Absence Of Cysteamine Research Articles

Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

BackgroundCystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H+ symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis.MethodsMorphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs).ResultsDespite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs.ConclusionsOur results indicate that, as compared to HD-MSCs, Cys-MSCs show reduced ability to differentiate into osteoblasts, which can be reverted after cysteamine treatment.

Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics

Optimization of ovum pick up (OPU) followed by in vitro embryo production (IVP) is strongly driven by the needs of both beef and dairy cattle breeders to enhance genetic improvement. The rapidly growing use of genomic selection in cattle has increased the interest in using OPU-IVP technology to increase the number of embryos and offspring per donor, thus allowing enhanced selection intensity for the next generation. The aim of this study was to optimize embryo production through supplementation of cysteamine during in vitro maturation (IVM) and in vitro culture (IVC) of both slaughterhouse- and OPU-derived oocytes. The effects on embryo production and on embryo cryotolerance, post-transfer embryo survival, and calf characteristics, including gestation length, birth weight, perinatal mortality, and sex ratio were studied. In study 1, immature slaughterhouse-derived cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with or without 0.1 mM cysteamine, fertilized and cultured for 7 days in 0.5 ml SOFaaBSA. In study 2, cysteamine was present during both IVM (0.1 mM) and IVC (0.01, 0.05, 0.1 mM) from Days 1 to 4. In study 3, OPU-derived COCs were matured in medium supplemented with or without 0.1 mM cysteamine in a 2 × 2 factorial design (OPU week and cysteamine treatment). Embryos were evaluated for stage and grade on Day 7 and, depending on the number of transferable embryos and recipients available, the embryos were transferred either fresh or frozen-thawed at a later date. The presence of cysteamine during IVM significantly increased the embryo production rate with slaughterhouse-derived COCs (24.0% vs. 19.4%). The higher number of embryos at Day 7 was due to an increased number of blastocysts, whereas the distribution of embryos among different quality grades and cryotolerance was not affected. Embryo production rate was negatively affected when cysteamine was present during both the processes of IVM and IVC during Days 1 to 4 of culture (13.2%–19.3% vs. 26.4%). The presence of cysteamine during IVM of OPU-derived COCs also significantly increased the embryo production rate (34.4% vs. 23.4%). The higher number of embryos was again totally due to an increased number of blastocysts, whereas cryotolerance was not affected. The relative increase in embryo production rate was higher with OPU-derived oocytes compared with slaughterhouse-derived COCs (47% vs. 24%). This improvement resulted in a mean of 1.73 transferable embryos per OPU session compared with 1.06 in the absence of cysteamine. The presence of cysteamine did not affect pregnancy rate, gestation length, birth weight, perinatal mortality, and sex of calves born from either fresh or frozen-thawed embryos. This study reported that cysteamine supplementation during IVM greatly improved the efficiency and affectivity of an OPU-IVP program.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário

Complexos cumulus-oócito (COC), oócitos desnudos (DO) e DO cocultivados com células do cumulus em suspensão (DO+CC) foram maturados in vitro (MIV) na presença ou ausência de cisteamina (50mM). Observou-se efeito benéfico da cisteamina durante o cultivo de MIV, pois a maturação nuclear no grupo COC cisteamina foi maior do que a do COC controle (P<0,05). No grupo sem a adição de cisteamina, foi observado que a ausência de CC durante o cultivo de MIV prejudicou a maturação nuclear em DO, em relação ao COC (P<0,05), todavia a cisteamina restaurou a capacidade de progressão da meiose em DO, tornando-os semelhantes aos COC (P>0,05). O acoplamento entre oócitos e CC durante MIV demonstrou ser essencial para aquisição da competência do oócito para suportar o desenvolvimento embrionário inicial, pois COC apresentaram maior porcentagem de blastocistos e eclosão quando comparados a DO e DO+CC (P<0,05). A inclusão de cisteamina no cultivo de MIV não restaurou a aquisição da competência em DO e DO+CC, que permaneceram semelhantes aos do grupo-controle (P>0,05). Conclui-se que a cisteamina no meio de MIV melhora as taxas de maturação nuclear em COC e restaura a capacidade de progressão da meiose em DO. Todavia, na concentração utilizada neste estudo, não promove efeito benéfico no desenvolvimento embrionário.

