Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.     

Geminate structures of African cassava mosaic virus

Two types of geminate structures were purified from African cassava mosaic geminivirus (ACMV)-infected Nicotiana benthamiana plants and analyzed by electron cryomicroscopy and image reconstruction. After cesium sulfate density gradient centrifugation, they were separated into lighter top (T) and heavier bottom (B) components. T particles comigrated with host proteins, whereas B particles were concentrated in a cesium density typical for complete virions. Both particles were composed of two incomplete icosahedra of 11 capsomers each, but T particles were slightly larger (diameter, 22.5 nm) and less dense in the interior than B particles (diameter, 21.5 nm). T particles were frequently associated with small globules of approximately 14 nm diameter of unknown origin. The overall structure of ACMV, a begomovirus transmitted by whiteflies, was similar to that of Maize streak virus (MSV), a mastrevirus transmitted by leafhoppers, although the vertices of the icosahedra were less pronounced. Models of ACMV coat proteins based on Satellite tobacco necrosis virus support the exposure of parts of the molecule essential for transmission specificity by whiteflies and provide possible structural explanations for the smaller protrusion of the ACMV capsid relative to MSV. The differences of ACMV and MSV virion shapes are discussed with reference to their different animal vectors.     

Profile of resistance of human immunodeficiency virus to mannose-specific plant lectins

The mannose-specific plant lectins from the Amaryllidaceae family (e.g., Hippeastrum sp. hybrid and Galanthus nivalis) inhibit human immunodeficiency virus (HIV) infection of human lymphocytic cells in the higher nanogram per milliliter range and suppress syncytium formation between persistently HIV type 1 (HIV-1)-infected cells and uninfected CD4(+) T cells. These lectins inhibit virus entry. When exposed to escalating concentrations of G. nivalis and Hippeastrum sp. hybrid agglutinin, a variety of HIV-1(III(B)) strains were isolated after 20 to 40 subcultivations which showed a decreased sensitivity to the plant lectins. Several amino acid changes in the envelope glycoprotein gp120, but not in gp41, of the mutant virus isolates were observed. The vast majority of the amino acid changes occurred at the N glycosylation sites and at the S or T residues that are part of the N glycosylation motif. The degree of resistance to the plant lectins was invariably correlated with an increasing number of mutated glycosylation sites in gp120. The nature of these mutations was entirely different from that of mutations that are known to appear in HIV-1 gp120 under the pressure of other viral entry inhibitors such as dextran sulfate, bicyclams (i.e., AMD3100), and chicoric acid, which also explains the lack of cross-resistance of plant lectin-resistant viruses to any other HIV inhibitor including T-20 and the blue-green algae (cyanobacteria)-derived mannose-specific cyanovirin. The plant lectins represent a well-defined class of anti-HIV (microbicidal) drugs with a novel HIV drug resistance profile different from those of other existing anti-HIV drugs.     

Site-specific labeling of ‘second generation’ annexin V with 99mTc(CO)3 for improved imaging of apoptosis in vivo

In this study ‘second generation’ AnxV was specifically labeled with ^99mTc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged ‘second generation’ AnxV labeled with ^99mTc(CO)₃ in comparison to ^99mTc-HYNIC-cys-AnxV and ^99mTc(CO)₃-DTPA-cys-AnxV.     

Improved automated monitoring and new analysis algorithm for circadian phototaxis rhythms in Chlamydomonas

Automated monitoring of circadian rhythms is an efficient way of gaining insight into oscillation parameters like period and phase for the underlying pacemaker of the circadian clock. Measurement of the circadian rhythm of phototaxis (swimming towards light) exhibited by the green alga Chlamydomonas reinhardtii has been automated by directing a narrow and dim light beam through a culture at regular intervals and determining the decrease in light transmittance due to the accumulation of cells in the beam. In this study, the monitoring process was optimized by constructing a new computer-controlled measuring machine that limits the test beam to wavelengths reported to be specific for phototaxis and by choosing an algal strain, which does not need background illumination between test light cycles for proper expression of the rhythm. As a result, period and phase of the rhythm are now unaffected by the time a culture is placed into the machine. Analysis of the rhythm data was also optimized through a new algorithm, whose robustness was demonstrated using virtual rhythms with various noises. The algorithm differs in particular from other reported algorithms by maximizing the fit of the data to a sinusoidal curve that dampens exponentially. The algorithm was also used to confirm the reproducibility of rhythm monitoring by the machine. Machine and algorithm can now be used for a multitude of circadian clock studies that require unambiguous period and phase determinations such as light pulse experiments to identify the photoreceptor(s) that reset the circadian clock in C. reinhardtii.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.     

Identical TCR beta-chain rearrangements in streptococcal angina and skin lesions of patients with psoriasis vulgaris

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR beta-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin address in "cutaneous lymphocyte-associated Ag" (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.