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PubMed2005

DNA double-strand breaks and homology search: inferences from a species with incomplete pairing and synapsis.

The relationship between meiotic recombination events and different patterns of pairing and synapsis has been analysed in prophase I spermatocytes of the grasshopper Stethophyma grossum, which exhibit very unusual meiotic characteristics, namely (1) the three shortest bivalents achieve full synapsis and do not show chiasma localisation; (2) the remaining eight bivalents show restricted synapsis and proximal chiasma localisation, and (3) the X chromosome remains unsynapsed. We have studied by means of immunofluorescence the localisation of the phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks; the SMC3 cohesin subunit, which is thought to have a close relationship to the development of the axial element (a synaptonemal complex component); and the recombinase RAD51. We observed a marked nuclear polarization of both the maturation of SMC3 cohesin axis and the ulterior appearance of gamma-H2AX and RAD51 foci, these being exclusively restricted to those chromosomal regions that first form cohesin axis stretches. This polarised distribution of recombination events is maintained throughout prophase I over those autosomal regions that are undergoing, or about to undergo, synapsis. We propose that the restricted distribution of recombination events along the chromosomal axes in the spermatocytes is responsible for the incomplete presynaptic homologous alignment and, hence, for the partial synaptonemal complex formation displayed by most bivalents.

Animals↗

PubMed1980

Karyotype analysis of the plant-parasitic nematode Heterodera glycines by electron microscopy. II. The tetraploid and an aneuploid hybrid.

In the plant-parasite nematode Heterodera glycines, 2 forms, in addition to the diploid (9 bivalents), have been isolated and analysed: a tetraploid (18 bivalents) and an aneuploid (14 bivalents, hybrid between the diploid and the tetraploid). Observations on the formation of their karyotypes indicates normal and non-homologous pairing. Eighteen normal synaptonemal complexes (SC) are present in pachytene nuclei of the tetraploid. Two of the SCs are enclosed in a large heterochromatin mass that is displaced to one side of the nucleus. Such a mass has not been observed in the diploid or the aneuploid. Another 2 normal SCs of the tetraploid have each a 'modified SC region' (MSC) within which the SC appears disorganized. The aneuploid has 14 SCs that are unattached at either end. Only 25% of the karyotype length is normal in the appearance of the SCs. The rest can be traced by the presence of disorganized SC material and condensed chromatin. Four MSCs are present in the hybrid nuclei. The possible role of the MSCs in the sex determination system is discussed.

Aneuploidy↗

PubMed2006

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains.

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.

Binding Sites↗

PubMed1983

Multivalent inactivated virus oil emulsion vaccines in broiler breeder chickens. II. Trivalent vaccines in breeders not previously vaccinated with live Newcastle disease, infectious bursal disease, and tenosynovitis vaccines.

Inactivated Newcastle disease (NDV), infectious bursal disease (IBDV), and viral arthritis/tenosynovitis (VA) viruses were incorporated into water-in-oil emulsion vaccines either alone, in bivalent combinations, or in a trivalent vaccine. Twenty-week-old broiler breeder chickens with no previous exposure to NDV, IBDV, or VA live virus vaccines were injected intramuscularly with the monovalent, bivalent, or trivalent vaccines. The antibody responses to NDV in all three vaccines were poor, and NDV-hemagglutination-inhibition (HI) geometric mean titers (GMTs) never rose above 20 during the 40-week trial. The antibody response to IBDV showed a strong primary response 4 weeks after vaccination, but IBD-VN geometric mean titers declined steadily to less than 100 by 4 months after vaccination. The antibody GMTs to IBDV continued to decline for the remainder of the trial. The antibody response to VA virus was biphasic, with peak VA-virus neutralization (VN) geometric mean titers occurring at 3 months and 6 months postvaccination. The amplitude of the response to the monovalent, bivalent, and trivalent vaccines was inversely proportional to the number of antigens incorporated into each vaccine. Maternal antibody titers in the progeny against each of the three antigens reflected those of the parents. In no case were maternal antibody titers detectable beyond 14 days of age.

