Ultrastructure of Salmonella typhimurium forms with unbalanced growth induced by lithium chloride

The action of 1.0 and 1.5 M LiCl on S. typhimurium induces the appearance of unbalanced growth forms capable of growing and multiplication, when subcultured in a medium with this preparation. In this culture the prevalence of cells differing in their structure from the initial Salmonella cells and from stable L-form cultures is observed. Cells characteristic of the initial culture and cells resembling the L-forms occur in lesser numbers. LiCl seems to affect peptidoglycan and the cytoplasmic membrane, which brings about disturbances in the permeability of the surface structures of the cell.     

Elevated yield of monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells treated by GSK-3 inhibitors

A study of the cartilage differentiation of mesenchymal stem cells (MSCs) would be of particular interest since one strategy for cell-based treatment of cartilage defects emphasizes the use of cells that are in a differentiated state. The present study has attempted to evaluate the effects of two well-known glycogen synthase kinase-3 inhibitors, including lithium chloride (LiCl) and SB216763 on a human marrow-derived MSC (hMSC) chondrogenic culture. Passaged-3 MSCs were condensed into small pellets and cultivated in the following groups based on the supplementation of chondrogenic medium: transforming growth factor (TGF)-β1, TGF-β1 + LiCl, TGF-β1 + SB216763, TGF-β3, TGF-β3 + LiCl, and TGF-β3 + SB216763. The cultures were maintained for 21 days and then analyzed for expression of Sox9, aggrecan, collagen II, β-catenin, and axin genes. Deposition of glycosaminoglycan (GAG) in the cartilage matrix was also measured for certain cultures. The presence of both LiCl and SB216763 along with TGF-β in the MSC chondrogenic culture led to the up-regulation of cartilage-specific genes. TGF-β3 appeared much better than TGF-β1. Based on our findings, SB216763 was more effective in up-regulation of cartilage-specific genes. These chondrogenic effects appeared to be mediated through the Wnt signaling pathway since β-catenin and axin tended to be up-regulated and down-regulated, respectively. In the culture with SB216763 + TGF-β3, significantly more GAG was deposited (P < 0.05). In conclusion, addition of either SB216763 or LiCl to hMSC chondrogenic culture up-regulates cartilage-specific gene expression and enhances GAG deposition in the culture.     

Growth effects of lithium chloride in BALB/c 3T3 fibroblasts and madin-darby canine kidney epithelial cells

The ability of LiCl to initiate DNA synthesis was studied in Madin-Darby canine kidney (MDCK) cells, and mouse BALB/c 3T3 fibroblasts. In a defined culture medium lacking serum, LiCl increased DNA synthesis in BALB/c 3T3 cells 100–200% over control values. Maximum DNA synthesis was observed with concentrations of LiCl between 10 and 25 mM and increases from 40–50% over control were observed with concentrations as low as 1 mM. Exposure of BALB/c 3T3 cultures to LiCl resulted in an increase in the percentage of cells initiating DNA synthesis, total DNA content and cell number. Lithium chloride, in combination with insulin or epidermal growth factor (EGF), had either an additive or synergistic effect upon the growth of BALB/c 3T3 fibroblasts. MDCK cells proved refractory to the growth actions of LiCl, although they responded to EGF and insulin with increased DNA synthesis. Lithium chloride appears to have a direct effect on cell proliferation in some but not all cell types.     

Evidence that lithium ions can modulate lectin stimulation of lymphoid cells by multiple mechanisms

Inhibition of PHA stimulation of hamster lymph node cells by theophylline, DBcAMP, or indomethacin or PHA stimulation of thymocytes by theophylline or DBcAMP was partially reversed by addition of 10 mM LiCl to the cultures. Addition of LiCl to Con A-stimulated lymphoid cells treated with the same reagents did not alter the inhibition. In contrast, addition of 10 mM LiCl to Con A-stimulated cultures enhanced the inhibition induced by the Na,K ATPase inhibitor, ouabain. Like LiCl, this latter inhibitor was found to be effective in modulating stimulation only if added early in the culture.These data support the hypothesis that LiCl can modulate lymphocyte responsiveness at the level of cyclic nucleotide metabolism, as exemplified by PHA stimulation, or at the level of the Na,K ATPase, exemplified by Con A stimulation. The site of involvement of Li⁺ ion would appear to be dependent on the biochemical nature of the stimulating signal.     

