Seroprevalence of Brucellosis in Human Immunodeficiency Virus Infected Patients in Hamadan, Iran

OBJECTIVES: Brucellosis is a systemic disease with a wide spectrum of clinical manifestations. This study aimed to determine the seroprevalence of brucellosis in human immunodeficiency virus (HIV) infected patients in Hamadan Province in the west of Iran. METHODS: A total of 157 HIV-infected patients were screened through standard serological tests, including Wright’s test, Coombs’ Wright test, and 2-mercaptoethanol Brucella agglutination test (2ME test), blood cultures in Castaneda media, and CD4 counting. Data were analyzed using Stata version 11. RESULTS: Wright and Coombs’ Wright tests were carried out, and only 5 (3.2%) patients had positive serological results. However, all patients had negative 2ME results, and blood cultures were negative for Brucella spp. Moreover, patients with positive serology and a mean CD4 count of 355.8 ± 203.11 cells/μL had no clinical manifestations of brucellosis, and, and the other patients had a mean CD4 count of 335.55 ± 261.71 cells/μL. CONCLUSION: Results of this study showed that HIV infection is not a predisposing factor of acquiring brucellosis.

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Soropositividade para brucelose em suínos em abatedouros / Seropositivity for brucellosis in pigs in slaughter houses

O presente trabalho teve por objetivo investigar a soropositividade para brucelose em suínos em abatedouros. Foram coletados 910 soros de suínos, procedentes de 30 propriedades, abatidos em frigoríficos da região central do estado de São Paulo, e submetidos às provas de soroaglutinaçao com antígeno tamponado acidificado (AAT) e 2-Mercaptoetanol (2-Me) objetivando determinar a ocorrência da enfermidade nesta espécie. Do total de soros avaliados foram observados 25 (2,7%) animais reagentes ao AAT pertencentes a 10 propriedades, caracterizando 36% de propriedades positivas. Dos animais positivos ao AAT, 16% apresentaram titulo de 25 (incompleto) e 52% titulo de 25 na soroaglutinação lenta (SAL), 8% apresentaram titulo de 50 incompleto na SAL e 25 incompleto no 2-ME e 8% apresentaram titulo de 50 na SAL e 25 no 2-ME. Estes resultados demonstram o elevado percentual de propriedades positivas para brucelose nesta região e ressaltam a necessidade de implementação de programas oficiais efetivos para o controle da brucelose suína.

The aim of this investigation was to evaluate the seropositivity for brucellosis in pig slaughterhouses. Sera of 910 pigs from 30 farms and slaughtered at slaughterhouses, located in the central region of the state of São Paulo, were tested with Buffered Plate Acidified Antigen (BPAA) and 2-mercaptoethanol (2-Me) as confirmatory test to determine the serological positivity in this species. The results demonstrated that 25 (2.7%) of the pigs from 10 farms reacted to BPAA, corresponding to 36% of positive properties. From BPAA positive animals 16% and 52% presented titers of 25 incomplete and 25 respectively in the Tube agglutination test (TAT), 8% presented 50 and 25 incomplete, respectively, in TAT and 2-ME, and 8% resulted 50 and 25 positive in TAT and 2-ME, respectively. These results demonstrate the high percentage of positive farms for swine brucellosis in this region and reinforce the need for official and effective program implementation for the swine brucellosis control.

Surface Polarity Dependent Solid-state Molecular Biological Manipulation with Immobilized DNA on a Gold Surface

As the demand for large-scale analysis of gene expression using DNA arrays increases, the importance of the surface characterization of DNA arrays has emerged. We compared the efficiency of molecular biological applications on solid-phases with different surface polarities to identify the most optimal conditions. We employed thiol-gold reactions for DNA immobilization on solid surfaces. The surface polarity was controlled by creating a self-assembled monolayer (SAM) of mercaptohexanol or hepthanethiol, which create hydrophilic or hydrophobic surface properties, respectively. A hydrophilic environment was found to be much more favorable to solid-phase molecular biological manipulations. A SAM of mercaptoethanol had the highest affinity to DNA molecules in our experimetns and it showed greater efficiency in terms of DNA hybridization and polymerization. The optimal DNA concentration for immobilization was found to be 0.5 microM. The optimal reaction time for both thiolated DNA and matrix molecules was 10 min and for the polymerase reaction time was 150 min. Under these optimized conditions, molecular biology techniques including DNA hybridization, ligation, polymerization, PCR and multiplex PCR were shown to be feasible in solid-state conditions. We demonstrated from our present analysis the importance of surface polarity in solid-phase molecular biological applications. A hydrophilic SAM generated a far more favorable environment than hydrophobic SAM for solid-state molecular techniques. Our findings suggest that the conditions and methods identified here could be used for DNA-DNA hybridization applications such as DNA chips and for the further development of solid-phase genetic engineering applications that involve DNA-enzyme interactions.

