Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase.

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.

Formation of mixed d- and f-block metal clusters in inert matrices: comparison of the observed and theoretical optical spectra of AgHo.

The UV visible spectra obtained after simultaneous cocondensation of silver and holmium atoms with argon matrices at 9 K have been studied in the 200-800 nm region. While no new feature can be observed upon deposition, selective irradiation into both silver or holmium atomic absorptions results in growth of a new band at 430 nm, associated with formation of a mixed silver holmium species, tentatively assigned as AgHo. To support the assignment of the observed bands ab initio quantum chemical calculations were carried out for the dinuclear and trinuclear silver and holmium species, using pseudopotential approaches. Results for the electronic excitation energies and corresponding transition dipole moments for the diatomic molecules Ag2, Ho2, AgHo provide evidence that the 430 nm band should be attributed to the mixed cluster AgHo (theoretical band position at 436 nm), while the doublets at 498/504 and 558/563 nm belong to the homonuclear species Ho2 (theoretical values are at 482 and 562 nm). First conclusions are drawn with respect to the formation of the metal trimers Ho3, Ag2Ho, AgHo2.

Nucleoli in a pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes.

OBJECTIVE: To investigate the arrangement of chromosomes within pronuclei-stage mouse zygotes. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Location of major alpha-satellite DNA, centromeres, and telomeres, and relative location of chromosomes. RESULT(S): Chromosomes appeared to be oriented inward by centromeres and to be interconnected by major alpha-satellite DNA, which appeared to be the sole DNA component of the nucleoli. This chromosomal arrangement persisted throughout interphase. Chromosomal painting failed to identify chromosomal ordering within pronuclei. CONCLUSION(S): Pronuclear nucleoli are represented by alpha-satellite sequences of interconnecting chromosomes that hold all chromosomes together during interphase. Chromosomes within the pronucleus are randomly positioned relative to each other.

Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation.

Ca(2+)-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca(2+) concentrations in photoreceptors decrease from 600 to 50 nM. RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R. K., Dizhoor, A. M., and Yamazaki, A. (1999) J. Biol. Chem. 274, 15547-15555). We have found that the Ca(2+)-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca(2+). The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca(2+) concentrations. A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca(2+) concentrations forms dimers both in the absence and in the presence of Ca(2+). Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca(2+) concentrations are also unable to dimerize in the absence of Ca(2+). Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca(2+). These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase. The Ca(2+)-free GCAP-1 is also capable of dimerization in the absence of Ca(2+), but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca(2+).

Culture media and their components differ in their ability to scavenge reactive oxygen species in the plasmid relaxation assay.

OBJECTIVE: To investigate the modulation of DNA-damaging effects of reactive oxygen species by media composition. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Plasmid relaxation. RESULT(S): Ham's F-10 medium, 1% Percoll, superoxide dismutase (1, 10, or 100 IU), and synthetic serum substitute did not affect DNA damage by reactive oxygen species and did not have any effect on plasmid DNA damage. Plasmid DNA damage was partially inhibited in the presence of P-1 and human tubal fluid media. Human serum albumin, phenol red, glucose, polyvinyl alcohol, polyvinylpyrrolidone, sucrose, and HEPES also were found to protect DNA from damage. CONCLUSION(S): In vitro fertilization media and their components vary widely in the way they affect DNA damage by reactive oxygen species.

Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.

Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

[Polymorphism of B1-associated DNA fragments on various stages of ontogenesis of mice in tissues of different histogenesis]. / Polimorfizm B1-assotsiirovannykh fragmentov DNK na raznykh stadiiakh ontogeneza myshi v tkaniakh razlichnogo gistogeneza.

Pattern of B1-associated DNA fragments was studied by means of polymerase chain reaction (PCR) in the mouse tissues of different histogenesis of 15 and 20 day old embryos and of adult mice C57B1/6. As many as 20 DNA fragments were revealed on electrophoregrams differing in their molecular masses (m. m.) and amounts of amplified products. DNA fragments varied within a 100-10,000 bp range. The clusters of B1-associated DNA fragments, containing 100-200, 300-400 and 800-1000 bp, were most intensive in all studied electrophoregrams. The B1-associated DNA fragments from muscles of adult mice differed from those of other tissues by the presence of a DNA fragment with 800 bp. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown their general similarities in m. m. values. But a significant distinction, that was found, involved the presence of a DNA fragment with m. m. approximately 6000 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The obtained results allow to use the B1-PCR method for studying genome recombination during ontogenesis, intraspecies divergence and also at malignant cell transformation.

Intracytoplasmic sperm injection in the rat.

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5-1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.

The impact of cellular fragmentation induced experimentally at different stages of mouse preimplantation development.

