X Chromosome Control of Meiotic Chromosome Synapsis in Mouse Inter-Subspecific Hybrids

Hybrid sterility (HS) belongs to reproductive isolation barriers that safeguard the integrity of species in statu nascendi. Although hybrid sterility occurs almost universally among animal and plant species, most of our current knowledge comes from the classical genetic studies on Drosophila interspecific crosses or introgressions. With the house mouse subspecies Mus m. musculus and Mus m. domesticus as a model, new research tools have become available for studies of the molecular mechanisms and genetic networks underlying HS. Here we used QTL analysis and intersubspecific chromosome substitution strains to identify a 4.7 Mb critical region on Chromosome X (Chr X) harboring the Hstx2 HS locus, which causes asymmetrical spermatogenic arrest in reciprocal intersubspecific F1 hybrids. Subsequently, we mapped autosomal loci on Chrs 3, 9 and 13 that can abolish this asymmetry. Combination of immunofluorescent visualization of the proteins of synaptonemal complexes with whole-chromosome DNA FISH on pachytene spreads revealed that heterosubspecific, unlike consubspecific, homologous chromosomes are predisposed to asynapsis in F1 hybrid male and female meiosis. The asynapsis is under the trans- control of Hstx2 and Hst1/Prdm9 hybrid sterility genes in pachynemas of male but not female hybrids. The finding concurred with the fertility of intersubpecific F1 hybrid females homozygous for the Hstx2^Mmm allele and resolved the apparent conflict with the dominance theory of Haldane''s rule. We propose that meiotic asynapsis in intersubspecific hybrids is a consequence of cis-acting mismatch between homologous chromosomes modulated by the trans-acting Hstx2 and Prdm9 hybrid male sterility genes.     

Amniotic membrane transplantation in the treatment of persistent epithelial defect on the corneal graft

It has been shown that amniotic membrane transplantation (AMT) improves healing of the epithelium defects as it serves as a basement membrane for endothelial cells growth, prevents inflammatory cell infiltration and reduces apoptosis in keratocytes. Having in mind the healing properties of AM we investigated the efficacy of AMT in persistent epithelial defect (PED) on the corneal graft. 80 corneal grafts were prospectively followed up for presence of PED 10 months after surgery. PED was detected in 12 cases (15%) having surgery for: rejected graft (n = 4), keratoconus (n = 3), keratoconus following PK on a second eye (n = 3), corneal perforation (n = 1) and Stevens-Johnson keratopathy (n = 1). Epithelial defect (ED) developed 14 +/- 7 days after surgery in 10 cases and 1.5 month in other two. All patients were primarily conservatively treated with subconjuctival steroids and artificial tears for 10 days and systemic steroid therapy if needed after, until the period of 2 weeks. 4 patients were healed. Since ED was unresponsive to all previous treatments for more than 2 weeks, one layer of AM was placed on the corneal lesion in 5 patients, and in 3 cases of deep PED several layers of AM were placed. Healing of the defect was obtained in 7/8 (87.5%) eyes. In 1 patient second AM transplantation was necessary. Mean epithelization time was 2 weeks (range 1-3 weeks) in monolayer and 3 weeks (range 2-4 weeks) for multilayer cases. 5 out of 8 patients retained the same best corrected visual acuity (BCVA) while 3/8 patients improved their vision more than 2 lines. Preoperative corneal thickness of 255 +/- 40 mm increased to 455 +/- 90 mm. AM transplantation facilitates healing of corneal epithelium. PED on the corneal graft unresponsive to conventional treatment can be effectively cured when covered with one or more amniotic membrane layers.     

Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G(4)T(4)G(4) fragment

Using circular dichroism spectroscopy, gel electrophoresis, and ultraviolet absorption spectroscopy, we have studied quadruplex folding of RNA/DNA analogs of the Oxytricha telomere fragment, G(4)T(4)G(4), which forms the well-known basket-type, antiparallel quadruplex. We have substituted riboguanines (g) for deoxyriboguanines (G) in the positions G1, G9, G4, and G12; these positions form the terminal tetrads of the G(4)T(4)G(4) quadruplex and adopt syn, syn, anti, and anti glycosidic geometries, respectively. We show that substitution of a single sugar was able to change the quadruplex topology. With the exception of G(4)T(4)G(3)g, which adopted an antiparallel structure, all the RNA/DNA hybrid analogs formed parallel, bimolecular quadruplexes in concentrated solution at low salt. In dilute solutions ( approximately 0.1 mM nucleoside), the RNA/DNA hybrids substituted at positions 4 or 12 adopted antiparallel quadruplexes, which were especially stable in Na(+) solutions. The hybrids substituted at positions 1 and 9 preferably formed parallel quadruplexes, which were more stable than the nonmodified G(4)T(4)G(4) quadruplex in K(+) solutions. Substitutions near the 3'end of the molecule affected folding more than substitutions near the 5'end. The ability to control quadruplex folding will allow further studies of biophysical and biological properties of the various folding topologies. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 797-806, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Detecting hydrogen peroxide in leaves in vivo – a comparison of methods

Four hydrogen peroxide detecting probes, 3,3′‐diaminobenzidine (DAB), Amplex Red (AR), Amplex Ultra Red (AUR) and a europium–tetracycline complex (Eu₃Tc) were infiltrated into tobacco leaves and tested for sensitivity to light, toxicity, subcellular localization and capacity to detect H₂O₂ in vivo. In the absence of leaves, in water solutions, AUR was very much sensitive to strong light, AR showed slight light sensitivity, while DAB and Eu₃Tc were insensitive to irradiation. When infiltrated into the leaves, the probes decreased the photochemical yield (Φ_PSII) in the following order of effect AR > DAB > AUR > Eu₃Tc. With the exception of Eu₃Tc, all probes stimulated the build‐up of non‐photochemical quenching either temporally (DAB, AUR) or permanently (AR), showing that their presence may already limit the photosynthetic capacity of leaves, even in the absence of additional stress. This should be taken into account when using these probes in plant stress experiments. Confocal laser scanning microscopy studies with the three fluorescent H₂O₂ probes showed that the localizations of Eu₃Tc and AUR were mainly intercellular. AR partly penetrated into leaf chloroplasts but probably not into the thylakoid membranes. Photosynthesis‐related stress applications of AR seem to be limited by the low availability of internal leaf peroxidases. Applications of AR for kinetic H₂O₂ measurements would require a co‐infiltration of external peroxidase, imposing another artificial modifying factor and thus taking experiments further from ideal, in vivo conditions. Our results suggest that the studied H₂O₂ probes should be used in leaf studies with caution, carefully balancing benefits and artifacts.     

Microtubule-sliding modules based on kinesins EG5 and PRC1-dependent KIF4A drive human spindle elongation

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Quantitative characteristics of the conducting system in stem of winter wheat genotypes with spike fertility genes

Three groups of winter wheat (Triticum aestivum L.) genotypes having spike fertility genes (SFG) were used in field trials: (1) Tetrastichon sessile spikelets (TSS), (2) Normal spikelets (NS), (3) Indeterminate rachilla spikelets (IRS). The capacity of conducting system of the peduncle and the ear sink capacity of the main stem have been measured. There was a highly significant positive correlation (r = 0.899 and higher) between peduncle diameter and parameters quantifying peduncle vascular system. Compared with the control cultivar Hana, the TSS and NSS genotypes had higher both the number of vascular bundles, phloem and bundle cross section area and kernel number per ear. However, the highest kernel number per ear was found in the IRS genotypes although their bundle and phloem area was only equal or even lower then that of the variety Hana. Further studies are needed in developmental anatomy of spikes and stems to elucidate also differences in the relationships between the conducting capacity and kernel number per spikes in the TSS, NS and IRS genotypes.     