The Anti‐oxidant Cysteamine Confers a Protective Effect Against Oxidative Damage in Saccharomyces cerevisiae in the Absence of Endogenous Repair Machinery

In vitro chemical studies have shown that natural anti‐oxidants are beneficial compounds that prevent and may help repair DNA damage caused by intracellular reactive oxygen species (ROS), such as hydrogen peroxide. Thiols (RSH), naturally‐occurring, electron‐rich, biological compounds, donate electrons to neutralize free radicals, removing them from the body. Here we seek to elucidate the biochemical pathway by which the thiol cysteamine protects against oxidation by hydrogen peroxide in the absence of endogenous oxidative stress or DNA repair machinery. Previous investigations have shown that Saccharomyces cerevisiae gene deletion strains lacking the ctt1 gene exhibit increased growth in the presence of cysteamine as compared to their untreated counterparts when assaulted with hydrogen peroxide. The current study investigates the potential protective effects of cysteamine in other S. cerevisiae knock‐out strains. Western Blot analysis is used to test protein levels in the oxidative response and DNA repair pathways in the presence and absence of cysteamine.

Contributions of mechanical and sonochemical effects to cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles

The objective was to investigate the contributions of mechanical effects due to kinetic force induced by the dynamic behavior of microbubbles and sonochemical effects due to free radicals produced by inertial cavitation to cell membrane damage under sonoporation conditions in which cells with adjacent microbubbles were irradiated with single-shot pulsed ultrasound. The free radical scavenger cysteamine was used to control the occurrence of sonochemical effects, and the ratios of cells with membrane damage to intact cells were compared in the presence and absence of cysteamine. To determine the optimal dose of cysteamine, free radical production on exposure to burst pulse ultrasound was investigated using KI-starch solutions with different concentrations (0-5 mM) of cysteamine. High-speed observation of the dynamic behavior of Levovist microbubbles during ultrasound exposure was also carried out in the presence and absence of cysteamine, and the difference in the ratios of the maximum bubble diameter to the initial diameter was evaluated. Next, human prostate cancer cells with adjacent Levovist microbubbles were exposed to single-shot pulsed ultrasound with a center frequency of 1 MHz, a peak negative pressure of 1.1 MPa, and a pulse width of 3 μs, and the percentages of cells with membrane damage were evaluated by fluorescent microscopy using propidium iodide in the presence and absence of cysteamine. It was confirmed that cysteamine at a concentration of 5 mM completely suppressed sonochemical effects without causing a change in the dynamic response of microbubbles to pulsed ultrasound. The percentages of cells with membrane damage in the presence and absence of cysteamine (5 mM) were 10.3% ± 4.1% (n = 13) and 8.7% ± 3.9% (n = 9), respectively. No significant difference was found (P = 0.36). The results indicate that cell membrane damage induced by single-shot pulsed ultrasound with adjacent microbubbles was due mainly to mechanical effects, not to sonochemical effects.

Effect of Thiol‐Containing Molecule Cysteamine on the Induction of Inducible Nitric Oxide Synthase in Hepatocytes

