An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.     

Functional analysis of a complex oncogene arrangement in biotype III Agrobacterium tumefaciens strains

The ubiquitous grapevine-associated octopine/cucumopine Ti plasmids of biotype III Agrobacterium tumefaciens strains carry two T regions, TA and TB, with a complex oncogene arrangement. Within the octopine/cucumopine group, two main strain types were identified: large TA strains with a TA region resembling the TL region of the biotype I octopine strain Ach5 and small TA strains with a similar T region organization as the large TA strains but with a large internal TA deletion. Structural and functional studies of the representative large TA strain Tm4 revealed six oncogenes. Each oncogene was inserted in a disarmed vector and tested for biological activity using the corresponding oncogenes of Ach5 as standards. Five Tm4 oncogenes, TA-iaaM, T-ipt, T-6b, TB-iaaH and TB-iaaM, were shown to be active, the IS-interrupted TA-iaaH gene was inactive. To study the role of each gene in the pTiTm4 context, several single and multiple pTiTm4 mutations were constructed. It was shown that whereas TA-iaaM and TB-iaaH are essential for tumour formation on grapevine, T-ipt, T-6b and TB-iaaM are not. The avirulence of the TA-iaaM ^- mutant was shown to be due to an inhibitory effect of the T-ipt gene, since a TA-iaaM ^- /T-ipt ^- double mutant was fully virulent. We conclude that the TA-iaaM gene of large TA strains is specifically required to counteract the tumour growth inhibiting activity of the T-ipt gene. Both TA-iaaM and T-ipt are absent from the small TA strains. A model on the roles and interactions of the different oncogenes in large TA and small TA strains is presented.     

Involvement of Two Types of TNF Receptor in TNF-α Induced Neutrophil Apoptosis

We previously demonstrated that tumor necrosis factor-α (TNF-α) induces rapid human neutrophil apoptosis. In this paper, we examined which of the TNF receptors, p55 kDa TNF receptor (55-R) or p75 kDa TNF receptor (75-R), or both are involved in this process using specific rabbit antisera. Antibodies to 55-R (anti55-R) or 75-R (anti75-R) alone did not induce neutrophil apoptosis. Further addition of cycloheximide and anti-rabbit immunoglobulin to anti55-R but not to anti75-R accelerated apoptosis, although anti75-R augmented the capacity of anti55-R to do so. These results suggest that 55-R is a prerequisite for TNF-α induced neutrophil apoptosis.     

Sexual behavior of donkey jacks: influence of ejaculatory frequency and season

The courtship of 5 jacks was evaluated weekly for 12 mo. The characteristics of sexual behavior were recorded before the collection of 2 ejaculates, at 4-h intervals, into an artificial vagina in the presence of a female in either natural or induced estrus. The maximum time given to the jack to perform the ejaculatory mount was 1 h. If the jacks did not ejaculate, another attempt was made the following week. At the time of collection, the male and female remained free in a paddock (20 m2). The data presented in this study is based on results in which the first and second ejaculates occurred successively. Statistical analysis was done by a 2 x 2 factorial design in randomized blocks. The mean +/- SEM of sexual behavior characteristics for the first and second ejaculate were, respectively: time until first mount = 0.7 +/- 0.2 and 0.9 +/- 0.2 min; time until first erection = 11.9 +/- 1.1 and 11.6 +/- 1.1 min; time until ejaculation = 15.0 +/- 1.2 and 13.6 +/- 1.2 min; frequency of partial exposure of penis = 3.4 +/- 0.4 and 3.2 +/- 0.4; frequency of total exposure of penis = 1.0 + 0.2 and 1.1 +/- 0.2; frequency of flehmen responses = 6.6 +/- 0.5 and 4.6 +/-0.5; frequency of erections = 1.3 +/- 0.1 and 1.2 +/- 0.1; frequency of retreats away from the female = 1.8 +/- 0.2 and 1.0 +/- 0.2; frequency of mounts with erection but without ejaculation = 0.3 +/- 0.1 and 0.1 +/- 0.1; frequency of mounts without erection = 2.0 +/-0.1 and 1.2 +/- 0.1 ; and frequency of pelvic copulatory movements = 4.8 +/- 0.4 and 4.4 +/- 0.4. Individual differences were observed (P<0.05) for partial and total exposure, flehmen responses, mounts without erection and pelvic copulatory movements. The variables flehmen responses, retreats away from the female and mounts without erection showed significant differences (P<0.05) between ejaculates. Seasonal effects on sexual behavior characteristics were not found. However, a monthly effect was noted for flehmen responses, partial exposure of the penis, mounts without erection, retreats away from the female and pelvic copulatory movements when the 2 ejaculates were combined.