M. S.

M. S.

In the present study the effect of diets restricted in either zinc or pyridoxine upon estrogen directed gene expression in the mature rat uterus was tested. Sexually mature female rats were maintained on zinc-adequate (40 mg/kg diet) <i>ad libitum</i> or restricted-fed, pyridoxine-deficient, or zinc-deficient ( < 1 mg/kg diet or 3mg/kg diet) <i>ad libitum</i>-fed diets for 35 days. All animals were bilaterally ovariectomized and used for experimentation at 14 days post ovariectomy. On day 35 each rat was injected intraperitoneally with estrogen. They were killed at different times post injection and thymidine kinase (TK, EC 2.7.1.21) or creatine kinase (CK, EC 2.7.3.2) activity was assayed in uteri cytosol fractions. In addition the steady state level of <i>ckb</i> mRNA in uteri cytosol fractions was measured following estrogen administration. The weight gain of the rats fed the low zinc and low pyridoxine diets was significantly lower than those fed the zinc-adequate diet ad libitum. The consumption of the zinc-deficient diet resulted in a significant decrease in plasma zinc while a pyridoxine deficient diet produced a significant reduction in plasma pyridoxine. Vehicle-injected uterine TK activity was 2-3 pmoles of d-TMP/min/mg protein. The TK activity was significantly increased (p < 0.05) 42 h post estrogen injection on the zinc-adequate diet <i>ad libitum</i> and pair-wt fed rats. This activity was sustained at 48 h post injection prior to declining to control values within 60 h. The maximum (4-fold) increase occurred at 36 h post estrogen injection in pyridoxine-deficient rats which was sustained at 42 & 48 h. The increase in uterine TK activity was 3-fold at 42 hand 48 h post injection. However this increase was not significantly different from the peak value seen in zinc-adequate and pyridoxine-deficient diet fed rats. No measurable effect of estrogen on CK activity was observed on the zinc adequate or zinc-deficient diet fed rats using a coupled enzyme assay. However the time course of ckb mRNA induction on the zinc-adequate pair-wt fed rats following estrogen administration paralleled the time course of estrogen induced protein (IP) synthesis previously observed by Gorski et al. (1970). IP is now known to be the brain type isoenzyme of creatine kinase. An induction of <i>ckb</i> mRNA between 0-3 h post estrogen injection was not observed on the zinc-deficient diet fed rats. However in a subsequent experiment an induction of uterine ckb mRNA 2 h following estrogen administration was observed in zinc-deficient rats. The possible reasons for this discrepancy are discussed. Zinc ions are known to be required to enable the estrogen receptor complex to bind to DNA and initiate transcription. It has been hypothesized that inadequate provision of dietary zinc may therefore reduce compliance to estrogen directed gene expression by limiting the efficiency of recruitment of zinc ions for stabilization of the zinc finger of the steroid receptor. The results of the present study failed to support this hypothesis at this moderate level of zinc depletion. / Ph. D.