Effects of fibroblasts conditioned media on differentiation of programmable cells of monocytic origin to insulin-producing cells

The characteristic of stem cells in self renewal and differentiation to different types of cells has stimulated the interests for using stem cells as a starting material for generating insulin secreting cells. We've evaluated the differentiation potential of Programmable cells of monocytic origin [PCMOs] into insulin producing cells effected from the growth factors and fibroblasts conditioned media [FCM]. Peripheral blood monocytes of rat were cultured for 6 days in RPMI with 15% FBS, beta- mercaptoethanol, MCSF and interleukin-3. Then, these cells were incubated in differentiation media with HGF, EGF, Nicotinamide, 15% fibroblasts conditioned media and glucose for 15days. Morphological differences of cells were studied by invert microscope. In several stages, the amounts of insulin in supernatant of cells were measured by radioimmunoassay kit. Also productions of insulin from differentiated cells were studied with DTZ special staining. In response to MCSF and IL-3, monocytes dedifferentiated. These programmable cells of monocytic origin [PCMOs] were capable of differentiating into insulin producing cells in differentiation media. The morphology of differentiated cells was similar to Beta cells and the amount of insulin in supernatant of differentiated cells was much higher than PCMOs [P<0.05]. HGF, EGF, Nicotinamide and fibroblasts conditioned media are differentiation factors of PCMOs into insulin producing cells. According to the results insulin producing cells can be differentiated from programmable cells of monocytic origin in presence of fibroblasts conditioned media

Induction of bone marrow stromal cells into cholinergic-like cells by nerve growth factor

Bone marrow stromal cells [BMSC] are used as a source for cell therapy in different model for neurological disorder such as stroke and spinal cord injury. However, the transdifferentiation of BMSC into cholinergic phenotype requires more investigation. BMSC were isolated from adult rats, pre-induced with [beta-mercaptoethanol [BME] and followed by nerve growth factor [NGF] induction. Neurofilaments of 68 kDa, 160 kDa and 200 kDa [NF-200, NF-160 and NF-68, respectively] immuno-staining were used for evaluating the transdifferentiation of BMSC into neuronal phenotype. The percentage of neurofilaments immuno-reactive cells was applied in order to evaluate the results at the pre-induction and the induction stages. Also, NeuroD and Oct-4 expressions, using RT-PCR, were used in assessing the progression of BMSC into neuronal lineage. Choline acetyltransferase immuno-reactive cells were used for estimating the percentage of cholinergic neuronal phenotype. Immuno-staining with anti-microtubule-associated protein-2 [MAP-2] and anti-synapsin-I antibodies was done in order to evaluate cell tendency for synaptogenesis. The yield of cholinergic neurons with BME as pre-inducer and NGF as inducer was 80%. Also, NF-200, NF-160, NF-68, MAP-2 and synapsin-I were detected in the transdifferentiated cells. RT-PCR showed the expression of NeuroD, while Oct-4 was not detected. BME as pre-inducer and NGF as inducer for BMSC transdifferentiation into cholinergic phenotype are potential sources in traumatic injury therapy in the central nervous system

The drug-resistant mechanism of clinical non-fermenting bacilli producing IMP-1 metalloenzyme / 生物医学工程学杂志

A total of 50 clinical imipenem-resistant isolates of Pseudomonas aeruginosa and Acinetobacter baumannii were subjected to the ceftazidime-2- mercaptoethanol -double-disk synergy test and to the PCR assays with primers specific for bla(IMP-1). After the process of sequencing the positive one to identify the results, PCR analysis was conducted with primers specific for class 1 integrons. For synergy test, 28 isolates gave positive results, among which were 27 Pseudomonas aeruginosa and Acinetobacter baumannii. Only one Pseudomonas aeruginosa was found to carry bla(IMP-1), and bla(Int1) at the same time. This is the first ascertainment of IMP-1 producing Pseudomonas aeruginosa isolate carrying bla(IntI1) in West China, which is of significance to the research on the clinical spread of these drug-resisitant genes.