Cysteamine, which is a known antioxidant and anti-inflammatory agent, is believed to be a key regulator of essential metabolic pathways in organisms. Cysteamine has beneficial effects in liver damaged by a variety of insults. During liver injury, inducible nitric oxide synthase (iNOS) is induced by lipopolysaccharide or proinflammatory cytokines, leading to excessive nitric oxide (NO) production. Accumulated evidence indicates that NO is an important factor associated with hepatic dysfunction. We examined whether cysteamine influences the induction of iNOS in hepatocytes. Primary cultured rat hepatocytes were treated with interleukin (IL)-1beta in the presence and absence of cysteamine. NO production, iNOS induction, and iNOS signal were analyzed. IL-1beta stimulated the inhibitory protein kappaB (IkappaB)/nuclear factor kappaB (NFkappaB) pathway, resulting in the activation of NFkappaB (nuclear translocation and DNA binding), which was followed by the induction of iNOS and NO production. The addition of IL-1beta and cysteamine (1-4 mmol/L) markedly inhibited NO production, with a maximal effect at 4 mmol/L (80%-90% inhibition). Cysteamine also decreased the levels of iNOS protein and mRNA. Transfection experiments revealed that cysteamine decreased the transactivation activity of the iNOS promoter. An electrophoretic mobility shift assay demonstrated that cysteamine inhibited the activation of NFkappaB. Furthermore, cysteamine decreased the mRNA levels of the NFkappaB subunit p65 but increased those of the inhibitory protein IkappaB. These findings suggest that cysteamine inhibits iNOS induction at the step of NFkappaB activation. Further study is necessary to define the molecular basis of this effect of cysteamine on the regulation of NFkappaB and its pharmacologic implications.

Enrichment of in vitro maturation medium for buffalo ( Bubalus bubalis) oocytes with thiol compounds: Effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus–oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 μg/ml FSH, 5 μg/ml LH and 1 μg/ml 17β-estradiol in the absence or presence of cysteamine (50 μM), with or without 0.3 mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine + cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.

Glutathione synthesis during in vitro maturation of buffalo ( Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis. Cumulus–oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 μg/ml FSH, 5 μg/ml LH and 1 μg/ml 17-β-estradiol in the absence or presence of cysteamine (50 μM), with or without 5 mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions. In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.

In vitro maturation and glutathione synthesis of porcine oocytes in the presence or absence of cysteamine under different oxygen tensions: role of cumulus cells.

The present study was conducted to evaluate the effect of cumulus cells on the in vitro maturation (IVM) and glutathione (GSH) synthesis of porcine oocytes cultured in the presence or absence of cysteamine under different oxygen tensions, and on their subsequent male pronucleus formation after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 45 h in modified TCM-199 supplemented with or without 150 microM cysteamine under a humidified atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2. When cultured in medium supplemented with cysteamine under 20% O2 tension, the rates of COC maturation to the metaphase II (MII) stage were significantly higher than those of DOs (P<0.05). Regardless of the addition of cysteamine and oxygen tension, the rates of male pronucleus formation in COCs after IVM and IVF were significantly higher than in DOs (P<0.05). The GSH content of oocytes was significantly increased by the addition of cysteamine to the maturation medium (P<0.05), with significantly higher GSH content in COCs than in DOs (P<0.05). However, the GSH content of COCs and DOs was not significantly different when cultured in medium without cysteamine. These results indicate that cumulus cells play an important role in nuclear maturation to MII, GSH synthesis in porcine oocytes cultured in the presence of cysteamine, and subsequent male pronucleus formation after IVF.

Effect of the sulfhydryl compound cysteamine on gamma-radiation-induced mutations in double-stranded M13 DNA