Thirteen trials (n=930) were conducted to investigate the supplementation of weanling pigs with high levels of Zn and to compare the availability of Zn from several Zn sources for weanling pigs. In the first four trials, supplementing Zn by injecting Zn acetate either i.m. or i.p. at various times near weaning did not improve postweaning growth performance compared with pigs that were not injected. Additionally, stressing pigs by regrouping and then injecting Zn acetate did not improve growth performance. Serum Zn concentrations were increased in all of the trials by the injection of Zn. In the next five trials, feeding 3,000, 2,000 or 1,000 mg Zn/kg of diet from ZnSO₄, Zn-lysine or Zn-methionine did not improve growth performance immediately after weaning compared with pigs fed diets with 105 mg Zn/kg of diet. Feeding 3,000 mg Zn/kg of diet as ZnO (P < .05) improved growth performance above that of pigs fed 3,000 mg Zn/kg of diet from the other sources, but did not improve growth performance compared to controls. Lower tissue Zn concentrations suggested a lower availability of Zn from ZnO compared with ZnSO₄, Zn-lysine and Zn-methionine. There was little difference in Zn availability among the other sources. In the next three trials, feeding diets with different levels of lysine had little influence on the availability of Zn from Zn-lysine compared to ZnSO₄. Results indicate that Zn from Zn-lysine is not absorbed in conjunction with the lysine component of the complex. Additionally, there were no differences in the availability of Zn from ZnSO₄ compared to Zn-lysine. In the last trial, Zn from ZnO was less available (P < .05) to Zn deficient pigs than ZnSO₄, Zn-lysine or Zn-methionine when rib bone Zn concentration was used as an indicator of Zn availability. In summary, supplementing weanling pigs with high levels of Zn immediately before or after weaning does not appear to improve growth performance. Furthermore, Zn from ZnO is less available to weanling pigs than Zn from ZnSO₄, Zn-lysine or Zn-methionine. / Ph. D.

Manipulation of dietary zinc produced a different body zinc status in male Japanese quail during 3 weeks of treating period. After intramuscular injection with 8μmol/100g body weight of estradiol-17β, Japanese quail were sacrificed and vitellogenin (phosphoprotein) production was assessed in these birds by analyzing plasma for protein-bound phosphorus concentrations (PBP). Plasma PBP concentrations of estrogen-injected male quails increased from undetectable values for control birds to O.66mg/ml for high-zinc birds and O.42mg/ml for low-zinc birds. About 36% loss of vitellogenin synthesis was associated with the consumption of the zinc-deficient diet. Plasma zinc concentrations also increased on the estrogen injection because of the zinc-binding property of vitellogenin. The extra zinc ions in the plasma were considered to be come from the body zinc pools. Liver as well as other major tissues was believed to act as a reservoir of zinc for egg development but it was not observed that liver was the major source of the extra zinc in the plasma. / Master of Science

Thesis (MTech (Agriculture))--Cape Peninsula University of Technology, 2018. / Zinc deficiency on various soil types have been reported in arable soils of sub Saharan Africa (SSA) including South Africa. A pot trial was conducted at the Cape Peninsula University of Technology, Wellington campus to investigate the interaction of different application rates of Zn at various soil pH on the grain yield and quality of spring wheat in a completely randomized factorial design replicated three times. The four soil pH tested were: pHA: 5.1, pHB: 5.6, pHC: 6.1, pHD: 6.6 which correspond to lime application at 0, 0.5, 1 and 1.5 t/ha. Five Zn rates (Zn1: 3.5; Zn2: 4.5; Zn3: 5.5 Zn4: 6.5, and Zn5: 7.5 mg /kg soil which correspond to Zn1: 7; Zn2: 9; Zn3: 11; Zn4: 13 and Zn5: 15 kg /ha) were applied at two (planting and flowering) growth stages. Yield and yield component data collected were analyzed using SAS version 9.2 and means were separated by Duncun’s Multiple Range Test (DMRT). The results showed that grain yield and yield components were significantly affected by lime application pHC (6.1): 1t/ha at planting. Zn application at planting had no significant effect on the grain yield and yield components. However, at flowering, the simultaneous increase of Zn along with increase in lime positively affected grain yield and yield components. Plant analysis showed that at both stages (planting and flowering), Zn application, especially at pH 6.6, significantly increased P, K, Ca, Na, Mg Fe, Cu and B concentrations in wheat grain, but the concentrations of N, Mn, Zn and protein remained unaffected. Zn application had no effect on most nutrients due to the presence of lime. While the absence of lime, Zn4: 6.5mg/kg (corresponding to 13kg/ha) significantly increased the nutrients. In addition, Zn3: 5.5mg/kg (corresponding to 11kg/ha) promoted Zn absorption by grain in all treatments.

