Effects of dietary zinc deprivation on zinc concentration and ratio of apo/holo-activities of angiotensin converting enzyme in serum of mice

Objective

The aim of the present study was to evaluate the effects of dietary zinc deprivation on zinc concentration, the activity of angiotensin converting enzyme (ACE) and ratio of apo/holo-activities of ACE (ACE ratio) in the serum of mice.

Methods

Twenty-two male mice were deprived of dietary zinc for up to 9 days. Zinc concentration in the serum was measured by means of atomic absorption spectrometry. Also, the activity of ACE in the serum was measured by HPLC as the activity of holo-ACE. After addition of zinc to the serum in vitro, the increase of ACE activity over the initial value was determined as that of apo-ACE. Finally, the ratio of apo/holo-ACE activities was calculated.

Results

There was a significant decrease of zinc concentration, but a slight decrease of the activity of ACE in the serum of mice by intake of zinc-deficient diet for 9 days. ACE ratio tended to increase on day 5 and was significantly increased on day 9 in mice deprived of dietary zinc.

Conclusion

These findings suggest that ACE ratio is as sensitive as serum zinc concentration for the evaluation of zinc deficiency and can be used for the biochemical diagnosis of zinc nutritional status in patients with zinc deficiency-induced taste impairment.     

Increase in synaptic hippocampal zinc concentration following chronic but not acute zinc treatment in rats

Electroconvulsive seizures (ECS), one of the most effective treatments of depression, induce mossy fiber sprouting (when assayed by means of synaptic zinc method), and this indicates an increase in the synaptic zinc level in the hippocampus following such therapy. The aim of the present study was to investigate the influence of acute and chronic zinc hydroaspartate administration on the synaptic and total zinc level in the rat hippocampus. We used two methods of zinc determination: (1) zinc-selenium method, which images the pool of synaptic zinc, and (2) flame atomic absorption spectrometry, which assays the total concentration of zinc. Our results indicate that chronic (14 x 65 mg/kg), but not acute, zinc hydroaspartate administration intraperitoneally (i.p.) increases the pool of synaptic zinc in the majority of rat hippocampal layers (by 72-190%), except for the stratum moleculare and stratum radiatum CA, and perforant path DG. On the other hand, no changes were found in total hippocampal zinc level, measured by flame atomic absorption spectrometry. These data suggest that chronic zinc treatment increases the pool of synaptic zinc in the hippocampus, and this effect is similar to that observed following chronic ECS treatment. The measurement of zinc concentration in the whole hippocampus by the flame atomic absorption spectrometry method is not sensitive enough to detect such subtle alteration.     

Localization of ZnT7 and zinc ions in mouse retina--immunohistochemistry and selenium autometallography

Zinc transporter 7 (ZnT7, Slc30a7), a member of the Slc30 family, is involved in mobilizing zinc ions from the cytoplasm into the Golgi apparatus. In the present study, we examined the distribution and localization of ZnT7 and the labile zinc ions in the mouse retina using immunohistochemistry and in vivo zinc-selenium autometallography (ZnSe(AMG)). Our results showed that ZnT7 is abundantly expressed in the ganglion cells and pigment epithelial cells of the mouse retina. ZnT7 is also expressed in the amacrine cells and the layer of optic fibers of the mouse retina, but to a lesser extent. Weak staining of ZnT7 was detected in the inner plexiform layer, outer plexiform layer, and outer segment of the photoreceptors. However, ZnT7 was not detected in the outer nuclear layer and inner segment of the photoreceptors. A high level of labile zinc pool was detected in the pigment epithelial cells, the inner segment of the photoreceptors, and the marginal region of the inner nuclear layer. Less amount of labile zinc ions were detected in the ganglion cells of the retina. These observations strongly suggest that ZnT7 may play critical roles in retinal zinc homeostasis and that chelatable zinc pools may have multiple functions in the retina.     

