatory process from turning into a latent form, it is necessary to detect acute postpartum endometritis promptly using diagnostic methods taking into account the obtained parameters of the dynamics of structural changes in the uterine tissues.

[Prolonged interferon papillomavirus infection treatment].

Prolonged interferon papillomavirus infection treatment. In patients with recurrent genital HPV caused by human papillomavirus types 6 and 11, the use of genferon suppository according to prolonged scheme (500 000 IU 2 times a day for 10 days and further 500 000 IU 2 times a week for 3 months) in comparison with a short scheme (500 000 IU, 2 times a day for 10 days) is more effective in preventing recurrence of infection due to the normalization, an immune and cytokine status, as well as the elimination of the infectious agent from the affected tissues. Prolongation of the drug use did not cause undesirable side effects. The effectiveness of prolonged treatment regimes is determined by the pharmacological properties genferon components that provide systemic and local effects.

[Evaluation of clinical efficiency of viferon gel for the treatment of acute rhinopharyngitis].

Because numerous studies have demonstrated that the majority of acute inflammatory diseases of the upper respiratory tract are associated with viral infection, interferon-based medicines are being extensively developed for non-specific therapy and prevention of acute respiratory viral infections (ARVI). The objective of the present study was to evaluate the efficiency of local application of viferon gel for the treatment of acute rhinopharyngitis from the results of examination and treatment of a group of outpatients with this pathology (n = 90). Viferon gel was introduced into each half of the nose at a dose of 0.5 ml 4-5 times daily. It was shown that timely prescription of this preparation accelerates the achievement of positive results of AVRI therapy and thereby reduces duration of the disease and prevents the development of bacterial complications in the upper respiratory tract.

[Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection].

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tickborne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 --> Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 --> Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.

[Rezonizat and cycloserine in complex therapy of drug resistant tuberculosis (comparative studies)].

An open comparative investigation of the Russian drugs rezonizat and cycloserine was performed. The studies made it possible to recommend the use of rezonizat in complex therapy of patients with drug resistant tuberculosis. Higher activity and better tolerability of rezonizat vs. cycloserine was shown.

[Detection of Borrelia DNA in the Borrelia burgdorferi sensu lato complex in the blood of patients with Ixodes tick-borne borrelios].

Borrelia DNA was detected by polymerase chain reaction in the blood of patients who had suffered from tick suction. DNA was revealed in 28.7 of the blood samples from patients with the erythematous form of Ixodes tick-born borrelioses (ITBB) and in 14.3% of those diagnosed as having tick-borne encephalitis. Blood Borrelia DNA was not detected in patients with end-stage and chronic ITBB. Comparing the results of detection of DNA with those Borrelia protein antibodies has shown that the antibody titers detectable in the patients having Borrelia DNA are lower than those in the patients with the same form of ITBB and without DNA. The detection of DNA should be accomplished in the first 4 weeks after tick bite.

[Morphofunctional characteristics of the retina and optic nerve in endocrine ophthalmopathy].

Optic neuropathy is a serious complication of endocrine ophthalmopathy (EOP). The morphological characteristics of the optic nerve (provided by a Heidelberg, HRT II retinotomograph) were compared with the functional impairments of various canals of light, color, and contrast sensitivities (detected by an Offon programme complex, MBN, Moscow). There were decreases in contrast and color sensitivities just at the early stages of the disease and deteriorated functions of contract and color sensitivity canals as optic neuropathy progressed. The HRT II retinotomograph has shown a significant change in the thickness of optic nerve fibers, the area of the optic disk (OD), and the volume of the neuroretinal ring, which corresponds to the magnitude of clinical manifestations and the severity of OD lesion. The first retinal changes appear long before optic nerve compression at the orbital apex (orbital apex syndrome), which are caused by ischemic and hypoxic processes due to orbital soft tissue edema. The most sensitive test in detecting the latent stage of optic neuropathy in EOP is a color and contrast sensitivity topography test. In EOP, the morphological optic nerve changes correlate with functional impairments in different canals of the visual system, reflecting the stage of the disease.

[Detection of DNA of borrelia circulating in Novosibirsk region].

Detection of DNA of Borrelia burgdorferi sensu lato was performed by PCR in taiga ticks Ixodes persulcatus, in blood samples and skin bioptates of small forest mammals, and in blood and urine samples of humans after attaching of ticks events. In Novosibirsk region both in natural reservoir and in patients with Ixodes ticks-borne borreliosis DNA of Borrelia garinii and Borrelia afzelii are detected. DNA of these borrelia were detected in 8 from 72 of taiga ticks, in 36 from 298 of blood and skin samples of small forest mammals, and in 32 from 102 of human blood and urine samples. In all studied samples DNA of B. garinii from NT29 subgroup was predominated. Borrelia DNA in which sequence of intergenic spacer region was homologous to sequence Chy13p first detected in China has been detected in one blood sample from red-backed vole (Clethrionomys rutilus).

Direct assay of Aplysia tissues and cells with laser desorption/ionization mass spectrometry on porous silicon.

Desorption/ionization on porous silicon (DIOS) is a form of laser desorption mass spectrometry that allows for the direct mass analysis of a variety of analytes without the addition of organic matrix. Protocols are described for the direct analysis of exocrine tissue and single neurons using DIOS-MS. The atrial gland of Aplysia californica was blotted on to porous silicon and analyzed with DIOS-MS in the range m/z 1000-4000. The ability to culture invertebrate neurons directly on porous silicon is also presented. Isolated bag cells regenerated neuronal processes in culture on porous silicon. DIOS-MS allowed the direct detection of the peptides contained in individual cultured neurons indicating that with appropriate protocols, DIOS can be used with biological samples with considerable thickness.