Modification of maturation condition improves oocyte maturation and in vitro development of somatic cell nuclear transfer pig embryos

This study examined effects on the developmental competence of pig oocytes after somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) of : 1) co-culturing of oocytes with follicular shell pieces (FSP) during in vitro maturation (IVM); 2) different durations of maturation; and 3) defined maturation medium supplemented with polyvinyl alcohol (PVA; control), pig follicular fluid (pFF), cysteamine (CYS), or beta-mercaptoethanol (beta-ME). The proportion of metaphase II oocytes was increased (p < 0.05) by co-culturing with FSP compared to control oocytes (98% vs. 94%). However, blastocyst formation after SCNT was not improved by FSP coculture (9% vs. 12%). Nuclear maturation of oocytes matured for 39 or 42 h was higher (p < 0.05) than that of oocytes matured for 36 h (95-96% vs. 79%). Cleavage (83%) and blastocyst formation (26%) were significantly higher (p < 0.05) in oocytes matured for 42 h than in other groups. Supplementation of a defined maturation medium with 100 micrometer CYS or 100 micrometer beta-ME showed no stimulatory effect on oocyte maturation, embryo cleavage, or blastocyst formation after PA. beta-ME treatment during IVM decreased embryo cleavage after SCNT compared to pFF or PVA treatments, but no significant difference was found in blastocyst formation (7-16%) among the four treatment groups. The results indicated that maturation of oocytes for 42 h was beneficial for the development of SCNT embryos. Furthermore, the defined maturation system used in this study could support in vitro development of PA or SCNT embryos.

[Seroepidemiological study of brucellosis in high risk groups in Boyerahmad 1384]

Brucellosis is a zoonotic disease that may have a major public health and economic impact in most countries. The disease appears as a Malt fever in humans and abortion in animals. This study was designed to determine the serologic titer of Brucella in high risk and non high risk people in Boyerahmad. A retrospective seroepidemiological study was performed on samples collected from 604 high risk and non high risk people using Rose Bengol test, tube standard test as a rapid test and 2 mercaptoethanol [2ME] and comb's wright as a confirmatory test. The data collected were analyzed by X[2] test via SPSS. Seroprevalence of Brucellosis in high risk people appeared to be high in the Rose Bengal and tube standard test [TST] 6.62 at titer >/= 1/40 whereas for non high risk it was 0%. Confirmation test in high risk people was shown with 2ME in four people. Brucellosis is a major cause of disease in high risk people which can be due to direct or indirect contact with diary products of the related animals

significance of modified latex agglutination with 2-mercapto-ethanol [2ME] test in differentiation between acute and chronic toxoplasmosis

Different serological tests often measure different antibodies that posess unique patterns of rise and fall with time after infection. A combination of serological tests is frequently required to establish whether an individual has been more likely infected in the distant past or has been recently infected. This study was carried out in Kirkuk Hospitals and Primary Health Care Centers to detect Toxoplasma antibodies among 319 pregnant women aged from less than 18 to more than 35 years old. The period of study was from beginning of November 2003 to end of May 2004. The study showed that 117 cases out of 319 were positive for Toxoplasma gondii [36.6%] by using LAT, 54 cases out of 319 were positive for IgM- ELISA [16.9%] and 66 out of 117 were positive for 2ME test [56.6%]. The total serum protein and serum albumin did not vary significantly between seropositive and seronegative toxoplasmosis, but the serum globulin level was elevated in Toxoplasma seropositive cases