Animals↗

PubMed1983

Multivalent inactivated virus oil emulsion vaccines in broiler breeder chickens. III. Trivalent Newcastle disease, infectious bursal disease, and arthritis/tenosynovitis viruses vaccine in primed breeders.

Animals↗

PubMed1985

Field trials to test the efficacy of polyvalent Marek's disease vaccines in layer and broiler breeder chickens.

As a follow-up to earlier trials to evaluate the efficacy of polyvalent Marek's disease (MD) vaccines in broilers, four trials were conducted with layer or broiler breeder flocks. Chickens caccinated with trivalent (Md11/75C plus SB-1 plus HVT) and bivalent (SB-1 plus HVT) vaccines were compared with those vaccinated with turkey herpesvirus (HVT) alone. Data from three of the four trials indicated polyvalent vaccines were more efficacious than HVT. However, critical interpretations were confounded by low MD lesion frequencies, unequal exposure in different houses on a farm, and difficulty in the differential diagnosis of mortality due to neoplasms. The data confirmed our earlier observation that trivalent and bivalent vaccines were equally effective. Broiler progeny of breeders vaccinated or contact-infected with Serotype 2 MD virus were well protected by either bivalent or HVT vaccines against challenge with very virulent MD virus strains in the laboratory. A high incidence of lymphoid leukosis was observed in two trials and may have been due to post-hatch exposure to an unidentified environmental source of avian leukosis virus.

Animals↗

PubMed2005

A DNA-based vaccine for the prevention of human cytomegalovirus-associated diseases.

Multiple lines of evidence indicate that in the transplant population human cytomegalovirus (HCMV) infection and its associated diseases are controlled by humoral and cellular immune responses similar to those that arise in asymptomatic, healthy individuals during a naturally-acquired infection. The dominant antibody response to HCMV is to the major surface glycoprotein B (gB) and the dominant cellular immune response is to the tegument phosphoprotein (pp65). We propose that an immunotherapeutic plasmid DNA (pDNA) vaccination approach that induces the requisite responses to major immunological targets of HCMV may provide relief from HCMV-associated diseases in the transplant setting. We have developed gene-based immunotherapeutic products consisting of pDNAs encoding gB and pp65 of HCMV. When tested individually in mice, both pDNAs were highly immunogenic. Relative to vaccination with either gB or pp65 pDNA delivered alone, vaccination with gB and pp65 pDNAs delivered together in phosphate-buffered saline (PBS) elicited reduced antibody and T cell responses to each antigen. Formulating this bivalent vaccine with a poloxamer-based delivery system (VF-P1205-02A), however, significantly increased the antigen-specific immune responses relative to those induced with the bivalent vaccine in PBS, and completely abrogated the decrease in pp65-specific T cell responses observed in mice covaccinated with the pDNAs in PBS. Based on these data, and a favorable safety and toxicity profile in preclinical studies, the bivalent HCMV vaccine consisting of gB and pp65 pDNAs delivered with VF-P1205-02A has advanced to human clinical trials.

Animals↗

PubMed1998

Immunogenicity of a combined hepatitis B and Haemophilus influenzae type b conjugate vaccine in Alaska Native infants.

UNLABELLED: Hepatitis B and Haemophilus influenzae b (Hib), prevalent diseases in Alaska Native infants, have been reduced 95% following universal vaccination. Therefore, we were interested in studying a new combined Hib and hepatitis B vaccine in this population. METHOD: Healthy Alaska Native infants free from Hib and hepatitis B exposure were randomized into four groups, three with different lots of bivalent Hib and hepatitis B vaccine, and one with Hib and hepatitis B monovalent controls. The Hib component had 7.5 micrograms of polyribosylribitol phosphate polysaccharide (PRP) bound to 125 micrograms of Neisseria meningitidis outer membrane protein complex (OMP); the hepatitis B component was 5 micrograms of recombinant hepatitis B surface antigen. The vaccines were given at 2 months, 4 months, and 12-15 months of age. RESULTS: There were no differences in the responses to the bivalent compared to the monovalent controls except for a lower Hib anti-PRP level at 12-15 months in the bivalent recipients; this did not persist after the booster. CONCLUSION: A combined Hib and hepatitis B vaccine appears immunogenic. The recommended schedule for the Hib component (PRP-OMP) of 2 months, 4 months, and 12-15 months appears to promote an optimal response to the hepatitis B component as well.

