Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea.     

Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage

Proliferating plant cells treated during the late S period with 5-aminouracil (AU), give the typical response that DNA-damaging agents induce, characterized by: an important mitotic delay, and a potentiation of the chromosome damage by caffeine post-treatment. The study of labelled prophases, after a tritiated thymidine pulse, allowed evaluation of the mitotic delay induced by AU as well as its reversion by caffeine, while chromosome damage was estimated by the percentage of anaphases and telophases showing chromosomal aberrations. Post-treatment with adenosine alone has shown no effect on mitotic delay or chromosomal damage. However, when cells after AU were incubated in caffeine plus adenosine, the chromosome damage potentiation was abolished without affecting the caffeine action on mitotic delay. As a consequence, we postulate that caffeine could have two effects on G2 cells with damaged DNA: the first, to cancel their mitotic delay and the second to inhibit some DNA-repair pathway(s). Only this last effect could be reversed by adenosine.     

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway

BackgroundWhen redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis.PurposeThe present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H₂O₂)-induced injury in SH-SY5Y cells in vitro.MethodsSH-SY5Y cells were simultaneously treated with AU and H₂O₂ for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry.ResultsThe results showed that AU can significantly increase the H₂O₂-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H₂O₂ stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H₂O₂-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways.ConclusionThese results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.     

Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Cândida albicans cells

Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)₂K and E(AU)₂, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)₂ has a “coiled coil” structure in water while (AU)₂K has an α-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of α-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus.     

Chromosomal aberrations induced by caffeine in somatic ganglia of Drosophila melanogaster

Nerve ganglia of third-instar larvae were treated with various doses of caffeine (5×10^?4, 10^?3, 5×10^?3, 10^?2 and 2×10^?2 M) for 2 h at 25±1°C. The ganglia were fixed at set time intervals after treatment so that the effect of caffeine in different stages of the cell cycle could be observed. Chromatid aberrations were induced only when the caffeine was administered in G₂ or approaching mitosis. No aberrations were observed after treatment in S or early G₂. In relation to the different doses administered, a threshold effect was evidenced, the number of aberrations increasing in a marked way at doses exceeding 5×10^?3 M. These data indicate, that the effect observed in Drosophila melanogaster is similar to that described by Kihlman in animals and plants treated with caffeine at temperatures below 30°C.Results obtained in non-cytological tests (non-disjunction, chromosome loss, lethal recessives, dominant lethals) have so far given incomplete indications as to the mutagenicity of caffeine in Drosophila. The results we have obtained with the cytological test seem to contribute to a better definition of the mutagenecity.     

Defective removal of O6-methylguanine (O6-MeG) from the DNA of lymphocytes treated with N-methyl-N-nitrosourea (MNU) in autoimmune diseases

Asynchronously growing Chinese hamster cells (M3-1) were UV-irradiated (λ = 254 nm) and then incubated with/without caffeine (2 mM) for 20 h. Microscopic evaluation of metaphase spreads revealed that after UV-irradiation alone (5.0 J/m²) and caffeine treatment alone, the percentage of cells with condensed chromatin appearing fragmented and/or pulverized (‘GCS-like’ cells; GCS, Generalized Chromosome Shattering) was very low while it was high following the combined treatment. Cytogenetic and flow cytometric analysis of cells obtained by mechanical shaking cultures treated with UV and caffeine indicated that ‘GCS-like’ cells have the same DNA content as untreated cells in G2 phase and mitosis.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea. Received: 16 June 1998 / Accepted: 13 July 1998     

A genome scan for quantitative trait loci affecting the Salmonella carrier-state in the chicken

Selection for increased resistance to Salmonella colonisation and excretion could reduce the risk of foodborne Salmonella infection. In order to identify potential loci affecting resistance, differences in resistance were identified between the N and 6₁ inbred lines and two QTL research performed. In an F2 cross, the animals were inoculated at one week of age with Salmonella enteritidis and cloacal swabs were carried out 4 and 5 wk post inoculation (thereafter called CSW4F2 and CSW4F2) and caecal contamination (CAECF2) was assessed 1 week later. The animals from the (N × 6₁) × N backcross were inoculated at six weeks of age with Salmonella typhimurium and cloacal swabs were studied from wk 1 to 4 (thereafter called CSW1BC to CSW4BC). A total of 33 F₂ and 46 backcross progeny were selectively genotyped for 103 and 135 microsatellite markers respectively. The analysis used least-squares-based and non-parametric interval mapping. Two genome-wise significant QTL were observed on Chromosome 1 for CSW2BC and on Chromosome 2 for CSW4F2, and four suggestive QTL for CSW5F2 on Chromosome 2, for CSW5F2 and CSW2BC on chromosome 5 and for CAECF2 on chromosome 16. These results suggest new regions of interest and the putative role of SAL1.     