value of 2-mercaptoethanol test in diagnosing recurrent active brucellosis

Brucellosis is endemic in Iraq; particularly in Northern parts. Several problems are encountered in diagnosing the disease depending on serological tests; especially among cases suspected to have reactivation of the disease. To evaluate the value of 2-Mercaptoethanol [2ME] test in diagnosing recurrent active brucellosis. The study population consisted of 466 patients who had been treated and cured from a previous attack of brucellosis [diagnosed clinically or serologically], who returned suffering from symptoms suggesting a new attack of brucellosis with positive slide agglutination [SAT] test and on whom a 2ME test was conducted. Patients were collected from A1-Salam teaching hospital in Mosul during 2 years period [1999-2000]. The 2ME test was positive in 184/408 [45%]. The probability of having a positive 2ME test increased if the SAT was positive at high titers. The study also revealed high lgG antibody titers, mainly ranged between 1:160 and 1:640, with doubled positive rate among females in comparison to males. The study revealed that 2ME test is useful in diagnosing about 45% of suspected cases with activation of previously treated and cured brucellosis. A direct SAT should first be conducted for suspected cases and the probability of having a positive 2ME test has increased if the primary SAT was positive at higher titers

Successful cryopreservation of in vitro derived bovine blastocysts in microcapillary pipette tips

The open pulled straw has been used effectively to vitrify preimplantation embryos because of geometric features that allow rapid rates of temperature exchange. One possible inexpensive alternative to the open pulled straw are commercially-available microcapillary pipette tips commonly used for electrophoresis. The main purpose of this study was to compare the survival rates of in vitro produced blastocysts following vitrification in microcapillary pipette tips and open pulled straws. Two experiments were conducted to investigate the effect of type of carrier, age of exp and ed blastocyst, and addition of beta-mercaptoethanol to post-warming culture medium on survival of vitrified in-vitro derived blastocysts. Exp and ed blastocysts [Day 7, 0900H and 1900H after insemination; insemination = Day 0 at 0900H] were vitrified while loaded randomly in groups of 4-10 into open pulled straws or pipette tips. Following warming, embryos were cultured in groups of up to 20 in 20 microl microdrops of modified KSOM or modified KSOM containing 100 micro M beta-mercaptoethanol at 38.5°C in 5% CO[2] in air. Survival after warming was assessed as the percentage of vitrified embryos that re-exp and ed and the percent that hatched after 48 hrs culture. Post-warming survival rates were not affected by type of carrier or age of the exp and ed blastocyst [P>0.05]. The proportions of embryos that re-exp and ed [55.5%] and hatched [25.7%] were higher [P<0.01] for those cultured with beta-mercaptoethanol than for those cultured without [re-expansion: 42.4%; hatching: 12.6%]. In conclusion, the microcapillary pipette tip represents an inexpensive alternative to the open pulled straw for cryopreservation

Comparison of the Widal test and a modified 2-mercaptoethanol Widal test for the diagnosis of enteric fevers

1. Develop a modified version of slide Widal test [2- mercaptoethanol modified Widal test] to differentiate between current/recent and previous typhoid and paratyphoid [enteric] fevers. 2. Evaluate the utility of the 2ME-modified Widal test [2ME-MWT]. A comparative study between conventional Widal test, and 2ME-MWT in the light of bacterial culture of clinical specimens. Three hundred and fifty individuals suspected to have enteric fevers [ENFS] were studied in Mosul City, Iraq. These patients were 246 [70.3%] females and 104 [29.7%] males with age ranged from 5- 84 years [X +/- SD =32 +/- 0.8]. They were all tested by conventional Widal and 2ME-MWT as well by culture of their blood, stool and urine. It was found that 200/350 [57.1%] of tested patients had positive Widal test [titer [3]

Differentiation of human fetal liver CD34+ cells into neuronal cells induced by beta-ME and BHA in vitro / 中国应用生理学杂志

<p><b>AIM</b>To establish model of differentiation of fetal liver stem cells induced by beta-ME + BHA into neural cells in vitro;</p><p><b>METHODS</b>CD34+ cells from naturally aborted human fetal liver were isolated with MACS Kit, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). After confluent more than 80%, the 5 passage cells were induced by 10(-3) mol/L beta-mercaptoethanol (beta-ME) and 2 x 10(-4) mol/L BHA for 24 hours, and washed with PBS, and then incubated in serum-free medium for 5 hours to 5 days. The characteristics of treated cells were assayed by immunocytochemistry staining analysis.</p><p><b>RESULTS</b>Cells treated by beta-ME+ BHA exhibited neuronal phenotype, and expressed neuronal specific proteins such as nestin, NeuN, TrnJ-1, and NF-M, which were not found in control cells. Statistic analysis showed that 81% cells were NeuN-positive, 75% cells TuJ-1-positive, 47% cells NF-M-positive, 90% cells NSE-positive.</p><p><b>CONCLUSION</b>beta-ME + BHA can induce human fetal liver CD34+ cells to produce neuronal specific antigens and proteins in vitro and become neuronal cells. CD34+ cells from human fetal liver possess potentials of differentiation into neural cells.</p>

Effect of beta-mercaptoethanol or epidermal growth factor supplementation on in vitro maturation of canine oocytes collected from dogs with different stages of the estrus cycle

Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.