Alaska↗

PubMed2000

Targeting and therapy of carcinoembryonic antigen-expressing tumors in transgenic mice with an antibody-interleukin 2 fusion protein.

The purpose of this study was to engineer a bivalent single-chain anticarcinoembryonic antigen (CEA) antibody and an interleukin 2 (IL-2) fusion protein derivative for selective tumor targeting of cytokines. The variable domains of a high affinity anti-CEA antibody, T84.66, were used to form a single-gene-encoded antibody [single-chain variable fragment joined to the crystallizable fragment, Fc (scFvFc)]. The fusion protein (scFvFc.IL-2) consisted of mouse IL-2-fused to the COOH-terminal end of the scFvFc. The engineered proteins were assembled as complete molecules and were similar to the intact anti-CEA monoclonal antibody (Mab) in antigen-binding properties. Based on IL-2 content of the fusion protein, its ability to support proliferation of CTLL-2 cells was identical with that of IL-2. Despite a molecular size similar to that of the intact Mab, the blood clearance of the fusion protein was markedly faster than that of the intact Mab or scFvFc. Incubation of radiolabeled scFvFc.IL-2 but not the intact or scFvFc antibodies in mouse serum was accompanied by the appearance of complexes, suggesting that the latter may contribute to the accelerated clearance of the fusion protein. Biodistribution and tumor targeting studies were carried out in CEA-transgenic mice bearing CEA-positive murine tumors as well as the antigen-negative parental tumor. The bivalent anti-CEA scFvFc had tumor localization properties similar to those of the intact Mab. Although fusion of IL-2 to the COOH-terminal end of the bivalent scFvFc altered its pharmacokinetic properties, the fusion antibody was able to target tumors specifically. Maximum uptake of the intact Mab, scFvFc, and scFvFc.IL-2 in CEA-positive tumors was 29.3 +/- 5.0, 19.5 +/- 2.1, and 6.6 +/- 0.9% injected dose/g, respectively. Maximum tumor localization ratios (CEA-positive/CEA-negative tumor) were similar for all three antibody types (4.6-6.0), demonstrating the antigen specificity of the tumor targeting. Significant antigen-specific targeting to CEA-positive normal tissues of transgenic mice was not observed. Although the tumor-targeting properties of the fusion protein were low, the growth of CEA-expressing (P = 0.01) but not antigen-irrelevant (P = 0.22) syngeneic tumor cells was inhibited after treatment of transgenic mice with the anti-CEA-IL-2 antibody. Therapy of CEA-expressing tumors was improved after i.v. administration of the fusion protein (P = 0.0001). These studies indicate that anti-CEA antibody-directed cytokine targeting may offer an effective treatment for CEA-expressing carcinomas. The availability of an immunocompetent CEA transgenic mouse model will also help to determine the immunotherapeutic properties of these fusion proteins.

Animals↗

PubMed2000

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Amino Acid Sequence↗

PubMed2001

[Damaging effect of antibiotics on the structure of synaptonemal complexes of meiotic chromosome of mice].

The structure of synaptonemal complexes (SCs) of chromosomes of mouse primary spermatocytes were studied using electron microscopy on days 1, 10, and 36 after the completion of per os administration of drugs belonging to three groups of antibiotics: tetracyclins, macrolides, and fluoroquinolones. The antibiotics were administered to mice during ten days. At the substages of early and middle pachytene, heteromorphic SC bivalents and fragments of chromosome-core elements were detected in spermatocytes at all times studied after the administration of the antibiotics of three groups. As cells passed through the period from early to middle pachytene, the number of cells containing heteromorphic SC bivalents and the fragments of chromosome cores gradually decreased, which could be an indication of selection of cells with chromosomal aberrations. A high level of associations between the X chromosome and autosome bivalents (including heteromorphic ones) also favors this suggestion. A gradual decrease in the number of chromosomal aberrations was detected, as time elapsed from the completion of antibiotics administration. The study of sperm obtained from epididymises of males did not reveal significant differences in both morphology and motility of sperm between males of the control and experimental groups.