The tightly regulated openings of the cardiac ryanodine receptor (RyR2) help to ensure that intracellular Ca2+- release from the sarcoplasmic reticulum (SR) can only occur when heart contractions are required. Usually this process is self-regulatory, where Ca2+ both activates and inhibits release of further Ca2+ from the SR. In the progression of heart failure some of this control is lost and in rest periods Ca2+ can leak from the SR into the cytosol. Recent evidence has suggested that Zn2+- dyshomeostasis may contribute to SR Ca2+- leak but the underlying mechanism is unclear. Using single channel electrophysiological studies in combination with live cell imaging of HEK 293 and fibroblasts, this study reveals that Zn2+, along with Ca2+ and the inhibitor Mg2+, plays a physiological role in the grading of Ca2+- release via RyR2. Importantly the data reveal that pathophysiological concentrations of Zn2+ (> 100pM) within the cytosol remove the requirement of Ca2+ to activate RyR2, resulting in irregular channel activity even in the presence of Mg2+. This increase in channel open probability due to Zn2+ is known to be associated with increased Ca2+- release events such as Ca2+ sparks suggesting that Zn2+ is a regulator of the SR Ca2+-leak current. A potential source of releasable Zn2+, which could modulate RyR2 activity in cardiomyocytes, are the acidic organelles (endosomes and lysosomes). This study provides key evidence that the two pore channels (TPCs), which are expressed on the surface of these organelles, are candidate channels for ligand-gated release of Zn2+. Importantly this research demonstrates that dysregulated Zn2+ homeostasis, resulting in elevated Zn2+ within the lysosome, has severe consequences upon cellular Ca2+- release from fibroblasts, which is primarily the result of Zn2+ acting as a pore blocker of TPC2. Together these data reveal a key role of Zn2+ as a second messenger which can regulate intracellular Ca2+- release in both health and disease.

Splenic and lymph node lymphocytes from Balb/C mice were activated in vitro by the heavy metal cations, Zn⁺⁺ and Hg⁺⁺, as noted by the several-fold increases in ³H-thymidine incorporation at 144 hours of culture. Optimal mitogenic concentrations of Zn⁺⁺ and Hg⁺⁺ were 200 μM and 10 μM, respectively. Data from experiments in which three different methods were used to enrich for either T or B splenic lymphocytes, i.e. cell passage over nylon wool columns, use of athymic Nu/Nu mouse spleen cells, or cell lysis with monoclonal anti-Thy-1 antibody plus complement, suggested that Zn⁺⁺ and Hg⁺⁺ were mitogens for T cells. Removal of macrophages from spleen cells by treatment with carbonyl iron followed by cell passage through nylon wool eliminated the lymphocyte responses to Zn⁺⁺ and to Hg⁺⁺. Moreover, addition of these macrophage-depleted lymphocytes to monolayers of resident peritoneal macrophages restored the lymphocyte responses to these mitogens. Both Zn⁺⁺ and Hg⁺⁺ activated splenic lymphocytes to display lectin-dependent cytotoxicity and to produce gamma interferon. Furthermore, Zn⁺⁺ induced low levels of natural killer activity in spleen cells. In contrast to spleen and lymph node cells, thymocytes and bone marrow lymphocytes did not respond to either cation under standard culture conditions. However, when cultured in the presence of E. coli-derived lipopolysaccharide (LPS) and 2-mercaptoethanol for 144 hours, thymocytes were activated by Zn⁺⁺ (200 μM) but not by Hg⁺⁺. Quantities of LPS as low as 1.0 ng/ml satisfied this culture requirement. Purified interleukin 1 could not replace the helping activity mediated by LPS. Thymocyte subpopulation studies showed that Zn⁺⁺ activated enriched peanut lectin receptor-negative mature thymocytes, but LPS was required for the response. Spleen cells from mice, intraperitoneally injected with ZnCl₂ for 7 to 14 days, were not activated in vivo as assessed by ³H-thymidine incorporation in vitro, nor did they display enhanced responses to T-cell or B-cell mitogens. However, zinc administration had negative effects by decreasing spleen cell numbers by 31% and thymic weight by 59%. A theoretical model is presented in which Zn⁺⁺ and Hg⁺⁺ may mediate their stimulating effects in vitro by altering histocompatibility "self" structures on the surface of lymphocytes and macrophages via interactions with sulfhydryl groups on these structures to which T lymphocytes with receptors for "altered self" structures respond with proliferation or cytotoxicity.