It has been demonstrated previously that removal of acellular debris from the preimplantation mouse embryo is beneficial for subsequent development to the hatched blastocyst stage. We have studied the impact of cellular fragmentation induced in the mouse embryo during the late pronuclei and 8-cell stages on the hatching frequency and total cell number at the blastocyst stage. At the late pronuclei stage about one-quarter of the cytoplasm was removed from embryos in the experimental group, in four to six steps, thus creating four to six cytoplasts that were subsequently returned as anucleated fragments under the zona pellucida. Embryos with one-quarter of the cytoplasm removed and with intact cytoplasm after partial zona dissection (PZD) served as controls. At the 8-cell stage, embryos with their nucleoplast removed from two blastomeres served as an experimental group. Groups of embryos with part of the cytoplast removed from two blastomeres (nucleated fragments), embryos with two blastomeres removed and embryos after PZD alone served as controls. After manipulation all embryos were left in culture and analysed at about 100 h after human chorionic gonadotrophin administration. Fragments induced at the late pronuclei stage did not participate in compaction and were often spontaneously expelled from the embryo during hatching. Neither embryo hatching rate nor total cell number was affected when compared with zygotes with reduced cytoplasm. Although both nucleated and anucleated fragments induced at the 8-cell stage participated in recompaction, hatching was not compromised and there was no interference in further development as assessed by the cell number or hatching rate at the blastocyst stage, as compared with embryos with blastomeres removed. We conclude that anucleated cellular fragments formed in an otherwise healthy embryo, both before and after acquisition of the ability for compaction, are benign and that their removal provides no benefit for embryo development, at least to the hatched blastocyst stage.

[Phenotypic variability in gag-myc transgenic mice]. / Fenotipicheskaia variabel'nost' u transgennykh myshei gag-myc.

The study of neoplastic growth capacity in gag-myc F3-F4 transgenic mice showed that they retain the capacity to develop hyperplasia and benign and malignant tumors same as F0-F2 transgenic mice do. However, the histological spectrum of tumors was much narrower, chiefly, due to the absence of rare patterns. The tumors in F3-F4 transgenic mice were mainly lymphomas and epithelial tumors of endocrine or exocrine glands. The incorporation of gag-myc in the genome of F3-F4 transgenic mice failed to tell on the variation of such quantitative polygenic characteristics as body mass and growth.

[Polymorphism of B1-associated DNA fragments from normal and transform ed murine hepatocytes]. / Polimorfizm B1-assotsiirovannykh fragmentov DNK v normal'nykh i transformirovannykh gepatotsitakh myshi.

Our study of B1-associated DNA fragment polymorphism in murine hepatocytes by means of polymerase chain reaction (PCR) allowed to reveal as many as 20 DNA fragments differing in their molecular masses (m. m.) and amount of amplified products varying within the range of 100-1000 bp. Within the same inbred strain of mice (C57B1/6), spectra of B1-associated DNA fragments were similar in different periods of ontogenesis (embryos of 15 and 20 days, adult mice), in different mice of the same or different litters. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown in general their similarities in m. m. values. But a significant distinction, that was found, involved the presence of DNA fragments with m.m. approximately 600 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The spectra of B1-associated DNA fragments from transformed hepatocytes of murine hepatoma MH-22a, in general, were the same as those from hepatocytes of C3HA strain mice. At the same time, a DNA fragment with m.m. of 450 bp, not detected in normal hepatocytes, was revealed in transformed ones. Nevertheless a DNA fragment with m.m. of 600 bp, characteristic of normal hepatocytes, was not observed in the transformed hepatocytes. The B1-PCR method can be used for studying genomic polymorphism both in different populations of mice, and during malignant growth.

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Some characteristics of neoplastic cell transformation in transgenic mice.

The role of the expression of different cellular genes and viral oncogenes in malignant cell transformation is discussed. We pay special attention to the role of the genes for growth factors and their receptors and homeobox genes in oncogenesis. Based on both the literature and our own data, specific features of tumors developed in transgenic mice are discussed. All of these data are used to analyze current theories of multistep oncogenesis and the stochastic component in this process. We suggest that all known evidence about the mechanisms of oncogenesis be used in studying the problem at various structural and functional levels in an organism. The chapter shows that transgenic mice are a most suitable model for studying various aspects of malignant transformation from the molecular to the organismal and populational levels.

[The characteristics of homologous DNA recombination in somatic cells. III. Intrachromosomal recombinations of the aminoglycoside phosphoribosyltransferase gene in transgenic mice]. / Izuchenie osobennostei gomologichnoi rekombinatsii DNK v somaticheskikh kletkakh. III. Vnutrikhromosomnye rekombinatsii gena aminoglikozidfosforiboziltransferazy u transgennykh myshei.

Intrachromosomal homologous recombination has been revealed in the DNA from transgenic mice of three pedigrees. The recombination DNA of aminoglycosid phosphoribosiltransferase (neo) gene was observed in liver, kidney, spleen, heart, skeletal muscle, germinal glands and tail. It is concluded that the constructed model can be used for studying recombinations in cells of various organs and tissues both in the course of embryogenesis and during their malignant transformation and tumor progression.