Role of modified Widal test in the diagnosis of enteric fever.

OBJECTIVES: To evaluate the diagnostic specificity of modified Widal for recent infection in comparison with conventional Widal test. METHOD: Modified widal test was simultaneously done along with conventional Widal test in serum samples obtained from 50 bacteriologically positive cases of Salmonella typhi infection as well as 50 healthy individuals. RESULTS: A four-fold difference in the titres was noticed in the 50 sera of the test group and no charge in the titres of the control group. The early rising O antibodies which are predominantly IgM in nature. These are due to recent infection and are inactivated by 2-mercaptoethanol. On the other hand H is a mixture of IgG and IgM hence IgM portion gets inactivated giving rise to fall in titre. By inactivating IgM antibodies in modified Widal test, the agglutination would be brought about only by specific IgG while in the conventional Widal test agglutination is due to specific IgG and IgM. The difference in the titres indicates specific IgM class of antibodies which is the hallmark of recent infection. CONCLUSION: If conventional Widal test and modified Widal test are simultaneously done, one can be definite about the diagnosis of enteric fever.

Selected epidemiological features of human brucellosis in Yazd, Islamic Republic of Iran: 1993-1998

Brucellosis is a significant health problem in countries where control of zoonoses is inadequate. During 1993-98, we analysed sera and cultures from 792 suspected brucellosis patients who presented with histories of fever, chills, night sweating, weakness, malaise and headache to the referral hospital in Yazd. Cases were investigated by tube agglutination test [TAT] and 2-mercaptoethanol test [2-MET] and a questionnaire was completed for each.TAT titre was > / = 1:1 60 for 745 patients [94.1%] and 2-MET was positive for 42 [5.3%]. Of 745 confirmed cases, 460 were from 1996-1997. Prevalence was highest in summer [39.5%] and more common males than among females. Prevalence was highest among those aged 10-19 years [27.7%]. Most patients had a history of infected cheese, milk and milk product consumption [98%]

The effect of antioxidants on the in vitro life-span of keratinocyte / 生物工程学报

The effect of antioxidants on the in vitro life span of mouse keratinocytes was investigated in this work. It was found that the life span of the keratinocytes cultured in the medium supplemented with antioxidants was extended significantly. The most beneficial antioxidant used in this work was the mercaptoethanol, followed by the catalase and SOD. However, the growth rates of keratinocytes in vitro under all the experimental conditions still declined with the culture time. It was also found that the antioxidants added in the medium were also helpful to enhance the keratinocyte colony formation. In addition, the aging kinetics of the mouse epidermal keratinocytes in vitro were analyzed, and finally the aging rate constants corresponding to antioxidants used were calculated.

Neuronal Differentiatin of P19 Cells / 대한해부학회지

P19, murine embryonal carcinoma (EC) cells can be induced to differentiate into neurons in the presence of retinoic acid (RA). To investigate neuronal differentiation of P19 cells in details, P19 aggregates were obtained in the presence or absence of RA, ascorbic acid (AA) and 2-mercaptoethanol (2-ME) in bacteriological Petri dishes. When the aggregates were transferred into the serum depleted medium, P19 cells exhibited dramatic morphological changes. Cells contained long and thin processes as detected in differentiated neurons. Western blot analysis showed that treatment of RA and AA induced expression of neuron-specific markers such as NCAM, NSE and Tuj1. Expression of GFAP was not detected, suggesting that P19 cells differentiate into neurons under our experimental condition. Immunocytochemical studies also revealed that treatment of RA and AA increased expression of NCAM and Tuj1. On the contrary, 2-ME was ineffective in the neuronal differentiation of P19 cells, which is consistent the results from the western blot analysis. These results suggest that differentiated P19 cells have similar characteristics to those of typically differentiated neurons. This study also suggests that P19 cells may provide useful tools to study neuronal differentiation in vitro.