This study was conducted to compare the availability of Zn from two Zn sources, an amino acid chelate and ZnSO₄. In three experiments, 78 Zn depleted and 24 Zn adequate pigs were used. Pigs were depleted of Zn by feeding an isolated soy protein, semi purified diet containing 17 ppm Zn. Of the 78 depleted pigs, 60 pigs were Zn repleted. During Zn repletion in Exp. 1, depleted pigs were fed the low Zn diet supplemented with 5, 15, or 45 ppm Zn either as ZnSO₄ or as Zn amino acid chelate (ZnAAC). In Exp. 2 and 3, low Zn diets were only supplemented with 45 ppm Zn. Zinc adequate pigs, used in Exp. 1 and 2, were fed the 45 ppm supplemental Zn diets. To evaluate differences in site and rate of apparent Zn absorption, chromic oxide was added to the diets of depleted pigs in Exp. 1 and 3. In all experiments, a 24-d period was sufficient to severely deplete the porcine body Zn stores, and to cause parakeratosis and growth retardation. Serum Zn concentrations and serum ALP-activities of depleted pigs dramatically decreased (P < .01) during the first 14 d of Zn depletion. At the end of Zn depletion, Zn contents in liver, kidney, pancreas, brain, and small intestine tissues of pigs fed the low Zn diet were reduced (P < .01) by 10 to 40 % compared with the adequate pigs fed the ZnSO₄ and ZnAAC diets. In Exp. 2, the growth retardation was associated with a low (P < .05) serum mitogenic activity and pituitary RNA content of depleted pigs compared with pair-fed adequate pigs. Moreover, the growth hormone mRNA fraction tended to be reduced (P < .10) for the Zn depleted pigs. In Exp. 1, the apparent absorption of Zn was higher (P < .01) for pigs fed ZnAAC compared with the ZnSO₄ group; however, this was not confirmed in Exp. 3 unless coefficients were corrected for Cr recovery. Furthermore, absorption of Zn occurred primarily within jejunal and distal segments of the small intestine. In the balance of Exp. 3, disappearance rates of Zn, Cu, Fe and DM were higher (P < .01) in depleted pigs fed ZnAAC compared with ZnSO₄. The recovery of Cr also was different (P < .01) between pigs fed the ZnSO₄ (87 %) and ZnAAC (70 %) diets. Moreover, the moisture content of the fecal matter was 11 % higher (P < .01) for the ZnAAC group compared with pigs fed ZnSO₄. In Exp. 1, depleted pigs fed the 15 ppm ZnSO₄ and ZnAAC diets regained their ability to grow, however, replenishment of body fluid and tissue Zn pools did not occur within the 24-d Zn repletion period. Both the 5 ppm ZnSO₄ and ZnAAC groups did not respond to Zn repletion within a 12-d period. In all experiments, the rate and degree of repletion of body fluid and tissue Zn stores was not different between pigs fed the 45 ppm ZnSOq and ZnAAC diets, although a higher (P < .05) serum mitogenic activity was observed for the adequate pigs fed ZnAAC compared with ZnSO4. In conclusion, an amino acid chelate did not improve growth, or rate and degree of replenishment of body fluid and tissue levels of Zn compared with pigs fed ZnSO₄. However, ZnAAC may have influenced intestinal luminal conditions since a higher rate of disappearance of Zn, Cu, Fe, Cr, and DM was measured. / Ph. D.