血清前白蛋白检测对儿童体内锌营养状况的评价

目的探讨血清前白蛋白(PA)检测对儿童锌缺乏监测的意义,以及PA水平与儿童体内锌含量的关系。方法对292例门诊体检无感染及肝炎儿童进行血清PA及锌检测。根据锌含量将儿童分为锌正常组(254例)、轻度缺锌组(26例)、中度缺锌组(7例)和重度缺锌组(5例),比较各组血清PA和锌检测的结果。结果以锌<11.6μmol/L、PA<125mg/L为阳性判定标准,锌正常组儿童血清PA阳性率为5.9%,锌缺乏组儿童PA阳性率为78.9%,两组PA阳性率间差异有统计学意义(P<0.01)。轻、中、重度缺锌组儿童血清PA水平比较,差异亦有统计学意义(P<0.05);轻度缺锌组儿童PA阳性数为19例,中度缺锌组儿童为6例,重度缺锌组儿童为5例,组间比较差异均有统计学意义(P<0.05)。结论血清PA是儿童锌缺乏的一个很有价值的监测指标,与儿童血清锌水平存在相关性。     

Expression, localisation and synthesis of versican by the enamel organ of developing mouse molar tooth germ: An in vivo and in vitro study

Objective

Versican is a large, aggregating chondroitin sulphate proteoglycan. In dental tissue, versican expression occurs primarily in mesenchymal tissue but rarely in epithelial tissue. We investigated the expression, localisation and synthesis of versican in the enamel organ of the developing tooth germ.

Design

To elucidate versican localisation in vivo, in situ hybridisation and immunohistochemistry were conducted in foetal ICR mice at E11.5–E18.5. Epithelium and mesenchyme from the lower first molars at E16.0 were enzymatically separated and versican mRNA expression was investigated by semi-quantitative RT-PCR. Organ culture of the separated samples combined with metabolic labelling with [³⁵S], followed by gel filtration, was performed to analyse secreted proteoglycans.

Results

Versican mRNA was first expressed in the thickened dental epithelium at E12.0 and continued to be expressed in the enamel organ until the bell stage. Versican immunostaining was detected in the stellate reticulum areas from the bud stage to the apposition stage. The enamel organ at E16.0 expressed versican mRNA at a level comparable to that in dental mesenchyme. Furthermore, when compared to dental mesenchyme, about 1/2–3/4 of the [³⁵S]-labelled versican-like large proteoglycan was synthesised and released into tissue explants by the enamel organ.

Conclusions

The dental epithelium of developing tooth germ is able to synthesise significant amounts of versican.     

Persistent zinc depletion in the mossy fiber terminals in the intrahippocampal kainate mouse model of mesial temporal lobe epilepsy

Purpose:   Zinc is released in synaptic vesicles with glutamate, and modulates glutamatergic neurotransmission. In brain, the highest amount of zinc, detected by Timm staining, is in the mossy fiber (MF) system in the hippocampus. In the intrahippocampal kainate (KA) mouse model of mesial temporal lobe epilepsy, which is elicited by intrahippocampal KA, prominent MF sprouting develops rapidly within 2 weeks post-KA. However, the intensity of Timm staining is reduced gradually thereafter. The present study is designed to determine the mechanisms underlying this reduction of Timm staining.
Methods:   The changes in Timm staining, and VGluT1, Synapsin-1, and zinc transporter 3 (ZnT3) immunoreactivity (IR) were examined from 4–56 days post-KA. An analysis of glutamate release in the KA-injected hippocampus was conducted by microdialysis before and during the continuous injection of midazolam (MDZ).
Results:   At 56 days post-KA, Timm staining disappeared completely, whereas VGluT-1-, Synapsin-1-, and ZnT3-IR were increased in the sprouted MF boutons. However, when the seizures were suppressed by a continuous perfusion of MDZ, the glutamate release in the hippocampus decreased and Timm staining was recovered.
Discussion:   This study showed that the reduction of Timm staining is the result of decreased zinc content but not the loss of MF itself. The reduction is the result of the enhanced release of zinc relative to storage, and it should facilitate the glutamate excitation that might be related to the epileptogenesis and rapid advancement of the morphologic changes in this model.     

