The efficiency of somatic cell nuclear transfer (SCNT) cloning remains low, thus limiting the applications of this technique. In this study, we used immunochemistry and confocal microscopy to detect the microtubule component, β-tubulin, in SCNT, parthenogenetic (PA), and intracytoplasmic sperm injection (ICSI) embryos before the first mitotic division. β-Tubulin is the component subunit of microtubule, which plays critical roles in regulating localization of cellular organelles, and the growth, maturation and fertilization of oocytes. Our results demonstrated similar changes of spindle patterns in PA and ICSI embryos. The second meiotic division resumed 1 h post-treatment, and the cytoplasmic asters (CAs) disappeared. After about 4–6 h of treatment, pronuclei formed with the midbodies connecting each other. Meanwhile, the CAs reappeared and a microtubule network developed in the cytoplasm. However, SCNT embryos showed abnormal multipolar spindles, and the pseudopronuclei that contained many nucleoli existed after 6 h of SrCl2 activation. Enucleated oocytes alone did not form spindle-like structures when they were artificially activated for 6 h, indicating that somatic cell chromosomes might be necessary for spindle formation in SCNT embryos. These results demonstrated abnormal changes of β-tubulin in mouse SCNT embryos, compared with PA and ICSI embryos.

Seeds of neem (Azadirachta indica A. Juss.), a multipurpose tropical tree, have limited desiccation tolerance. Whether their intermediate storage behaviour might be caused by an elevated percentage of 4C nuclei in the embryo at maturity was investigated. Morphological development of neem seeds was monitored on selected trees in Ouagadougou, Burkina Faso. Developing seeds were tested for germinability, and cell cycle events were determined by using flow cytometry and analysing the level of β-tubulin. Germination could occur after 8 weeks of seed development, but normal seedlings resulted only after 10 weeks or more. The change in fruit colour from green to yellow began after approximately 12 weeks of seed development. Immature, 4-week-old embryos about 2 mm in length had 15% of cells in the G2 phase and 60% in the G1 phase of the cell cycle, as indicated by 4C and 2C nuclear DNA levels, respectively. During maturation, the proportion of G2 cells declined to 3% at full maturity and slightly further after drying, and the proportion of G1 cells increased to approximately 90%. A strong β-tubulin signal was observed in tips of young embryonic radicles and cotyledons, but a weak or non-detectable signal was found in 9-week-old ones and in those from green-mature and yellow fruits. Because DNA replication and β-tubulin level were almost negligible at seed maturity, as in orthodox tomato seeds, it is suggested that these factors are not involved in the intermediate storage behaviour of neem seeds.

Links between germination, DNA replication and β-tubulin accumulation were studied with cabbage (Brassica oleracea L.) seeds, by using flow cytometric analysis of nuclear DNA content and immunodetection of β-tubulin levels. The seeds were incubated in water or 0.1–500 mM hydroxyurea solutions. Radicle tips isolated from dry cabbage seeds revealed most 2C (Go or G1 stage) and some 4C (G2 stage) signals of nuclear DNA contents and a constitutive level of β-tubulin. The onset of DNA replication in the radicle tip was observed between 12 and 24 h of imbibition in water and was preceded by an increase of β-tubulin levels. Incubation of the seeds in 1 mM hydroxyurea retarded DNA replication, whereas an arrest of DNA replication occurred upon incubation in 10 mM hydroxyurea or higher concentrations. The arrest of DNA replication and cell division did not block radicle protrusion and allowed some extension of the radicle. This demonstrated that DNA replication is not a prerequisite for radicle protrusion and initial extension. However, further seedling development, including root growth and root hair development, was dependent on DNA replication. Accumulation of β-tubulin was not affected by hydroxyurea. Thus, it can be deduced that both DNA replication and β-tubulin accumulation are two parallel and independent cell cycle events during seed germination.

Macrocyclic lactones (MLs) are highly lipophilic anthelmintics which are known to bind to and open ligand-gated ion channels. However, these anthelmintics, and particularly the avermectin members of the ML class of endectocides, are potent substrates for ABC transporters and these transporters may regulate drug concentration in both the host and the parasite. There is accumulating evidence that ivermectin (IVM), and to a lesser extent moxidectin (MOX), selects for certain alleles of P-glycoprotein and other ABC transporter genes, selects for constitutive overexpression of some of these gene products, and induces overexpression of some P-glycoproteins in nematodes. However, such mechanisms of ML resistance do not easily lend themselves to the identification of SNP markers for resistance because of the diversity of ABC transporters in nematodes, the apparent diversity of effects of different MLs, and because regulatory elements for ABC transporter gene expression are not well understood in nematodes. Another non ligand-gated ion channel gene which appears to be under IVM selection, at least in Onchocerca volvulus and Haemonchus contortus, is β-tubulin, and a simple genetic test for this selection has been described in O. volvulus. However, further work is required to elucidate a reliable marker associated with this gene in H. contortus or other parasitic nematodes of livestock. The possible involvement of ABC transporter genes and β-tubulin in ML resistance provides a start in developing our understanding of this phenotype and markers for its detection in field populations of parasitic nematodes. However, more work is required before these leads can provide practical SNP markers for ML resistance.