Cerebrin prohormone processing, distribution and action in Aplysia californica.

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic purification guided by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F- and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These findings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda.

[Psychophysical symptoms of demyelinating diseases of the optic nerve in multiple sclerosis]. / Psikhofizicheskie simptomy demieliniziruiushchikh porazhenii zritel'nogo nerva pri rasseiannom skleroze.

Visual system channels were examined in 25 patients with multiple sclerosis. A new complex of symptoms was detected, reflecting the acute stage and the chronic demyelinizing process, which includes disorders of contrast, color, and spatial contrast sensitivity and stereoscopic vision. This can be useful in differential diagnosis of optic nerve diseases and evaluation of the visual system function in multiple sclerosis and prediction of the disease course.

Peptide profiling of cells with multiple gene products: combining immunochemistry and MALDI mass spectrometry with on-plate microextraction.

Due to the intracellular chemical complexity and a wide range of transmitter concentrations, the detection of the complete set of peptide transmitters in a single cell is problematic. In the current study, a multidisciplinary approach combining single-cell MALDI-MS peptide profiling, northern analysis, in situ hybridization, and immunocytochemistry allows characterization of a more complete set of neurotransmitters than individual approaches in the Aplysia californica B1 and B2 motor neurons. Because different results were obtained using both in situ and immunohistochemical techniques compared to previous reports, MALDI-MS assays have been used to examine CP1-related gene products in these cells. However, MALDI with standard sample preparation does not detect the presence of the CP1 gene products. A novel on-plate microextraction approach using concentrated MALDI matrix 2,5-dihydroxybenzoic acid with a mixture of acetone and water as the solvent has been developed to allow the detection of trace-level gene expression products. Both neuropeptide precursors in the B1 and B2 neurons-the SCP and CP1 prohormones-end with large peptides that have multiple cysteine residues. For SCP, MALDI-MS verifies the presence of a novel 9325 Da SCP-related peptide. In the case of CP1, a disulfide-bonded homodimer is detected and the disulfide bonding pattern elucidated using MALDI-MS coupled with on-plate enzymatic digestion.

In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis.

The ability to directly sequence peptides from biological cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with postsource decay (PSD) and collision-induced dissociation (CID) fragment ion mass analysis is explored. Three different sample preparation methods are described for sequencing peptides in tissue samples and in single neurons from the invertebrate model Aplysia californica. To characterize peptides from the atrial gland, MALDI-PSD/CID is applied directly to a tissue blot covered with the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA). The resulting fragment ions combined with database searching confirm the structure of several novel peptides encoded by egg-laying hormone genes. Moreover, MS profiling of a single unidentified neuron detects peptides with molecular weights of myomodulins C and E; this assignment is confirmed using MALDI-PSD with the matrix 2,5-dihydroxybenzoic acid (DHB). DHB does not always provide adequate fragmentation for PSD experiments; therefore, a unique dual-matrix sampling method, employing both DHB and CHCA, is developed to directly sequence a decapeptide from a single cerebral ganglion B cell. Mass accuracy of fragment ions from cellular samples is typical for the instrument employed and is not deleteriously affected by the morphology and complexity of the samples.

Behavioral changes induced by GABA-receptor agonists in Lymnaea stagnalis L.

1. GABA, the GABAA receptor agonist, muscimol, and the GABAB receptor agonist, baclofen, were tested to study the involvement of the GABA neurotransmitter system in control of behaviour in the freshwater pulmonate snail, Lymnaea stagnalis L. Single injections of GABA (1-10 micrograms/ gbw) into the haemocoel of intact snails elicits a sequence of behavioural changes subsequently affecting feeding, locomotion, escape reactions, male mating behaviour and respiration. 2. Both muscimol and baclofen mimic distinct aspects of GABA action implying that the GABA action is mediated by both types of receptors. 3. The modulatory actions of GABA, muscimol, and baclofen on feeding were manifested as opening of the mouth and triggering of specific radular movements (e.g., protraction, retraction, rasping). 4. Baclofen (1-10 micrograms/gbw) evoked the full erection of the penis for a time long enough for natural copulation, while GABA itself caused only partial eversion of the preputium. Muscimol was less effective than GABA. The latency to penis eversion varied in a dose-dependent manner. The data emphasize the participation of baclofen-sensitive receptors in control of reproduction. 5. GABA and baclofen induced withdrawal of the head and anterior part of the foot accompanied by arrest of locomotion and respiration could be considered to be an escape reaction. 6. Muscimol (1-5 micrograms/gbw) failed to elicit withdrawal reactions but caused the loss of normal orientation and longitudinal contractions of the foot. 7. All the agents tested inhibited locomotion in a dose-dependent manner, for a substantial period of time. A decrease in total locomotor activity lead to the failure of animals to attach to the underlying surface, to have foot contractions and also to diminish locomotor velocity. 8. The majority of animals maintained the stereotyped complex reactions of respiration, but with restricted clockwise turning of the shell after drug treatment. 9. All the substances were shown to shift the background behavioural state characterised by high BSSs to a state similar to "rest" which was awarded a low BSS. It is concluded that the GABA neurotransmitter system can play an important role in activation and coordination of neuronal ensembles underlying behaviour and behaviour selection in Lymnaea stagnalis L.