反复呼吸道感染患儿锌治疗前后红细胞CD35分子表达和血清炎性因子的变化

目的检测反复呼吸道感染（recurrent respiratory tract infection,RRTI）患儿红细胞表面CD。分子的表达,研究循环免疫复合物（CIC）和血清炎性因子在感染反复发生中的机制,并观察锌治疗的临床效果。方法将116例RRTI患儿根据不同感染部位分为上呼吸道感染组和下呼吸道感染组,随机选择同期发病的急性呼吸道感染患儿40例和50名健康儿童作为对照,检测红细胞膜CD35分子表达、CIC阳性率,以及IL-6、IL-8和TNF-α的含量。从116例RRTI患儿中选取68例患儿,随机分成锌治疗组（38例）和对照组（30例）,治疗结束时和结束后12周再次检测上述指标。结果RRTI患儿（上呼吸道感染组和下呼吸道感染组）红细胞膜CD35分子表达明显低于健康对照组（t值分别为6．17和6．46,P值均〈0．01）,而CIC阳性率及其他炎性因子则较健康对照组明显升高,且这些指标在下呼吸道感染中变化更为明显。感染缓解期的RRTI患儿较急性呼吸道感染患儿红细胞CD35表达明显降低（t=20．307,P〈0．01）。经过锌治疗后,RRTI患儿的各项指标明显改善。结论红细胞膜CD,,分子表达低下和CIC等血清炎性因子的过量产生可能是RRTI患儿反复呼吸道感染的重要免疫病理机制之一。锌治疗对上述指标的改善有一定的作用。     

Thrombin regulates vascular smooth muscle cell proteoglycan synthesis via PAR-1 and multiple downstream signalling pathways

Introduction

Atherosclerosis is the underlying pathological process of most cardiovascular disease. Thrombin is a serine protease which can activate protease activated receptors (PAR) on vascular smooth muscle cells (VSMC) to elicit cellular responses that can contribute to the pathogenesis of atherosclerosis. Human atherosclerosis commences with the binding and retention of lipoproteins by the glycosaminoglycan (GAG) chains of chondroitin/dermatan sulfate proteoglycans. The potential effects of thrombin on the synthesis and structure of CS/DS proteoglycans produced by VSMCs was investigated.

Materials and methods

VSMCs were derived from human internal mammary arteries. Proteoglycan synthesis was assessed by [³⁵S]sulfate and [³H]glucosamine incorporation. Proteoglycan size was assessed by SDS-PAGE and size exclusion chromatography.

Results and conclusion

Thrombin caused a dose-dependent increase in [³⁵S]sulfate and [³H]glucosamine incorporation with maximum effects of approximately 150% at the highest doses tested. This increase was associated with increased size of biglycan and decorin assessed by SDS-PAGE. Chemically cleaved glycosaminoglycan (GAG) chains analyzed by SDS-PAGE and size exclusion chromatography were larger for proteoglycans from thrombin treated cells. VSMCs synthesize small GAGs when provided with exogenous xyloside and thrombin treatment also increased the size of the secreted xyloside GAGs. The effect of thrombin was not mimicked by the catalytically inactive FPRCK-HCT and was blocked in a concentration- dependent manner by the PAR-1 antagonist, JNJ5177049. Inhibition of PK C with GF 109203X resulted in concentration dependent but partial inhibition of [³⁵S]sulfate incorporation accompanied by a reduction in the size of biglycan and decorin. Epidermal growth factor (EGF) stimulated [³⁵S]sulfate incorporation and increased proteoglycan size and this was completely blocked by the EGF receptor tyrosine kinase inhibitor AG1478. AG1478 partially (32%, p < 0.01) blocked the effect of thrombin. Thrombin treatment of VSMCs increased the proportion of disaccharides sulfated at the 6 position of the GalNAc residues. Thus, thrombin has actions on VSMCs which increase the length and modify the sulfation pattern of GAG chains on proteoglycans in a manner that would enhance the binding of LDL. If manifest in vivo, this effect on proteoglycan synthesis and structure represents a new biochemical mechanism through which thrombin contributes to the development of atherosclerosis.