Combinatorial treatment with lithium chloride enhances recombinant antibody production in transiently transfected CHO and HEK293E cells

Lithium chloride (LiCl), which induces cell cycle arrest at G2/M phase, is known as a specific production rate (q _p)-enhancing additive in recombinant Chinese hamster ovary (CHO) cell culture. To determine the potential of LiCl as a chemical additive that enhances transient gene expression (TGE), LiCl was added to the CHO-NK and human embryonic kidney 293E (HEK293E) cell cultures before and/or after transfection with polyethylenimine as a transfection reagent. The effect of this addition on transfection efficiency (pre-treatment) and q _p enhancement during TGE (post-treatment) was examined. For the TGE of monoclonal antibody (mAb) in CHO-NK cells, pretreatment alone with 10 mM LiCl and post-treatment alone with 5 mM LiCl resulted in 1.2- and 3.4-fold increase of maximum mAb concentration (MMC), respectively, compared with the TGE without LiCl treatment. Furthermore, combinatorial treatment with LiCl (10 mM for pre-treatment and 5 mM for post-treatment) synergistically increased the TGE of mAb (5.3-fold increase in MMC). Likewise, combinatorial treatment with LiCl (10 mM for pre-treatment and 15 mM for post-treatment) in HEK293E cells synergistically increased the TGE of mAb (4.9-fold increase in MMC). Taken together, the data obtained here demonstrate that combinatorial treatment with LiCl is a useful means to improve TGE in CHO as well as HEK293 cells.     

Comparison of the instrumental reinforcer devaluation effect in two strains of rats (Wistar and Lister)

In this experiment, the effect of the reinforcer devaluation upon instrumental performance was analysed in two strains of rats (Wistar and Lister): Food deprived rats were trained to press a lever for sucrose pellets in a single session. Immediately after the fulfilment of this session, half of the Wistar and Lister rats received an injection of lithium chloride (LiCl), while the remaining animals were injected with a sodium chloride (NaCl) solution. A subsequent extinction test showed that the subjects who had received immediate LiCl did not press the lever as often as those injected with NaCl. No differences in response suppression were found between the two strains of rats. These results also show that a single devaluation experience is sufficient for an impact on instrumental performance.     

瑞拉菌素产生菌RL-2的诱变育种

本研究以委内瑞拉链霉菌秦岭变种RL-2(Streptomyces venezuelaevar. qinlingensis RL-2)为出发菌株, 分别采用紫外线、氯化锂及紫外线加氯化锂的复合诱变方式对其孢子悬液进行诱变处理。在复合诱变的紫外线照射时间为45 s和氯化锂浓度为0.4%的情况下, 获得一株瑞拉菌素的高产突变菌株UVL-108且连续传接6代其遗传特性较稳定。采用双向培养及生长速率法对其进行初筛和复筛, 结果表明: UVL-108的拮抗性能较出发菌株提高了77%, 其发酵液对稻瘟病菌的抑菌毒力是出     

Altered glycoprotein synthesis in mouse epidermal cells treated with retinyl acetate in vitro

Retinyl acetate alters glycoprotein synthesis in mouse epidermal cells in culture. Epidermal glycoproteins were enzymatically digested to glycopeptides and separated on DEAE Sephadex A50 columns using different concentrations of LiCl. There was a two-fold increase in incorporation of fucose and glucosamine in the 0.2 M LiCl fraction from cells treated for 3 weeks with 12.5 μg/ml retinyl acetate and 1.25% DMSO as compared with DMSO controls. No changes were noted in other fractions. The glycopeptide from A treated cells isolated on 0.2 M LiCl had a higher molecular weight than glycopeptides from that same fraction eluted by control cells. This isolated newly synthesized glycopeptide from vitamin A treated cells appears to be a single product by rechromatography on DEAE Sephadex A50 and Sephadex G100 columns.     

Induction of the Synthesis of Melanin and Pteridine in Cells Isolated from the Axolotl Embryo

It has previously been reported that when LiCl and tyrosine is added to ectodermal cells isolated from the blastula of Ambystoma mexicanum , then the synthesis of melanin is initiated in cells not normally engaged in this activity (mesenchyme cells, nerve cells and undifferentiated animal cells). In the present paper it has been shown that to obtain this effect tyrosine (0.02 mM) has to be present in the culture medium during at least one of the first seven days of culture, thus several days before melanin is produced. It is concluded that the added tyrosine is acting as an inductor of, and not as a substrate for the synthesis of melanin.
In the normal cultures it is possible to observe the spontaneous formation of yellow cells, indicating that they have produced pteridine. These cells are spherical, suggesting that they are undifferentiated embryonic cells. GTP is a precursor in the synthesis of pteridine, and in analogy with the observations made with tyrosine it was found that in the presence of LiCl a number of different cell types elaborate pteridine when GTP (0.1 mM) is added to the medium. Also in this case was it possible to show that GTP acts as an inductor, not as a substrate.     