Sulfhydryl compounds can protect DNA against free-radical-induced DNA damages not only by scavenging of radicals, but also by chemical non-enzymatic repair or modification of such damages by hydrogen-donation. To investigate the influence of chemical repair and modification on mutations, induced by gamma-radiation-generated free radicals (·OH, ·H), phosphate-buffered aqueous solutions of double-stranded (ds) M13 DNA were exposed to gamma-rays under N2 in the presence of 5 mM cysteamine. The exposed DNA was subsequently transfected to wild-type E. coli and mutations in the mutational target were characterized. This target in fact contains three different target sequences, i.e., the lac promoter/operator, the lacZα gene and a 144 bp inframe insert. The mutation spectrum obtained was compared with those in the absence of cysteamine under N2 and N2O. In the latter case, the ratio of ·OH and ·H available for reacting with DNA is about the same as under N2 + cysteamine. The results show that chemical repair and/or modification by cysteamine of potentially lethal lesions takes place, leading to a much higher survival of ds M13 DNA in the presence of cysteamine than could be expected on basis of scavenging of ·OH and ·H alone. This higher survival appeared to be accompanied with a higher mutation induction. However, the N2 + cysteamine mutation spectrum shows a remarkable resemblance with the N2O-spectrum. This holds for the total mutation target, as well as each of the three targets, although the mutations obtained in each of the three targets under the same irradiation conditions are quite different. Thus, it can be concluded that cysteamine is mainly effective on radiation-induced potentially lethal DNA lesions, and not so much on (pre)mutagenic damages. Moreover, the type of mutation appeared to be strongly dependent on the mutational target sequence.

Confirmation of the protective effect of cysteamine in in vitro ultrasound exposures

Armour and Corry (Radiat. Res. (1982) 89 369–380) reported that ultrasound-induced damage to in vitro Chinese hamster ovary cells was significantly reduced in the presence of cysteamine. The objective of this study was to attempt verification of this result. Four series of experiments were undertaken using in vitro cell suspensions, namely: (1) determination of the effect of cysteamine concentration on cell growth; (2) determination of the temperature dependence of ultrasonically induced cell damage; (3) determination of a dose-response relationship for the cytotoxicity of cysteamine; and (4) assessment of cell integrity and reproductive capacity in the presence or absence of cysteamine during ultrasonic exposure. Ultrasound parameters included a resonance frequency of 1 MHz, a continuous wave exposure duration of 5 min, and intensities from 0 to 21.6 W cm −2. The results indicated a dependence of ultrasound's efficacy on the medium's temperature during insonation and a significant reduction of ultrasound efficacy in compromising cellular integrity in the presence of cysteamine.

Radiation protection of E. coli strains by cysteamine in the presence of oxygen.

The survival of various E. coli K12 strains with defects in the rec system have been measured after gamma-irradiation in air in the presence (0.1 mol dm-3) or in the absence of cysteamine. The results confirm those of Bresler et al. (1978) indicating that the protection by cysteamine in the presence of oxygen is due to an influence on enzymatic repair. The low protection by cysteamine of wild-type cells pretreated with chloramphenicol which prevents protein synthesis, supports the above conclusion. The reason for the absence of a protective effect by OH radical scavenging and H-atom donation is discussed. It is proposed that DNA peroxyl radicals are formed during irradiation in the presence of oxygen and that they are transformed into hydroperoxides by H-atom donation from the intracellular glutathione and the added cysteamine. These hydroperoxides are still dangerous for the cell as indicated by the protective action of glutathione peroxidase observed by Marklund et al. (1984).

Einfluß von Cysteamin auf die UV-Empnndlichkeit von λ-Phagen mit variablem Bromuracil-Gehalt / Influence of Cysteamine on the UV-Sensitivity of λ-Phages with Various BU-Contents

λ-Phages with different amounts of BU in their DNA-strands were irradiated with long wave­ length UV light (302/313 nm) and short wavelength UV-light (254 nm) in the presence and ab­ sence of cysteamine. It was found that the dose reduction factor is dependent on the percentage BU-substitution only after irradiation with UV-light of 254 nm, not after long wavelength UV-irradiation. Furthermore, the inactivation cross section is proportional to the BU-content of the DNA after irradiation with long and with short wavelength UV-light in the presence of cysteamine. The results lead to the conclusion that the presence of cysteamine inhibits energy transfer processes which occur after irradiation with UV-light of 254 nm in the absence of cysteamine.

Influence of cysteamine on intramolecular energy transfer in 5-bromouracil-substituted phage-DNA.