Animals↗

PubMed1975

A field study of persistence of antibodies in California horses vaccinated against western, eastern, and Venezuelan equine encephalomyelitis.

As a result of the continuing threat of Venezuelan equine encephalomyelitis (VEE), a study was made to determine if revaccination against VEE (TC-83 vaccine) was feasible and if revaccination could be incorporated into other routine vaccination practices. Of the horses given annual vaccination with bivalent western equine encephalomyelitis (WEE) and eastern equine encephalomyelitis (EEE) vaccine, 57% retained detectable serum-neutralizing (SN) antiboyd titers for VEE 18 months after the initial VEE vaccination was given. Of horses with no record of WEE-EEE vacinnation, 100% retained detectable VEE SN antibody titers over the same period. The VEE geometric mean titer was 25 times greater for horses not previously vaccinated against WEE-EEE than for horses given annual WEE-EEE vaccination at the time of VEE vaccination. In horses vaccinated against VEE 18 months previously, the geometric mean titer increased from 4 to 70 at 48 days after the intitial WEE-EEE vaccination. This increase indicated that similar antigenic factors for VEE are possibly present in bivalent WEE-EEE vaccine. In horses previously vaccinated against WEE-EEE and VEE, the best SN antibody response to VEE revaccination occurred when VEE vaccine was given simultaneously with the bivalent WEE-EEE vaccine. Of 150 serum samples tested by both the SN and the hemagglutination-inhibiton tests, agreement between positive reactions at greater than or equal to 1:10 was 70% for VEE, 81% for EEE, and 87% for WEE.

Animals↗

PubMed1975

Reserpine and calcium: interaction at the injection site.

The subcutaneous administration of calcium prior to reserpine prevented the development of the well-known reserpine effects. Pretreatment with other bivalent cations induced a similar effect. The inhibitory effect of reserpine on the in vitro uptake of labelled catecholamines in brain homogenates was also prevented in rats pretreated with bivalent cations. On the other hand when calcium was injected at a site other than that of reserpine, there was no antagonistic effect. The latter results suggest an interaction between bivalent cations and reserpine at the injection site that prevents the systemic absorption of reserpine.

Animals↗

PubMed2002

[Lampbrush chromosomes in the japanese quail Coturnix coturnix japonica: cytological maps of macro chromosomes and meiotic crossover frequency in females].

Cytological maps of lampbrush macrobivalents of the Japanese quail (Coturnix coturnix japonica) were constructed. Investigation of chiasmata allowed determination of the meiotic frequency of reciprocal genetic recombination (crossing over) in Japanese quail females. The total chiasma number in bivalents of Japanese quail oocyte nuclei was determined to be 53-58. Macrobivalents 1-5 and Z of the Japanese quail had on average 3.3 chiasmata per bivalent, and microbivalents, 1.0-1.1 chiasmata per bivalent. The chiasmata (crossover) frequency in Japanese quail females was lower than in chicks. In macrochromosomes of Japanese quail females, one crossover occurred per 43.9 Mb, and in chicken, per 30.0 Mb. Judging from chiasma frequency, the genetic length of the Japanese quail genome is likely to be 2650-2900 cM. Crossover frequency in the species was 0.023 per Mb in macrobivalents and 0.07-0.08 Mb in microbivalents and for the total genome, 0.041 crossovers per Mb. The genetic length of one Mb (theta) in female Japanese quails was 1.14 cM in macrochromosomes, 3.60-4.12 cM in microchromosomes, and about 1.96-2.15 cM averaged over the genome.

Animals↗

PubMed2003

Chromosome pairing in tetraploid rye with monosomic-substitution wheat chromosomes.