Caffeine inhibition of postreplication repair of UV-damaged DNA in mouse epidermal cells

The effect of caffeine (0.25–1.5 mM) on UV-irradiated (5 and 10 J/m²) primary cultures of mouse epidermal cells (EPD) and an in vitro transformed cell line (PDV) was studied at the cellular and molecular levels. A synergistic reduction in cell survival induced by caffeine with UV-irradiation was found in the PDV cells at 10 J/m² but not at 5 J/m². When conversion of low molecular weight newly-synthesized DNA to high molecular weight DNA was studied in both cell types, caffeine at 1.5 mM had no effect on this conversion in unirradiated cultures. At 5 J/m², caffeine had a transitory inhibitory effect on this conversion. However, at 10 J/m² caffeine had a strong permanent inhibitory effect on this conversion at doses higher than 0.5 mM in PDV cells and higher than 0.25 mM in EPD cells. This apparent inhibition of elongation by caffeine in irradiated cells could not be accounted for by an effect on the rate of DNA synthesis. In PDV cells there was a direct correlation in terms of effective caffeine dose level between synergistic reduction in cell survival after UV and the effect on DNA elongation. Irradiated EPD cells were more sensitive to the inhibitory effect of caffeine on DNA elongation.     

Somatic crossing over in glycine max (L). Merrill: Possible potentiation of the recombinogenic effect of caffeine by 5-fluorodeoxyuridine and cytosine-β-D-arabinofuranoside

The leaves of variety T219 of Glycine max (soybean) exhibit yellow, dark green and twin or double spots on the two simple leaves and the first compound leaf of heterozygous Y₁₁y₁₁ plants. These spots mimic the phenotypes controlled by Y₁₁y₁₁, Y₁₁Y₁₁ and Y₁₁Y₁₁−y₁₁y₁₁ genotypes. It has been argued that p recombination is responsible for the origin of twin spots. At least some of the single spots appear due to failure of one of the components of the double spots.Treatment of seeds with caffeine solutions increases the frequency of all types of spots, particularly those of doubles. 5-Fluorodeoxyuridine (FUdR) and cytosine-β-D-aranibofuranoside (CA), both inhibitors of DNA synthesis, cause potentiation of the effects of caffeine by increasing all three types of spots. Since the relative frequencies of the different types of spots do not differ much in caffeine plus FUdR (or CA) treated materials from those observed in case of caffeine treatment alone, it is suggested that (1) the increase in the frequency of spots in caffeine plus FUdR (or CA) treated material is not due simply to additive effect of the two chemical, and (2) there is true synergism between caffeine and FUdR (or CA) which somehow leads to enhancement of the phenomenon of compementary reunions.     

Radiomimetic property of furazolidone and the caffeine enhancement of its lethal action on the vibrios

Sensitivities of the strains belonging to four vibrio biotypes to the action of furazolidone were investigated. Vibrio cholerae (classical) was most and Vibrio parahaemolyticus least sensitive to this drug. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect of their sensitivity to furazolidone. The drug was radiomimetic in action, the doses of UV light (D_UV) and furazolidone (D_f) required for 10% survival of the vibrios being correlated by the equation, D_f = 0.28 exp. (0.008 D_UV). Caffeine exhibited lethal synergism with furazolidone and the synergistic effect depended on the mode of caffeine treatment, the effect being maximum when caffeine was present along with and also after furazolidone treatment. UV spectrophotometric study revealed that caffeine did not bind with native DNA but did so with denatured DNA resulting in a bathochromic shift and a quenching of the caffeine absorption maximum at 209.4 nm. The binding isotherm (Scatchard plot) indicated the presence of a heterogeneity in the binding sites and that the parameters for the strongest mode of binding were n = 0.254 and k = 7.5 × 10⁵ M^?1.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

The influence of selenium and caffeine on chemical carcinogenesis in rats,mutagenesis in bacteria,and unscheduled DNA synthesis in human lymphocytes

