Gel filtration coupled with radioimmunoassay of fractions has demonstrated the existence of an 8000 dalton immunoreactive form of GIP (glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide), which may be a precursor in the biosynthetic pathway. A monoclonal antibody to GIP has been shown to have highly suitable characteristics for affinity purification of different species of IR-GIP. An enzyme-linked immunosorbent assay (ELISA) was developed for GIP, employing the monoclonal antibody and was used for screening fractions for peptides with the same antigenic determinant i.e. IR-foras of GIP. Classical strategy used in peptide purification may result in loss of related peptides if they are sensitive to the pH or temperature conditions used. Tissue from hog duodenal and jejunal mucosa was boiled and extracted into acetic acid. Peptides were then adsorbed to alginic acid, eluted with 200 mM HC1, precipitated with NaCl and desalted on Sephadex G-25. The desalted material was adjusted to pH 7.0 with 200mM ammonia and extracted with methanol. The methanol insoluble fraction demonstrated the highest content of IR-GIP₈₀₀₀⋅ The overall acidic charge on the larger IR-GIP oUUU moiety suggested the possibility that it might not be adsorbed to alginic acid. The monoclonal antibody to porcine GIP₅₀₀₀ was coupled to cyanogen bromide activated Sepharose -4B. The peptide fraction which was not adsorbed to alginic acid was applied to the column and the fraction which bound to the ligand was eluted with 100 mM HC1. The immunoreactive material was rotary evaporated to dryness and further purified to a monocomponent by HPLC. A µBondapak C₁₈ column and a linear gradient of acetonitrile in water containing 0.1% TFA was used for HPLC. Amino acid analyses revealed the following composition: Asx (6), Thr (2), Ser (3), Glx (3), Pro (3), Gly (4), Ala (8), Val (5), Met (1), He (0), Leu (7), Tyr (1), Phe (3), His (4), Lys (5), Arg (3), Trp (+). The N-terminal residue was identified as valine using the dansylation method. Cleavage of the molecule with trypsin and separation of the tryptic peptides on HPLC showed 2 peptides with elution times similar to tryptic peptides of GIP. Application of monocomponent IR-GIP designated IR-LGIP C, and GIP to the HPLC system confirmed the two peptides to be separate entities. Biological activity was assessed in the isolated perfused rat pancreas, a model used for measurement of the insulin releasing effect of GIP. IR-LGIP C did not demonstrate insulinotropic activity. It is unlikely that this polypeptide is a proform of GIP. It shares common immunoreactivity but lacks the necessary common core of amino acid residues. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Interdigestive myoelectric activity in rat intestine has been recorded and characterized. The interdigestive pattern of activity can be disrupted by oral glucose and high doses of the duodenal ulcerogen cysteamine. Intravenous glucose had no effect on the interdigestive myoelectric pattern, nor did high doses of porcine motilin. Attempts were made to develop a hybridoma cell line secreting antibodies that would recognize rat Intestinal immunoreactive motilin (IR-M). The murine myeloma cell line NS1 was fused with murine B-cells primed against porcine motilin. One hundred of the monoclonal cell lines produced secreted monoclonal antibodies that recognized porcine motilin. Attempts to identify a cell line secreting antibodies with the ability to stain rat intestinal tissue, however, produced only negative results. Rat intestinal IR-M has been characterized with respect to immunocytochemistry (ICC), radioimmunoassay (RIA), and chromatographic properties. The biological activity of partially purified rat intestinal IR-M has also been evaluated utilizing a rabbit isolated duodenal muscle strip preparation. Five different antisera and one monoclonal antibody directed against natural porcine motilin were utilized in an effort to detect IR-M containing cells in rat intestinal tissues. A variety of techniques were employed including tissue fixation with either Bouins, paraformaldehyde, or benzoquinone. In addition a variety of staining methods including, fluorescein conjugated second antibody, peroxidase-antiperoxidase or peroxidase conjugated second antibody techniques were used. All methods using these antibodies failed to detect IR-H in the rat small intestine. Porcine motilin was able to displace ¹²⁵I-motilin from antisera 13-3, 72X and M03. These antisera were utilized in a motilin RIA to evaluate acid extracts of rat intestinal tissue for IR-M. Only antisera 13-3 and 72X were capable of detecting IR-M in gut extracts, and these antisera gave different distributions of IR-M In the proximal small bowel. Rat intestinal tissue was extracted into 2% trifluoroacetic acid and the soluble fraction clarified by centrifugation. This acid extracted material was precipitated with sodium chloride then dissolved in methanol at pH 6.0. Methanol soluble material was precipitated with ether and the ether precipitate then dissolved in water and chromatographed on Sep-Pak C₁₈ cartridges (Waters). Sep-Pak cartridges were eluted with 50% acetonitrile: 0.1% TFA. The 50% eluate was then fractionated further using cation exchange, gel filtration and reverse phase high