Does follicular dominance occur in ewes?

The process by which a single follicle is selected to ovulate while others regress is unknown in ewes. If the dominant follicle secretes substances that directly inhibit the growth of other follicles, the superovulatory response to the administration of exogenous gonadotrophins may be blunted. Administration of 1250 iu pregnant mares' serum gonadotrophin (PMSG) before or after the emergence of the dominant follicle in the follicular phase, or 1000 iu PMSG in the presence or absence of a large healthy or atretic follicle during the luteal phase did not affect the induced ovulatory response. Comparisons between the ovary with or without the dominant follicle did not reveal any differences in ovulatory response to PMSG. The in-vitro features (i.e. mitotic index, oestradiol and testosterone production) of follicles ipsilateral or contralateral to the dominant follicle during the early and late follicular phases were also similar. If the dominant follicle secretes substances detrimental to the other follicles, this could be mimicked in vitro. Co-culture of small follicles with the largest follicles in a closed system did not reduce their incorporation of 3H thymidine in granulosa cells, compared with small follicles cultured alone. These data suggest that dominance is probably not operative in sheep. The administration of 500 iu of PMSG during the midfollicular phase increased ovulation rate in Merino ewes, indicating that dominance is essentially passive in ewes and can easily be overcome by raising gonadotrophin concentration.     

A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae

A strain of Vibrio cholerae, which had been engineered to express high levels of the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200-amino-acid exported protein, with a Cys-X-X-Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation in E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active-site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.     

Identification of a matrix-associated region 5'' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.     

Sensitivity and adrenoceptor affinity in the mesenteric artery of the deoxycorticosterone acetate hypertensive rat

This study examines vascular reactivity to alpha-adrenoceptor agonists in mineralocorticoid (deoxycorticosterone acetate (DOCA-salt) hypertensive and normotensive rats. The rats were anesthetized and the mesenteric artery was excised and cut helically into strips that were mounted in a muscle bath for the measurement of isometric force development. Addition of norepinephrine, epinephrine, phenylephrine, methoxamine, or clonidine to the bath caused contractions in all arteries. Arteries from hypertensive rats were more sensitive (lower ED50 values) to each of the agonists than arteries from normotensive rats. alpha-Adrenoceptor affinity for phentolamine (Schild analysis; norepinephrine as the agonist) in hypertensive arteries was not significantly different from that in normotensive arteries. Maximal force generation to clonidine was greater in hypertensive arteries than in normotensive arteries. These results demonstrate an augmented vascular sensitivity to several alpha-adrenoceptor agonists in DOCA hypertensive rats. This change in sensitivity is independent of a change in affinity for the adrenoceptor antagonist, phentolamine. It may be that a change in receptor number or an alteration in a post-receptor activation event accounts for this enhanced adrenoceptor responsiveness in mineralocorticoid hypertension.     

Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.     

M?ssbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe M?ssbauer spectroscopy. The M?ssbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.     

Structural analyses of a monoclonal heterodimeric suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10

A GAT-specific, MHC-restricted "second-order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 was biosynthetically radiolabeled with 35S-methionine and was isolated from cell extracts. The isolation procedure involved two-dimensional nonreducing/reducing SDS-PAGE and electroelution of the reduced off-diagonal polypeptide chains from the gel. Biochemical characterization studies revealed that TsF2 is a disulfide-linked heterodimer composed of a basic and an acidic polypeptide chain, both having m.w. of 30,000. Both chains are glycosylated and contain sialic acid residues. The basic polypeptide reacts with anti-I-J antisera, whereas the acidic chain contains the antigen-binding capacity. Monoclonal antibodies induced by immunizing rats with TsF2 purified from hybridoma supernatants were selected for the ability to block immunosuppression mediated by TsF2 in vitro. These antibodies, but not irrelevant antibodies, immunoprecipitated the 35S-methionine-labeled protein that migrates off the diagonal in two-dimensional gels. Thus, we have verified that the immunosuppressive protein that migrates off the diagonal in two-dimensional gels binds to antibodies that are known to inhibit the biologic activity of unpurified TsF2.     

Endocrine differences in rams after genetic selection for testis size

Testis diameter and body weight were recorded from 6 to 76 weeks of age in ram lambs from two established lines selected for high (H) and low (L) testis size. While testis growth was greater in the H line up to 14 weeks of age (P less than 0.001), body weight was significantly lower, with the L line rams being 10 kg heavier by 76 weeks. There were no differences in plasma LH up to 20 weeks of age, but FSH concentrations were significantly lower at 14 and 20 weeks in the H line. Testosterone concentrations were not significantly higher in the H line from 6 to 20 weeks. In lambs castrated at birth, significantly higher FSH values were recorded from 6 to 20 weeks of age in the H line (P less than 0.001) whereas there was no difference in LH concentration at 6 and 10 weeks of age between the lines. At 14 and 20 weeks, however, the concentrations of LH were greater in the H than L line lambs (P less than 0.05). After hemicastration at 6 weeks of age, the rate of growth of the remaining testis in the L line lambs was significantly faster than in entire lambs of that line from 10 to 20 weeks (P less than 0.05 at 10 weeks to P less than 0.001 at 20 weeks). There was no difference in the rate of testis growth between the the entire and hemicastrated lambs from the H line from 6 to 12 weeks of age. It can be concluded that there is an underlying genetic difference in pituitary gland and/or hypothalamic activity in ram lambs from the two selected lines.     

Is vegetation in equilibrium with climate? How to interpret late-Quaternary pollen data

Current methods for estimating past climatic patterns from pollen data require that the vegetation be in dynamic equilibrium with the climate. Because climate varies continuously on all time scales, judgement about equilibrium conditions must be made separately for each frequency band (i.e. time scale) of climatic change. For equilibrium conditions to exist between vegetation and climatic changes at a particular time scale, the climatic response time of the vegetation must be small compared to the time scale of climatic variation to which it is responding. The time required for vegetation to respond completely to climatic forcing at a time scale of 10⁴ yr is still unknown, but records of the vegetational response to climatic events of 500-to 1000-yr duration provide evidence for relatively short response times. Independent estimates for the possible patterns and timing of late-Quaternary climate changes suggest that much of the vegetational evidence previously interpreted as resulting from disequilibrium conditions can instead be interpreted as resulting from the individualistic response of plant taxa to the different regional patterns of temperature and precipitation change. The differences among taxa in their response to climate can lead a) to rates and direction of plant-population movements that differ among taxa and b) to fossil assemblages that differ from any modern assemblage. An example of late-Holocene vegetational change in southern Quebec illustrates how separate changes in summer and winter climates may explain the simultaneous expansion of spruce (Picea) populations southward and beech (Fagus) populations northward.