In tetraploid rye with single-substitution wheat chromosomes - 1A, 2A, 5A, 6A, 7A, 3B, 5B, 7B - chromosome pairing was analysed at metaphase I in PMCs with the C-banding method. The frequency of univalents of chromosome 1A was considerably higher than that of the other four wheat chromosomes of genome A (6A, 5A, 7A and 2A). Among chromosomes of genome B, the lowest mean frequency of univalents was observed for chromosome 5B. In monosomic lines, wheat chromosomes 1A, 2A, 5A, 6A, 7A and 5B paired with rye homoeologues most often in rod bivalents and in chain quadrivalents (also including 3B). The 47% pairing of 5B-5R chromosomes indicate that the rye genomes block the suppressor Ph1 gene activity. In monosomic plants with chromosomes 5A, 2A, 6A, 7A and 5B, a low frequency of rye univalents was observed. It was also found that the wheat chromosomes influenced the pairing of rye genome chromosomes, as well as the frequency of ring and rod bivalents and tri- and quadrivalents. However, the highest number of terminal chiasmata per chromosome occurred in the presence of chromosomes 5A and 2A, and the lowest - in the presence of chromosomes 3B and 7B. In the presence of chromosome 5B, the highest frequency of bivalents was observed. The results of the present study show that the rye genome is closer related to the wheat genome A of than to genome B. The high pairing of wheat-rye chromosomes, which occurs in tetraploid rye with substitution wheat chromosomes, indicates that there is a high probability of incorporating wheat chromosome segments into rye chromosomes.

Chromosome Banding↗

PubMed2003

The influence of rye cytoplasm on meiotic stability of tetraploid Secalotriticum.

Chromosome pairing in tetraploid Secalotriticum was analysed. In the studied plants wheat chromosomes in PMCs during metaphase I showed a higher degree of pairing, in comparison to the rye genome. This is reflected in a very low frequency of univalents and a higher frequency of ring bivalents. The occurrence of wheat univalents was dependent on wheat mixogenome. In plants with an unstabilized fourth homoeologous group, a heteromorphic bivalent 4A-4B was observed in 39.9% of PMCs, whereas in plants with an unstabilized seventh homoeologous group, chromosome 7A-7B pairing was found in all analysed cells. Rye univalents were present in all plants studied. The highest mean frequency of univalents and rod bivalents, both in wheat and in rye genomes, were recorded in plants whose first homoeologous group contained chromosome 1A. The mean number of terminal chiasmata per chromosome amounted to 1.78 in the wheat genome and 1.36 in the rye genome. It may be concluded that the plasmagenes in Secalotriticum did not increase the meiotic stability of the rye genome and also did not stabilize plant fertility.

Chromosomes↗

PubMedDate not supplied

[Effects of magnesium, manganese and cobalt ions on the activity of Ca2+-dependent ATPase of skeletal muscle plasma membranes].

Bivalent cations (Mn and Co) are found to activate ATPase activity of sarcolemma in the same degree, as Mg and Ca. The increase of the activity of Ca2+-dependent ATPase under the certain conditions of treatment of sarcolemma with KCl-Triton X-100 solution is not accompanied by the increase of ATPase activity in the presence of Mn and Co ions. ATPase activities in the presence of these ions correlate with the activity of Mg2+-dependent ATPase. Bivalent cations Mg, Mn and Co are competitive inhibitors for Ca2+-dependent ATPase. The apparent inhibition constants are determined to be 3,5-10(-5) M for Mg, 0,7-10(-3) M for Co and 1,5-10(-3) M for Mn. It is supposed that Ca2+-dependent ATPase has similar selectivity to bivalent cations as calcium influx that follows depolartization of plasma membrane.

Adenosine Triphosphatases↗

PubMed1992

Studies on the immunogenicities of intertypic chimeric polioviruses.

To study the relationship between structure and antigenicity, two hybrid viruses, XF414 and XF3, were constructed. In XF414, N-Agl of PV1 was replaced by N-Agl of PV2; while in XF3, N-Ag1 of PV1 was replaced by N-Ag1 of PV3. In this study, XF414 and XF3 were cultured on a large scale in primary monkey kidney cells. The viruses were concentrated with PEG, purified with chloroform and Sepharose 2B and inactivated with formaldehyde. Their bivalent antigenicity was confirmed by neutralization inhibition test, immunoelectron microscopy, and rabbit immunization. It was also shown that both XF414 and XF3 can elicit bivalent neutralizing antibodies and that the bivalent antigenicities are not destroyed by formaldehyde inactivation. Such a study can provide a basis for the development of multivalent picornavirus vaccines.

Animals↗