pressure liquid chromatography (HPLC). Rat intestinal IR-M peaks from cation exchange chromatography on SP-Sephadex-C25 (Pharmacia) were concentrated and examined for contractility in a rabbit duodenal muscle strip preparation. Purification after SP-Sephadex-C25 was approximately 20 fold. Desensitization of rabbit duodenum to porcine motilin could be demonstrated by pre-treatment with motilin. Contractile activity of partially purified rat intestinal IR-M was not inhibited by pretreatment with motilin. Chromatography on Bio-Gel P-10 (Biorad) eluted with 0.2M acetic HPLC, using a linear gradient of water/acetonitrile (10-45% acetonitrile in 30 min), rat intestinal IR-M did not co-elute with natural porcine motilin. In conclusion, the molecular weight of rat intestinal IR-M appeared to be similar to porci ne motilin as these two substances demonstrated co-elution on gel permeation chromatography. The lack of co-elution with porcine motilin on HPLC indicates that other molecular characteristics of rat intestinal IR-M, such as hydrophobicity, are not similar to porcine motilin. Furthermore, partially purified rat intestinal IR-M did induce a contractile response in rabbit duodenal muscle strips but porcine motilin did not desensitize this preparation to the contractile activity of rat intestinal extracts. This suggests that the contractile activity of these two compounds is induced via different receptor mechanisms. It is concluded that the immunoreactive motilin found in rat intestinal extracts does not resemble natural porcine motilin in structure or biological activity. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The work on Spiraea in vitro shoot cultures was done to determine the feasibility of using light quality to modify endogenous phytohormone balances to decrease apical dominance. Such an effect would enable a reduction in the high levels of exogenous cytokinin benzyladenine (BA) applied in culture and thus reduce potential side-effects. The Spiraea in vitro light quality response was characterized by examining the effects of different light wavelengths on growth. A mixture of red/FR induced rates of shoot proliferation with 0.25 mg/1 BA that were as high as rates obtained under white light with 0.5 mg/1 BA. Shoot quality, as determined by the proportion of shoots 1 cm or longer (useful shoots), was highest under red/FR light. The lowest shoot proliferation rate was observed under blue light. When light wavelengths intermediate between blue and red light (green, yellow, and orange) were applied to explants only minor growth modifications occurred. Green light did not inhibit shoot initiation but inhibited shoot elongation at the 0.5 mg/1 BA level. The efficacy of the light source-filter combinations in the first experiment was studied in two further experiments. With the three light sources (tungsten filament, fluorescent, and metal halide) together with a blue filter, results supported the putative blue light inhibitory effect suggested in the first light quality experiment. Under the red filter, the tungsten filament source induced the highest shoot number means at both BA levels used (0.25 and 0.5 mg/1). Two factors may have contributed to the red/FR effect observed in the first experiment; the time under an incubation light regime before transfer to the treatment regime, and the photon fluence rate of each regime. In the subsequent study to examine these factors, shoot initiation was optimized at the lower BA levels of 0.25 and 0.4 mg/1 when cultures under low fluence red/FR were transferred after four weeks to white light of a higher fluence for one more week. Glyphosate, a known promoter of IAA oxidation, was used to investigate the presumed effect of lowered IAA-cytokinin interactions. Two types of responses to glyphosate occurred, each one dependent on the glyphosate concentration. At the lower glyphosate level (0.087 mg/1), cultures under both light regimes with 0.25 mg/1 of BA, showed a strong inhibition of shoot initiation. This inhibitory effect was overcome in cultures with 0.5 mg/1 of BA and an overall stimulatory response occurred as shoot initiation rates were as much as four-fold higher than in the previous experiments. For both BA levels, changes in shoot number were greater under white light than under red/FR. At the higher glyphosate level (0.2 67 mg/1), the shoot initiation rates were greater than glyphosate-free controls for both BA levels under white light although under red/FR the rates were virtually unchanged from controls. The glyphosate effect investigated for Spiraea cultures appears to be influenced by the levels of the cytokinin BA resulting in pleiotropic effects which depend on the specific concentrations of each component. / Land and Food Systems, Faculty of / Graduate