Amiloride is an ineffective conditioned stimulus in taste aversion learning in C57BL/6J and DBA/2J mice

The epithelial sodium channel (ENaC) blocker amiloride has been shown to increase the behaviorally measured NaCl detection threshold in mice. In this study, a conditioned taste aversion (CTA) paradigm was used to examine whether 100 microM amiloride has a perceptible taste that could contribute to this observed decrease in behavioral responsiveness. Eighty-four C57BL/6J (B6) and 64 DBA/2J (D2) mice were divided into eight groups (n=8-12 per group), in which half received an injection of 0.15 M LiCl (2 mEq/kg) and the other half an equivalent saline injection, in three conditioning trials. The four conditioned stimuli were 100 microM amiloride hydrochloride, water, 0.1 and 0.3 M NaCl. Neither strain demonstrated acquisition of a CTA to amiloride in a brief-access (BA) taste test (5 s trials in the gustometer). Although 0.3 M NaCl is inherently aversive, its pairing with LiCl led to significantly further decreases in licking during the BA test on salt trials in both strains. The D2 strain clearly avoided 0.1 M NaCl, whereas avoidance of this stimulus was more equivocal in B6 mice. The inefficacy of amiloride to serve as a conditioned stimulus in taste aversion learning involving three LiCl pairings suggests that the effects of this ENaC blocker on taste-related behavioral responses to NaCl are likely due to its pharmacological interference with sodium taste transduction.     

Role of apoptotic signaling pathway in metabolic disturbances occurring in liver tissue after cryopreservation: Study on rat precision-cut liver slices

Precision-cut liver slices in culture (PCLS) appears as a useful and widely used model for metabolic studies; the interest to develop an adequate cryopreservation procedure, which would allow maintaining cell integrity upon incubation, is needed to extend its use for human tissues. We have previously shown that cryopreservation of rat PCLS leads to caspase-3 activation and early alterations of their K+ content upon incubation. In this study, we tested the hypothesis that counteracting intracellular K+ loss and/or interfering with cell death signaling pathways could improve the viability of cryopreserved PCLS. PCLS were prepared from male Wistar rat liver and cryopreserved by rapid freezing before incubation. The addition of a caspase inhibitor-Z-DEVD-FMK (2.5 microM)-in the culture medium did not improve viability of cryopreserved PCLS. Incubation of cryopreserved PCLS in a K+ rich medium (135 mM) increased K+ content and avoided caspase-3 activation, but did not improve cell viability. Caspase-3 inhibition, a decrease in cell lysis, and improvement of glycogen content were observed in cryopreserved PCLS after addition of LiCl (100 mM) in the incubation medium. These results indicate that, even if caspase-3 activation is linked to the K+ loss in cryopreserved PCLS, its inhibition does not allow restoring the metabolic capacities. LiCl, acting on a target upstream of caspase-3 inhibition, improves cell viability and allows glycogen accumulation when added in culture medium of cryopreserved PCLS; and could thus be considered as an interesting adjuvant in the culture of cryopreserved PCLS.     

Inhibition of dimethyl sulfoxide-induced Friend erythroleukemia cell differentiation in vitro by lithium chloride

This report describes the ability of ultra-pure lithium chloride (LiCl) to influence the growth kinetics and differentiation of Friend erythroleukemia cells in vitro. LiCl (0.2-50 mEq/l) was effective in reducing the ability of Friend cells to grow in liquid suspension culture (p less than or equal to 0.001). In addition, the capacity of these erythroleukemic cells to respond to the inducing agent dimethyl sulfoxide (DMSO) was also significantly reduced (p less than or equal to 0.001). These results demonstrate that LiCl can influence not only the proliferation of erythroleukemia cells but also their subsequent differentiation after exposure to such chemical inducers.     

锂和三尖杉酯碱对HL—60细胞增殖,分化和c—myc表达的影响

本研究利用细胞培养技术观察了氯化锂和三尖杉酯碱（ＨＴ）对ＨＬ－６０细胞增殖的影响,不同浓度的氯化锂对ＨＬ－６０细胞的集落形成和３Ｈ－ＴｄＲ参入均呈剂量依赖式抑制;三尖杉酯碱亦有类似的作用。在培养体系中加氯化锂和三尖杉酯碱时,对ＨＬ－６０细胞数及集落形成抑制作用与单用二者相比较有明显增加。用ＮＢＴ还原试验,氯化锂和三尖杉酯碱均促进ＨＬ－６０细胞的分化,小剂量氯化锂还能加强三尖杉酯碱对ＨＬ－６０细胞诱导分化作用。从氯化锂和三尖杉酯碱处理的ＨＬ－６０细胞中提取总ＲＮＡ,应用ＲＴ／ＰＣＲ检测ｃ－ｍｙｃ的表达,结果表明经氯化锂和三尖杉酯碱处理的ＨＬ－６０细胞ｃ－ｍｙｃ表达均降低,与未处理的ＨＬ－６０细胞ｃ－ｍｙｃ比较,说明氯化锂和三尖杉酯碱均能抑制ｃ－ｍｙｃ的表达,提示ｃ－ｍｙｃ很可能在白血病细胞增殖、分化中起调控作用。     