SummaryWith the aid of an analytical ultracentrifuge, single- and double-strand breakage rates of 5-bromouracil (BrU)-substituted DNA of a defective B. subtilis phage (PBSH) were determined after irradiation with U.V. (302, 313 nm) in the presence and absence of cysteamine. The presence of cysteamine reduced the appearance of single-strand breaks (10 per cent) in the BrU-containing strands of hybrid and bifilarly-substituted DNA. The primary breaks lead to secondary breaks in the complementary thymine-containing strand of hybrid DNA or in the complementary BrU-containing strand of bifilarly-substituted DNA (intra-molecular energy transfer). Cysteamine strongly reduces (< 1 per cent) these secondary breaks, indicating that radical reactions are involved in the mechanism of intramolecular energy transfer.

Protection and host cell repair of irradiated lambda phage. 3. Ultraviolet irradiation of 5-bromouracil-substituted phage.

5-bromouracil-containing λ phage, irradiated with UV light in the presence and absence of cysteamine, were assayed on normal and DNA-repair deficient bacteria. It has been found that these phage suffer (1) lesions, of unknown nature, which are neither reparable by the bacterial DNA repair systems nor prevented by cysteamine, a free radical scavenger and proton donor; (2) lesions which are converted to reparable lesions by cysteamine, and which may be sugar damages (perhaps accompanied by pyridimine dimers) which are converted to simple dimers; and (3) irreparable lesions, which do not occur in the presence of cysteamine, and which are probably sugar damages. These latter are the most important, and in large part explain the increased sensitivity to irradiation of 5-bromouracil-substituted phage. © 1969 Springer-Verlag.

Protection and host cell repair of irradiated lambda phage. II. Irradiation of 5-bromouracil-substituted phage with near visible light.

Phage λ, in which thymine has been totally substituted by the analog, 5-bromouracil, have been irradiated with near visible light in the presence and absence of cysteamine, a free radical scavenger and proton donor. These phage were then assayed on normal bacteria and on mutants which are unable to repair DNA lesions. It is concluded that these phage suffer three different kinds of lesions: (1) a class which are not reparable, but which can prevented cysteamine, suggesting that they are sugar damages; (2) a class which are reparable but are not eliminated by cysteamine and (3) a class which can be neither eliminated nor repaired. The nature of the latter two types of lesions cannot be surmised at present.

Search for a mechanism for the increased sensitivity of 5-bromouracil-substituted DNA to ultraviolet radiation

1. 1. The 5-bromouracil-substituted DNA's of Escherichia coli 15T − and T3 coliphage were labeled in thymine and 5-bromouracil with different isotopes and exposed to 280 nm radiation. Acid and enzymic hydrolysates of the substituted DNA's of the above organisms were analyzed for photoproducts by paper chromatography. 2. 2. The same four or five photoproducts, derived from 5-bromouracil, were detected in the acid hydrolysate of the irradiated DNA's under all conditions tested, including irradiation in vivo and in vitro and in the presence and absence of cysteamine. 3. 3. No product containing both the 5-bromouracil and thymine labels could be detected. 4. 4. Quantitative differences in the distribution of photoproducts were obtained under the various conditions of irradiation. Cysteamine, when present during irradiation, caused a decrease in the formation of uracil but had little effect on the total photodecomposition of 5-bromouracil. Also, an increase in dose caused an increase in the contribution of uracil to the total photoproducts. 5. 5. At low doses there is relatively little difference between the photosensitivity of 5-bromouracil and thymine in DNA. 6. 6. Exposure in solution to 254 nm radiation only slightly affects the chromatographic mobility of the photoproducts derived from 5-bromouracil. 7. 7. The excision in vivo of thymine-containing dimers from the irradiated DNA is unaffected by the presence of 5-bromouracil in the DNA. 8. 8. The relatively smaller numbers of thymine-containing dimers in irradiated DNA substituted with 5-bromouracil are quantitatively explicable in terms of the random substitution of bromouracil for thymine. 9. 9. The implication of these data is discussed with reference to the suggested mechanisms of the radiation sensitization of DNA by 5-bromouracil substitution.