The influence of sodium selenite (Na₂SeO₃) and caffeine on chemical carcinogenesis induced in rats by diethylnitrosamine (DEN), N-nitrosomorpholine (NM), andN-methyl-N-nitro-N-nitrosoguanidine (MNNG) was investigated. A dose-dependent inhibitory effect of Na₂SeO₃ (l–10 ppm) on hepatocarcinogenesis induced by DEN was demonstrated. Na₂SeO₃ also increased the latency period for stomach tumor formation in rats treated with MNNG. Combined treatment of rats with Na₂SeO₃ plus vitamin C added to the diet resulted in a slight inhibition of NM-induced liver carcinogenesis. Supplementation of diet with Na₂SeO₃ plus butylated hydroxytoluene, vitamin C, and vitamin E did not reveal any additive inhibitory effect compared to the inhibitory effect of Na₂SeO₃ given alone. Caffeine (600 rag/L) reduced the number of liver tumors induced in rats by DEN. Preliminary experiments have indicated that combined treatment of rats with selenium and caffeine could result in more effective inhibition of DEN-induced liver carcinogenesis. Further experiments are being conducted to study the influence of selenium and caffeine on mutagenic activity of 1-methyl-l-nitrosourea (MNU) inSalmonella typhimurium TA 1535. The pretreatment of bacteria cells with Na₂SeO₃ (3–10 p.g/mL) increased the mutagenic response of bacteria to MNU. A synergistic stimulation of mutagenic activity of MNU was observed in bacteria pretreated simultaneously with Na₂SeO₃ and caffeine. In addition the influence of Na₂SeO₃ on UDS induced by DEN in human lymphocytes was investigated. The trace element inhibited the UDS up to 82%. The possible role of potentiation by NazSeO3 of the cell killing effect of DEN in inhibition of liver carcinogenesis was discussed.     

Antioxidant activity elicited by low dose of caffeine attenuates pentylenetetrazol-induced seizures and oxidative damage in rats

Although caffeine supplementation has a beneficial effect on people with neurological disorders, its implications for oxidative damage related to seizures are not well documented. Thus the aim of this study was to investigate the effects of two weeks caffeine supplementation (6 mg/kg; p.o.) on seizures and neurochemical alterations induced by pentylenetetrazol (PTZ 60 mg/kg i.p.). Statistical analyses showed that long-term rather than single dose caffeine administration decreased the duration of PTZ-induced seizures in adult male Wistar rats as recorded by cortical electroencephalographic (EEG) and behavioral analysis. The quantification of EEG recordings also revealed that caffeine supplementation protected against a wave increase induced by PTZ. Neurochemical analyses revealed that caffeine supplementation increased glutathione (GSH) content per se and protected against the increase in the levels of thiobarbituric acid reactive substances (TBARS) and oxidized diclorofluoresceine diacetate (DCFH-DA). Also, caffeine prevent the decrease in GSH content and Na⁺, K⁺-ATPase activity induced by PTZ. Our data also showed that the infusion of L-buthionine sulfoximine (BSO; 3.2 μmol/site i.c.v), an inhibitor of GSH synthesis, two days before injecting PTZ reversed the anticonvulsant effect caused by caffeine. BSO infusion also decreased GSH content and Na⁺, K⁺-ATPase activity. However, it increased DCFH-DA oxidation and TBARS per se and reversed the protective effect of caffeine. Results presented in this paper support the neuroprotective effects of low long-term caffeine exposure to epileptic damage and suggest that the increase in the cerebral GSH content caused by caffeine supplementation may provide a new therapeutic approach to the control of seizure.     

Properties of intratetanic individual contractile responses in rat slow skeletal muscles during modulation of sarcoplasmic reticulum Ca<Superscript>2+</Superscript> release

In control experiments (n = 16), during direct stimulation of m. Soleus by trains of 5, 10 and 50 stimuli at a rate of 20 Hz a biphasic change was detected in the amplitude of the last contractile responses (LCR_N) depending on N, where N is the number of individual contractile responses in the tetanus. Thus, an initial decrease in LCR_N amplitudes (down to 54 ± 8% for LCR₅) was followed by a subsequent increase (up to 218 ± 14% for LCR₅₀) and significant shortening of their half-relaxation time compared to the initial response (down to 44 ± 8% for LCR₅₀). Caffeine at concentrations of 5 (n = 6) and 10 (n = 4) mM exacerbated LCR₅ depression (down to 31 ± 8% and 15 ± 4%, respectively) against the background of arising characteristic stationary contracture responses. The subsequent increase in the LCR_N amplitude was substantially lower than in control experiments (114 ± 18% and 46 ± 9% for LCR₅₀ compared to the initial response at 5 and 10 mM of caffeine, respectively). The LCR₅₀ half-relaxation time during the effect of caffeine at both concentrations also remained considerably shorter than that of individual responses recorded both in the presence of caffeine and in control experiments. In contrast to the control and caffeine effects, LCR₅ and LCR₁₀ amplitudes during the effect of 10 μM of dantrolene (n = 5) remained at the level close to that of the first response (102 ± 7% and 106 ± 8%, respectively), while the LCR₅₀ amplitude displayed a considerably smaller increase (to 143 ± 14%) than observed in control muscles. Besides, dantrolene further enhanced muscle relaxation at rest. Caffeine (10 mM), as applied in the presence of dantrolene, restored the dynamics of changes in amplitude–temporal characteristics of last contractile responses to values approximating those in control. The amplitude–temporal characteristics of action potentials recorded extracellularly in individual m. Soleus muscle fibers did not change significantly during the transition from single to train stimulation under the same protocol as in mechanographic experiments. These data may be interpreted in support of the previously advanced hypothesis on the implication of Ca²⁺-induced Ca²⁺ release in skeletal muscles under their tetanic stimulation as an additional mechanism of excitation–contraction coupling [1, 2].     