Cell cycle activity in dry and germinating untreated and treated (soaked in water and subsequently in fungicide) seeds of two sugarbeet cultivars, collected at commercial harvest time (late mature seeds) and about 2 weeks before this (immature seeds), was investigated by flow cytometry, and by immuno-detection of β-tubulin and the B-subunit of the 11 S globulin. Germination capacity and field emergence were tested. With dry seeds of both cultivars, higher G2 / G1 ratios were observed in the radicle tips of late mature seeds, as compared with those from immature seeds. The late mature seeds contained more partly degraded (soluble) B-subunit of 11 S globulin, typical of germinating or primed sugarbeet seeds. Thus events associated with the onset of germination had occurred in the seed lots collected at commercial harvest time. The cytoskeleton protein β-tubulin was not detectable in dry seeds from either harvest. Western blotting revealed an accumulation of β-tubulin during germination and this was faster in the late mature harvested seeds which was correlated with the onset of DNA replication. Soaking enhanced the rate of cell cycle activation during germination as well as vigour, germination capacity, and field emergence. There was positive correlation between the G2 / G1 ratio and the traits examined in laboratory and field tests. It is concluded that a combined analysis of proteins and cell-cycle-related events can be used in understanding and predicting sugarbeet seed quality.

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sitw, was used. DNA replication, as measured by flow cytometry, and β-tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy.

In this study, the 27 kDa immunodominant antigen (CP23), 70 kDa heat shock protein (HSP70), actin and β-tubulin genes were amplified and sequenced for the first time from human isolates of Cryptosporidium cervine genotype. New primers were designed from reported sequences of other Cryptosporidium species and genotypes as well as the whole genome sequences of C. parvum and C. hominis, which enabled novel gene sequences and regions extending beyond those deposited in GenBank to be determined. In comparison with other species in the Cryptosporidium genus, multiple sequence alignment and phylogenetic analysis revealed that the Cryptosporidium cervine genotype isolates from humans clustered most closely with Cryptosporidium deer mouse genotype and C. suis (n. sp. formerly pig genotype I). The complete coding sequence of CP23 was determined to reveal low (72·4% and 68·0–69·8% respectively) identity to C. parvum and C. hominis sequences and the presence of a unique multiple proline-alanine-proline-valine (PAPV) repeat region.

Observations of low response of patients infected with Onchocerca volvulus to ivermectin suggest that the parasite may be under a selection process toward potential resistance. To limit the extension of this phenomenon, it is crucial to characterize the genes of O. volvulus that are involved. For this, O. volvulus adult worms collected before the introduction of ivermectin in an onchocerciasis endemic area of central Cameroon were genotyped for β-tubulin. To derive a baseline to investigate the selective pressure of ivermectin, we analysed (1) the frequency distribution of the β-tubulin alleles, and (2) the relationship between the different β-tubulin related genotypes and the fertility status of the female worms. The frequency of allele b of the β-tubulin gene was very low, as it was observed in West Africa. We observed a deficit of heterozygous female worms leading to Hardy Weinberg disequilibrium, which might be explained by a shorter life-span of these worms compared to the homozygous worms. Unexpectedly, our results also show that the heterozygous female worms were much less fertile than the homozygotes: more than two thirds of the homozygotes were fertile, whereas only 37% of the heterozygotes were fertile. These results will be further considered when analysing post-treatment data.

Mammalian homologues of the Drosophila canonical Transient Receptor Potential (TRPC) protein have been proposed to encode the store-operated Ca2+ influx (SOC) channel(s). This study examines the role of TRPC1 in the SOC mechanism of retinal cells. htrpc1 transcript was detected in bovine retinal and in human adult retinal pigment epithelial (ARPE) cells. Western blot analysis also confirmed the expression of TRPC1 protein in neuronal cells including retina and ARPE cells. To determine the role of TRPC1 protein in retinal cells, TRPC1 was recombinantly expressed in ARPE cells and changes in intracellular Ca2+ were analyzed. ARPE cells stably transfected with htrp1 cDNA displayed 2-fold higher Ca2+ influx with no significant increase in the basal influx. Consistent with this the overexpressed TRPC1 protein was localized in the plasma membrane region of ARPE cells. Interestingly, both bovine retinal tissues and ARPE cells showed that TRPC1 protein co-localizes and could be co-immunoprecipitated with β-tubulin. Disruption of tubulin by colchicine significantly decreased both plasma membrane staining of the TRPC1 protein and Ca2+ influx in ARPE cells. These results suggest that TRPC1 channel protein is expressed in retinal cells, further, targeting/retention of the TRPC1 protein to the plasma membrane in retinal cells is mediated via its interaction with β-tubulin.

TaqMan minor groove binder probes were evaluated as to their suitability for the real-time allelic discrimination of the β-tubulin codon 200 TTC/TAC single nucleotide polymorphism in cyathostomin species. Amplification of titrated cloned full-length β-tubulin cDNA revealed that the TaqMan minor groove binder PCR is capable of specifically detecting as few as 10 copies. Testing of DNA from single adult and larval stages of several different species of cyathostomin allowed reproducible genotyping of individual worms. Using the real-time PCR approach, the throughput of samples was considerably increased compared with conventional post-PCR readout procedure. Only 7·8% homozygous TAC L3 were found among 102 L3 which were genotyped from phenotypically BZ-resistant small strongyle populations. The percentages of the homozygous TTC and heterozygous TTC/TAC were 41·3% and 50·9%, respectively. This resulted in a total TAC-allele percentage of only 33·3%. These findings correspond to data obtained by genotyping of an experimentally selected BZ-resistant cyathostomin population. It is concluded that the β-tubulin codon 200 polymorphism is not the sole mechanism involved in the process of BZ resistance in cyathostomins.

Resistance in Fasciola hepatica to triclabendazole (‘Fasinex’) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the β-tubulin molecule. Sequencing of β-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 μg/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.