Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P. thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P. thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics.     

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.     

A review on disease transmission studies in relationship to production of embryos by in vitro fertilization and to related new reproductive technologies

This paper addresses the circumstances of germplasm contamination and updates on transmission of pathogenic agents by embryos produced in vitro and by associated techniques. It has been shown that some pathogenic agents might have been associated with the follicular oocytes and oviductal cells, collected for in vitro fertilization (IVF), resulting in infected embryos. Experimental introduction of pathogenic agent with oocytes or infected semen into the IVF system allows, in most cases, for the fertilization of eggs and for the production of some transferable quality embryos. Rendering of oocytes and embryos free of infectious pathogens, using the standard sequential washing or enzymatic treatment, is inconsistent and more difficult in the presently used in vitro fertilization system as compared to in vivo produced embryos.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.     

Specificity of the action of parathyroid hormone upon mitochondrial metabolism: Cyanogen bromide-derived parathyroid-hormone peptides

The mitochondrial response to cyanogen bromide-treated parathyroid hormone was studied as a means of testing further the relationship between the structure and the effects in vitro of this hormone. The treated hormone and appropriate control hormone were tested in a standard bioassay and in a mitochondrial assay system in vitro.

Reaction of more than 90 % of the methionine residues in the hormone resulted in total inactivation of the hormone both in vivo and in vitro. This result disagrees with previously published data.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

杂交瘤细胞体外大规模培养研究进展

单克隆抗体在生物学和医学研究领域中显示了极大的应用价值,是免疫检验中的新型试剂,是生物治疗的导向武器。作为医学检验试剂,单克隆抗体可以充分发挥其优势,如特异性好,灵敏度高,更便于质量控制,利于标准化和规范化。传统的方法是利用小鼠腹水制备单克隆抗体,但是近几十年杂交瘤细胞体外大规模培养制备单克隆抗体技术也在不断发展。特别是单克隆抗体在疾病诊断和治疗方面的需求,更进一步促进了杂交瘤细胞体外培养生产技术的发展,体外培养杂交瘤细胞生产的单克隆抗体已应用到许多方面。由于杂交瘤细胞的半贴壁性质,无论是悬浮培养还是贴壁培养,均可进行杂交瘤细胞的体外大规模培养。针对应用于体外诊断试剂的杂交瘤细胞体外培养制备单克隆抗体进行综述,主要包括中空纤维细胞培养和生物反应器细胞培养方法,以及不同培养方法优化的进展。     

A cell-free culture system for development of large numbers of Brugia pahangi larvae.

Techniques used in the in vitro culture of massive numbers of Brugia pahangi third-stage larvae (L3) are described. Procedures for larval preparation and four culture conditions, with or without animal cell co-cultures, were studied, resulting in the adoption of a relatively simple cell-free culture system for routine harvesting of larval moulting excretory/secretory product.     

Studies on the chromosomes of human lymphocytes treated with diazepam in vitro

Human lymphocytes were exposed in vitro to various concentrations of the psychotherapeutic agent diazepam, using two different techniques for culturing the lymphocytes, a whole-blood and a macro procedure. There was no evidence for a damaging effect of diazepam on the lymphoblast chromosomes at any concentration or exposure time studied with either technique of culture. The significance of results obtained in vitro on chromosome-breaking effects of chemical agents in commercial use is discussed, and the importance of some technical details in conducting such experiments is pointed out.     

Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor

CGS 20267 is a new non-steroidal compound which potently inhibits aromatase in vitro (IC₅₀ of 11.5 nM) and in vivo (ED₅₀ of 1–3 μg/kg p.o.). CGS 20267 maximally inhibits estradiol production in vitro in LH-stimulated hamster ovarian tissue at 0.1 μM with an IC₅₀ of 0.02 μM and does not significantly affect progesterone production up to 350 μM. In ACTH-stimulated rat adrenal tissue in vitro, aldosterone production was inhibited with an IC₅₀ of 210 μM (10,000 times higher than the IC₅₀ for estradiol production); no significant effect on corticosterone production was seen at 350 μM. In vivo, in ACTH-treated rats, CGS 20267 does not affect plasma levels of corticosterone or aldosterone at a dose of 4 mg/kg p.o. (1000 times higher than the ED₅₀ for aromatase inhibition in vivo). In adult female rats, a 14-day treatment with 1 mg/kg p.o. daily, completely interrupts ovarian cyclicity and suppresses uterine weight to that seen 14 days after ovariectomy. In adult female rats bearing estrogen-dependent DMBA-induced mammary tumors, 0.1 mg/kg p.o. given daily for 42 days caused almost complete regression of tumors present at the start of treatment. Thus compared to each other, CGS 16949A and CGS 20267 are both highly potent in inhibiting estrogen biosynthesis in vitro and in vivo. The striking difference between them is that unlike CGS 16949A, CGS 20267 does not affect adrenal steroidogenesis in vitro or in vivo, at concentrations and doses several orders of magnitude higher than those required to inhibit estrogen biosynthesis.     