Additive coclastogenicity of sodium selenite and caffeine in CHO cells treated withN-methyl-N′-Nitro-N-Nitrosoguanidine

The clastogenic effect ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster ovary (CHO) cells and its modulation by Na₂SeO₃ and caffeine were studied by metaphase analysis of chromosome aberrations (CA) as well as by measuring the formation and repair of single-strand (ss) DNA breaks employing hydroxylapatite chromatography. Treatment of CHO cells with MNNG (1.25 or 2.5 × 10-5M) for 3 h caused CA in 11 and 19% of metaphases scored, respectively. Pretreatment of cells with Na₂SeO₃ (1–5 μg/mL) or caffeine (0.2–2.0 mg/mL) for 2 h resulted in a 2–3.5-fold increase of CA frequency. Addition of both modulators during the mutagen exposure tended to cause a slight inhibition of clastogenic activity of MNNG (1.25 × 10⁻⁵ M) or had no effect on CA number when MNNG was used at a concentration of 2.5 × 10−5M. Posttreatment of CHO cells with Na₂SeO₃ for 20 h after MNNG was ineffective in influencing the number of metaphases with CA, whereas, at these conditions, caffeine enhanced up to 6-7-fold the clastogenic activity of MNNG. Addition of both modulators during the whole experiment, 2 h pretreatment included, resulted in a further significant increase of CA frequency up to the total pulverization of chromosomes in all metaphases scored. The coclastogenic effect of caffeine was greater in this case. The enhancement of chromosome-damaging activity of MNNG by selenite and caffeine was better expressed when this carcinogen was applied at the higher concentration used. An additive coclastogenic effect was observed in CHO cells treated simultaneously with Na₂SeO₃ and caffeine plus MNNG. In addition, the treatment of CHO cells with MNNG (5 × 10⁻⁶ M) caused a rapid increase of ssDNA breaks number reaching maximal values after 30–45 min. However, up to 50–60% of MNNG-induced ssDNA breaks were repaired during the first 60–150 min after the mutagen exposure. The 2 h pretreatment of CHO cells with Na₂SeO₃ (2 μg/mL) or the addition of this trace element after MNNG had no effect on formation and repair of MNNG-induced ssDNA breaks. The coclastogenic effect of Na₂SeO₃ in CHO cells treated with MNNG was not directly linked to the induction and disappearance of ssDNA breaks measured by hydroxylapatite chromatography.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

Caffeine has a dual influence on NMDA receptor–mediated glutamatergic transmission at the hippocampus

Caffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A₁R and A₂R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca²⁺ homeostasis. We found that caffeine (30–200 μM) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A₁Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A_2ARs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A_2AR antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca²⁺ transients in neuronal cell culture, an action mimicked by the A₁R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 μM). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca²⁺ homeostasis.

     

Caffeine and heat have additive but not interactive effects on physiologic strain: A factorial experiment

This study tested the interactive effects of heat and caffeine on exercise-induced physiological strain by using a 2x2 within-subjects factorial design. Thirty-five physically fit Caucasians underwent a bout of exercise under four conditions wherein ambient conditions (heat vs no heat) and caffeine (placebo vs caffeine; double-blinded) were manipulated. Exercise consisted of a 60-min walk and 5-min step/squat test while wearing weighted backpack. Primary outcomes include measures of physiologic strain (Core temperature [T_r] and heart rate [HR]). Secondary measures included blood pressure, markers of sweat loss, and creatine kinase (CK). Repeated measures models were created to evaluate the individual and combined effects of heat and caffeine. Key results indicated that heat and caffeine significantly increased T_r and HR after walking and stair-stepping. No significant heat by caffeine interactions were detected, and caffeine's main effects were relatively low (≤0.17 °C for T_r and ≤6.6 bpm for HR). Of note, heat and caffeine exhibited opposite effects on blood pressure: caffeine increased both systolic and diastolic blood pressure (by 6–7 mmHg) and heat decreased them (by 4–6 mm Hg; ps < 0.05). In summary, heat and caffeine affected physiologic strain during exercise but exhibited no synergistic effects. In contrast, neither factor affected muscle damage. Clinical implications for heat illness risk in the military are discussed.