Timing of breeding, habitat preference and reproductive success of marsh harriers (Circus aeruginosus)

During 2002–2005 we analyzed Lack’s Hypothesis about the timing of the breeding of marsh harriers (Circus aeruginosus) in the Poodří to the breeding period and preference of heterogeneous habitat. An analysis of 43 nests revealed quantitative differences. Birds which started breeding earlier, reached significantly higher reproductive success than later breeding birds regardless of nesting habitat (P = 0.003, n _nests = 43). Even when the birds invested into their offspring the same way, their breeding success was not always the same. When the females nested in the common reed (Phragmites communis), they reached higher reproductive output than females nesting in cattail (Typha sp.) (P = 0.01, n _cattail = 18, n _{common reed} = 25). The habitat of the common reed is characterized by higher and denser vegetation cover than cattail vegetation (P < 0.001, n _measurements = 174 for both variables).     

In vitro induction of polyploidy in yacon (<Emphasis Type="Italic">Smallanthus sonchifolius</Emphasis>)

In vitro chromosome doubling was induced in octoploid (2n = 58) yacon using oryzalin and colchicine as mitotic spindle inhibitors. Nodal segments of in vitro cultured plants, 5–15 mm long, were exposed to 20, 25, or 30 μM oryzalin and 1, 3, or 5 mM colchicine for 24 or 48 h. The resulting ploidy level was determined by chromosome counting and flow cytometry. Out of 240 nodal segments, 3.33% hexadecaploid (2n = 116) plants were regenerated after the application of oryzalin. The greatest proportions of hexadecaploid plants (1.6%) were obtained after 48 h of 25 μM oryzalin treatment. With the colchicine treatment, only 0.42% hexadecaploid plants were detected and their survival rate was significantly lower in comparison with the oryzalin treatment. In hexadecaploid yacon, significantly higher levels of saccharides were detected (FOS 13.9 g/100 g FM, fructose 4.6 g/100 g FM and glucose 2.1 g/100 g FM) compared to the octoploid control (FOS 5.3 g/100 g FM, fructose 2.9 g/100 g FM and glucose 1.0 g/100 g FM). These results indicate that in vitro treatment of nodal segments with oryzalin solution could be an effective procedure for chromosome doubling and the polyploidy breeding can help to increase the FOS content in the tuberous roots.     

Different affinity states of CCK1 receptors on pancreatic acini and gastric smooth muscle in the rat

It has recently been shown that—after chronic cholecystokinin (CCK) treatment—an adaptation of pancreatic secretory but not gastric motor function does occur. Recent studies indicate that the CCK₁-receptor exists in two (i.e. high and low) affinity states, which could be distinguished by the CCK-analogue JMV-180. CCK occupancy of high and low affinity sites is thought to be related to the initiation of different intracellular events and consequent biological responses. Affinity states of CCK₁-receptors on pancreas and gastrointestinal (GI) smooth muscle could be different and this can offer an explanation for the different effects of CCK on pancreatic and gastric growth. We therefore studied the affinity states of CCK₁-receptors on isolated rat pancreatic acini and gastric smooth muscle preparations. When acini were incubated with increasing concentrations of CCK-8, a biphasic (i.e. stimulation followed by inhibition) effect on amylase release was observed. JMV-180 caused only stimulation of enzyme release and combined JMV-180 and CCK stimulation (at submaximal doses) resulted in an additive secretory response. CCK-8 induced contractions of pyloric, antral and fundic muscle in a concentration-dependent manner. The response was monophasic, reaching a plateau. JMV-180 had only a very weak effect on these preparations. On the contrary, it inhibited CCK-induced contractions in a competitive manner, the concentration–response curve to CCK being shifted to the right by the CCK analogue. Our data suggest that the affinity states of CCK₁-receptors on rat pancreatic and gastric tissue are different. On pancreatic acini CCK₁-receptors exist in both high- and low-affinity states whose occupation is followed by the sequence of intracellular events leading to growth. In contrast, occupation of low affinity receptors (the only ones present in the GI smooth muscle) does not lead to cell proliferation. This difference therefore explains the different adaptive response of the pancreas and the stomach to chronic CCK administration. Furthermore, different affinity states of CCK₁-receptors may mediate different functions of the digestive tract.