Effects of Calpain on Antioxidant Enzyme Activities

The activities of superoxide dismutase, catalase and glutathione reductase were not affected by in vitro incubation with the intracellular proteinase calpain, suggesting that these enzymes are not in vivo substrates of calpain. In contrast, the activity of another important antioxidant enzyme, glutathione peroxidase, is stimulated in vitro by calpain. This may explain the correlation between elevations in glutathione peroxidase activity and calpain activity which occur in aging, exercised and dystrophic muscle. Calpain treatment in vitro caused a large decrease in the activity of carnosine synthetase which is involved in the synthesis of the putative antioxidant carnosine. This may be the reason for the in vivo correlation between elevated calpain and diminished carnosine levels in aging, hypertensive, denervated and dystrophic muscles.     

The chemiluminescence response of bivalve haemocytes: utility in screening for immunomodulators and as a biomarker

Resistance to infectious diseases in bivalves depends primarily on the vigour and efficacy of haemocyte-dependent antimicrobial defence mechanisms. Like other phagocytes, haemocytes seem to rely on oxygen-independent (lysosomal hydrolases, lysozyme) and oxygen-dependent (reactive oxygen species) mechanisms to destroy ingested microorganisms. The generation of cytotoxic oxyradicals by haemocytes can be precisely quantified by means of a simple chemiluminescence (CL) assay using luminol or other CL probes. Tributyltin (TBT), and other environmental contaminants, at sublethal levels, will produce dose-dependent suppression of CL activity in haemocytes exposed in short-term, in vitro assays. Presumably, this suppression would find expression as impaired host defence capability. In fact, TBT has been shown to exacerbate progression and lethality of Perkinsus marinus infections in the oyster, Crassostrea virginica. This suggests that CL assays on haemocytes exposed in vitro to single agents or complex mixtures might be useful in screening for aquatic immunomodulators. Statistically significant alterations in CL responses of haemocytes withdrawn from bivalves exposed to xenobiotics in the laboratory or field are more difficult to identify because of high inter-animal variation; however, the use of haemocyte CL as a biomarker of effect merits further investigation.     

Reconstituted viral envelopes — ‘Trojan Horses’ for drug delivery and gene therapy?

Reconstituted viral envelopes (RVEs) are formed by solubilizing intact virus in detergent and reassembling the envelope on removal of detergent. RVEs can be formed in the presence of agents that become encapsulated and can then be utilized in vitro and in vivo for drug delivery, cell destruction, transfer of membrane components, and as vectors for genetic engineering. The problems with biotechnological applications of RVEs and possible strategies for overcoming them are discussed in this article.     

Acyl-ACPs的规模化合成

脂酰-酰基载体蛋白(fatty acyl-acyl carrier protein, acyl-ACP)是多种生物合成途径中的酰基供体。因供给限制,体外研究常用类似物acyl-CoA替代,而CoA部分和ACP有较大差异,限制了相关酶对底物识别的认识。因此稳定获得大量acyl-ACP是体外研究相关酶的催化机制及其代谢途径的关键。研究以holo-ACP和C4~C18链长脂肪酸为底物,在哈氏弧菌acyl-ACP合成酶(Vibrio harveyi acyl-ACP synthetase, VhAasS)催化下合成不同碳链长度的acyl-ACP;通过高效液相色谱(HPLC)方法,确定不同碳链长度acyl-ACP的合成产率。结果表明:碳链为C4~C14的acyl-ACP产率均高于90.0%,16:0-ACP产率为85.9%,18:1-ACP产率仅为25.7%。通过加入Li ⁺优化反应体系,16:0-ACP、18:1-ACP的产率达90.0%。进一步优化扩大反应体系可稳定获得20mg以上acyl-ACP;最后,把合成的acyl-ACP应用到甘油-3-磷酸酰基转移酶催化的反应体系中。不同链长acyl-ACP的规模化合成研究,为体外研究相关酶的催化机制提供重要基础。     

In vitro simulation and quantification of wear within the patellofemoral joint replacement

Quantification of the wear rate in vitro is now considered an essential step in the development of a new joint replacement prior to clinical trials. However, little research exists around in vitro simulation of wear in the patellofemoral joint (PFJ) despite over 200,000 being implanted annually within the European Union. A method to simulate wear in the laboratory using four input degrees of freedom within the PFJ of total knee replacement (TKR) has been developed. Wear simulation was validated through comparison of functional kinematics and patellar surface damage modes produced in vitro to clinical outcomes. The technique has been shown to replicate the prescribed in vivo kinematics in a reproducible and repeatable manner. The wear scar areas were similar to those found in vivo. However, geometrical measurements of wear were not reliable due to creep and geometry changes. As has been found previously with tibial inserts, geometrical determination of wear volume was not found to be an effective method of comparing wear from simulators and retrievals. Change in volume calculated gravimetrically was seen to be the most repeatable measure of patellar wear in vitro.     

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for In vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in -glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the -glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.     

UK-73,093: A non-peptide neurotensin receptor antagonist

UK-73,093 was identified in a screening program as a compound able to displace [³H]-neurotensin from its bovine brain receptor. We describe the discovery of this compound, species differences in receptor affinity and its characterization as a functional neurotensin antogonist in vitro and in vivo.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.