The role of juvenile hormone (JH) in alate-apterous polymorphism was investigated in the green peach aphid, Myzus persicae. At higher concentrations (65 ppm), the juvenile hormone analogue (JHA), kinoprene, was immediately toxic to apteriform nymphs. At lower concentrations (10 ppm), the compound was non-toxic and exhibited no apparent morphological activity in apteriform stages. In contrast, 65 ppm kinoprene administered to alatiform nymphs had juvenilizing and apterizing effects. The extent of these effects depended upon when the kinoprene was applied. Fourth instar alatiforms were the least sensitive as kinoprene-treated nymphs developed into normal adults with reduced sclerotization and pigmentation. Kinoprene-treated third instars underwent a supernumerary moult before metamorphosing into adults with malformed wings. Sclerotization and pigmentation were also lacking in these insects. When first and second instar alatiformsiwere treated with kinoprene, they also underwent a supernumerary moult. The adults which emerged exhibited both larval and apterous characteristics. Wing development was almost totally inhibited; the cauda and genital plate were poorly developed. In addition, sclerotization and pigmentation were reduced and ocell lacking. The secondary antennal sensoria were also malformed. These findings clearly (1) demonstrate that kinoprene can be employed as a JH mimic to alter the normal programming of the epidermal cells in alatiform nymphs and (2) indicate that JH plays an important role in aphid morphogenesis and polymorphism. The differential responses of the four alatiform nymphal instars suggest that elevated JH levels during the first and second instars are particularly important in inhibiting wing development. To determine the prenatal effects of JH on wing development and morphogenesis, kinoprene was also administered to newly ecdysed apterous adults. Even though conditions favoured alate production, 75% of the offspring produced by kinoprene-treated virginoparae developed into normal apterae. This suggests that elevated JH titers in maternal haemolymph inhibit wing development and promote development of apterae. Topical application of the anti-allatotropin, precocene-II, had variable effects on apteriform nymphs and adults. In all stages, precocene produced a significant decline in larvi-position. The effects were more pronounced in first and second instar apteriform nymphs and apterous adults than in third and fourth instar nymphs. When kinoprene was applied to these insects, larviposition increased significantly after 2 days. The findings demonstrate that JH stimulates reproduction in viviparous morphs of persicae. / Land and Food Systems, Faculty of / Graduate

Over the past decade long distance marathon running has become an important recreational activity. There is evidence that males with high levels of physical activity have some impairment of fertility. In order to investigate this further, 24 male marathon runners were studied over a period of a year. Each runner was assessed at regular intervals using hormonal profiles, anthropomorphic indices and semen evaluation. The training time and distance run increased progressively over the first five months of the study as the runners prepared for the Two Oceans marathon. Analysis of the serum hormonal profiles in this longitudinal study showed that the prolactin level increased when comparing the initial study month with the rest of the year and the progesterone level decreased. However the luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone and estradiol (E2) levels remained unchanged. When the runners were divided into a high and low training group according to the distance run in the preceding week, the only significant difference was the lower mean serum FSH level in the high training group. A decrease in semen volume was demonstrated as the training time increased. This trend was reversed as the runners' training decreased after the Two Oceans marathon. The percentage of morphologically normal spermatozoa showed an initial significant decrease in the first month of training. However, no significant difference was observed throughout the rest of year. An overall downward trend in semen motility in the first 5 months of the study was shown but this was only significant if the first and fifth study months were compared. The decrease in semen motility coincided with the period of maximum training. Since patients with an adequate sperm count but decreased motility have impaired fertility this finding is of considerable importance. In addition to the decrease in motility, there was a decrease in the percentage of morphologically normal spermatozoa when the initial month of low physical activity (December) was compared to all of the subsequent months analysed. This, too, is an important finding as the percentage of morphologically normal spermatozoa correlates directly with fertilisation and pregnancy rates. When the results were analysed in the high and low training months there was a significant difference in mean semen count and semen morphology. The mean count was higher in the high training group and this group also had a significantly higher normal morphology. However, there was no significant difference in semen volume and motility in the high and low training groups.

Please read the abstract in the section 00front of this document / Dissertation (MMed Vet (FER))--University of Pretoria, 1999. / Veterinary Tropical Diseases / unrestricted

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The mechanism of steroid transport and corticosterone secretion in the zona fasciculata-reticularis of rat adrenal cortex was investigated by measuring the subcellular distribution and concentrations of steroids following ACTH stimulation.