Lithium chloride restores host protein synthesis in herpes simplex virus-infected endothelial cells

In previous studies we have shown that herpes simplex virus type 1 (HSV-1) infection suppresses host-cell protein synthesis in human endothelial cells (EC). It has been demonstrated that lithium salts prevent viral replication in HSV-1 infected cells. In the present study, we have measured host-cell protein synthesis in HSV-1 infected EC in the presence or absence of 20 and 30 mM LiCl. Although LiCl restored synthesis of almost all host-cell proteins, [35S]methionine incorporation was most pronounced in thrombospondin and plasminogen activator inhibitor 1 and least in fibronectin and type IV collagen. LiCl was more effective at the higher concentration (30 mM) and when the compound was added to the EC culture at the time of infection rather than after adsorption of HSV-1. Synthesis of virus proteins continued in LiCl-treated EC but at a reduced rate. The data suggest that LiCl not only interferes with virus replication, but may also, to some extent, interfere with the virion-associated inhibition of host protein synthesis.     

Lithium chloride-induced primary cilia recovery enhances biosynthetic response of chondrocytes to mechanical stimulation

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0–50 mM LiCl for 24 h, with different pre-culture durations (0 to 4 days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50 mM LiCl compared to other concentrations (P?<?0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50 mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P?<?0.1) and proteoglycan (1.5-fold, P?<?0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.

     

Accumulation of β-catenin by lithium chloride in porcine myoblast cultures accelerates cell differentiation

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation to determine cell fate during embryogenesis. Lithium chloride (LiCl) is known to activate canonical Wnt signaling by inhibiting glycogen synthetase kinase-3β and consequently stabilizing free cytosolic β-catenin. To understand the role of the Wnt/β-catenin pathway in the regulation of porcine myoblast differentiation, we studied the effects of LiCl on cultured porcine myoblasts and β-catenin expression. A supplementation of 25 mM LiCl induced myoblast differentiation into myotubes over 3 days of culture. By semi-quantitative RT-PCR analyses, levels of mRNA encoding MyoD, Myogenin, Myf5 and several Wnt-responsive genes in the cultured myoblast cells were significantly increased after LiCl treatment. Using Western blotting and immunofluorescence analysis, we found that the protein levels of β-catenin were consistently increased by LiCl. Meanwhile, phosphorylated GSK-3β at Ser9 levels were also increased as an indicator of GSK-3β inactivation. Additionally, the nuclear staining of endogenous β-catenin was also significantly increased in porcine myoblasts 48 h after LiCl treatment. These results provided additional evidence that Wnt/β-catenin is a significant pathway that regulates myogenic differentiation. An enhanced level of β-catenin plays a positive role in porcine myoblast differentiation.     

Phospholipase C activation by prostacyclin receptor agonist in cerebral microvascular smooth muscle cells

The mechanism through which iloprost permits cerebral vasodilation induced by specific stimuli is incompletely understood. Previous study suggests there might be interplay between the adenylyl cyclase and phospholipase C (PLC) systems. Coupling of the prostacyclin receptor with the PLC pathway system was investigated. Iloprost, a stable prostacyclin analog, was used as a prostacyclin receptor agonist. We investigated the effects of iloprost (10-12-10-6 M) on inositol 1,4,5-trisphosphate (IP3) production by piglet cerebrovascular smooth muscle cells in primary culture. Iloprost caused concentration- and time-dependent increases in IP3 production in control cells and in cells pretreated with LiCl (to prevent further IP3 metabolism). Iloprost treatment (10-12 M) of cerebrovascular smooth muscle cells, in the absence and presence of 20 mM LiCl, resulted in 2-fold and 4-fold increases in the formation of IP3, respectively. In contrast, 10-10 M to 10-6 M iloprost, either in the presence or absence of LiCl, induced moderate or no increase in IP3 formation. Iloprost (10-10-10-12 M) strongly stimulated diacylglycerol (DAG) generation, whereas higher concentrations (10-8 M) did not induce an increase. In conclusion, the results suggest that prostacyclin receptors on cerebromicrovascular smooth muscle can couple to PLC, generating the second messengers, IP3 and DAG.