[The use of B1-PCR method for studying the genomic polymorphism involved in malignant growth]. / Ispol'zovanie metoda v1-ptsr dlia izucheniia genomnogo polimorfizma pri zlokachestvennom roste.

The BI-PCT method showed the profile of BI-associated fragments of LNA in the cell line of the mouse hepatoma MH-22a to differ from that of the liver cells of C3HA mice, hepatoma cells incorporating the DNA fragments with 450 bp and those with 600bp disappearing. Application of the same method failed to reveal any differences in the profiles of BI-associated DNA fragments in the differentiated and non-differentiated cells of the embryonal carcinoma F9 induced by retinoic acid and cAMP dibutyryl treatment. It is suggested that the spectra of BI-associated DNA fragments might correlate with genetic stability in tumor cells.

[Oncogenesis in transgenic mice]. / Onkogenez u transgennykh myshei.

Oncogenesis in transgenic mice is at present a model, most adequately reflecting the natural conditions of tumor development. One of more important traits of this model is that it allows to study malignant growth simultaneously at all the structure-function levels in the context of the whole organism. This paper is a review of results of a series of experiments in which the localization of tumors was dependent or independent on the tissue specificity of a promoter, as well as development of multiple tumors with the use of viral regulatory sequences in genetic constructions. It has been shown that although a transgene is expressed in most of the tissues, tumors develop in some particular tissues only. These observations are interpreted by some authors in favour of the concept of multistep cancerogenesis. In this view, of primary importance are the results of studies on oncogenesis in transgenic mice, which contradict this concept and are regarded by their authors as an evidence of the possibility of a one-step transformation of normal cell into malignant one. The analysis of the obtained material enabled us to put forward an assumption that the key role in oncogenesis is played not only by certain genetic disturbances, but also by multi-level homeostatic mechanisms. Apparently, it is just the transgenic mice with cellular or viral oncogenes in their genome that represent a more adequate model for the detection of certain molecular-biological mechanisms underlying these disturbances. Also, of much importance is abundant material accumulated by now on oncogenesis of transgenic mice which shows a possibility of the effective use of various genetic constructions with prokaryotic and eukaryotic regulatory sequences, a possibility to induce not only tumors of some particular tissues, but also multiple hyperplastic and neoplastic changes in one and the same mouse. Development of tumors in such transgenic mice can be regarded as a model of different types of cancer disease.

[Leukemia in delta-GAG-MYC transgenic mice]. / Gemoblastozy u transgennykh myshei delta-GAG-MYC.

Transgenic mice carrying two delta-gag-myc genetic constructions were produced and kept under observation during their whole life. Nineteen out of 119 transgenic mice developed such hemopoietic diseases as lymphoid tissue hyperplasia, lymphoma, lymphosarcoma and myeloma. Lymphoid tissue hyperplasia and lymphoma generally involved multiple hyperplastic and neoplastic pathologies which were regarded, on the whole, as "malignant disease". In all cases, lymphosarcoma and myeloma were the only deadly pathologies. Lymphomas and myelomas were detected after 3-9 months, lymphosarcomas--18-29 months while lymphoid tissue hyperplasia occurred virtually throughout the entire life span--3-31 months. The study has shown that transgenic mice carrying delta-gag-myc gene in their genome can be used in the designing of special models for investigations of certain patterns of leukemia.

[The microcirculatory bed within the brain stem in the acute period of severe craniocerebral trauma]. / Vnutristvolovoe mikrotsirkuliatornoe ruslo v ostrom periode tiazheloi cherepno-mozgovoi travmy.

The authors studied changes of the intrastem microcirculatory channel in 72 patients who died in various terms of the acute period of severe craniocerebral trauma. According to the mechanism of the injury, an acceleration trauma had occurred in all cases. The microcirculatory channel was studied by filling it with an indicator mass (gelatin + india ink) and subsequent measurement of the length of the capillary network per 1 mm3 in various parts of the brain stem by the method suggested by S. M. Blinkov and G. D. Moiseev. As a control, a similar study was conducted in 20 individuals who died from extracranial injuries. Significant changes were revealed in the microcirculatory channel of the brain stem in the early period of craniocerebral trauma, and their periodization was established.

[Intravitally undetected tuberculosis and non-registered foci od tuberculous infection]. / Ne vyiavlennyi pri zhizni tuberkylez i neuchtennye ochagi tuberkuleznoi infektsii.

Active pulmonary tuberculosis was revealed in 1.55% of the cases registered in 11042 postmortem certificates representing forensic medical examination material accumulated at the archives for a period of 4 years. 66.35% of the cases had not been registered in a dispensary. Pulmonary tuberculosis was stated as a cause of death in 72.7% and as a concurrent condition in 27.3%. The basic activities of the antituberculosis service is to detect tuberculous infection and to rapidly eradicate its transmission. In addition, a close association and coordinated activities of forensic medical experts, phthisiologists and sanitary-and-epidemiologists will ensure an improvement of the epidemiological situation.