Effect of rosuvastatin on high glucose-induced endoplasmic reticulum stress in human umbilical vein endothelial cells.

It is well established that endothelial injury plays an essential role in atherosclerotic plaque formation. Accumulating evidence has shown that high glucose levels may detrimentally affect cultured endothelial cells through endoplasmic reticulum (ER) stress. In this study, we investigated the effect of rosuvastatin on high glucose-induced ER stress in human umbilical vein endothelial cells (HUVECs). HUVECs treated with 30 mM glucose were used to simulate high-glucose conditions, and rosuvastatin concentrations ranging from 0.1 to 10 nM were used. Cell viability was analyzed by thiazolyl blue tetrazolium bromide assay, and apoptosis rate was measured using flow cytometry. Expression of GRP78, IRE1α, XBP1s, and CHOP was quantified using western blot and real-time polymerase chain reaction. Compared to cells treated with high glucose alone, cell viability increased and apoptosis rate decreased significantly in the rosuvastatin + high-glucose groups. Furthermore, GRP78, IRE1α, XBP1s, and CHOP expression was downregulated as a result of rosuvastatin administration. These results suggest that rosuvastatin may protect HUVECs from injury induced by high glucose levels, through alleviation of ER stress.

Isolation and characterization of a novel paraffin wax-degrading bacterium, Pseudomonas sp strain PW-1, from petroleum-contaminated sites.

An isolate capable of degrading paraffin wax was isolated from petroleum-contaminated sites in Daqing, China, and identified as Pseudomonas sp strain PW-1 by analyzing the 16S rDNA sequence (GenBank accession No.: KF529529) as well as the biochemical and physiological characteristics. The optimized degradation conditions of the isolate were as follows: FeSO4 metal ion concentration of 0.01 g, temperature of 30°C, (NH4)2SO4 nitrogen source concentration of 1.5 g/L, and a carbon: nitrogen ratio of 10:1. Response surface methodology-based analysis of the culture time, inoculation amount, and initial pH of the medium revealed that the optimal theoretical conditions were a culture time of 11.16 days, inoculation amount of 3.13%, and an initial pH of 9.29. The theoretical degradation rate was up to 54.68% under the optimal conditions. Taking into account the experimental conditions of a laboratory, 11.2 days of cultivating time, 3% inoculum, and a medium initial pH of 9.3 were used in practical settings. Experimental results showed that the degradation rate of paraffin wax was 52.85%, which demonstrated that this strain could degrade 1050 mg paraffin wax, using it as the sole carbon source, in a 1000-mL minimal salts medium. These results indicate that the strain PW1 can be used for application in oil wells with paraffin deposition problems in order to enhance oil recovery.

Effects of probucol on cultured human umbilical vein endothelial cells injured by hypoxia/reoxygenation.

There is increasing evidence suggesting that endoplasmic reticulum stress (ERS) plays an important role in the initiation and development of atherosclerosis. This study was designed to examine the effect of probucol on cultured human umbilical vein endothelial cells (HUVECs) injured by hypoxia/reoxygenation (H/R) and the potential mechanisms involving ERS. Injured HUVECs induced by Na2S2O4 served as an H/R model in vitro. The concentration of probucol in this study ranged from 3 to 27 µM. Cell viability was analyzed using MTT and a lactate dehydrogenase (LDH) assay. The expression of GRP78, X-box-binding protein (XBP)-1, and CHOP (c/EBP-hemologous protein) were quantified using western blot. Compared to cells with H/R injury alone, the results showed that the cell viability increased significantly with probucol, while the LDH leakage rate was significantly lower as analyzed by the LDH assay. Furthermore, the expression levels of GRP78, XBP-1, and CHOP were significantly downregulated. These results indicated that probucol effectively protected HUVEC from injury induced by H/R and that the mechanism might be related to attenuation of ERS.

Analysis of imprinted messenger RNA expression in deceased transgenic cloned goats.

Genomic imprinting is an important epigenetic mechanism that has vital effects on fetal growth and development. We observed the differences in four tissues (heart, spleen, liver, and kidney) from dead transgenic cloned goats using hematoxylin and eosin (H&E) staining. Eight imprinted genes in the tissues of dead transgenic cloned and normal goats were analyzed using reverse transcription polymerase chain reaction. H&E staining results from the abortion group indicated the lack of obvious morphological changes in heart and spleen tissues, while inflammatory cell infiltration and glomerular nephritis characteristics were observed in liver and kidney tissues, respectively. Compared to the control group, CDKN1C, H19, IGF2R, and SNRPN were significantly (P < 0.05) overexpressed in the heart tissue of the abortion group, while XIST was significantly reduced. In the liver tissues, CDKN1C and DLK1 expression decreased, while GNAS, H19, IGF2R, PEG3, and XIST expression increased significantly. In the spleen tissues, DLK1 expression increased, while GNAS, H19, IGF2R, PEG3, SNRPN, and XIST expression decreased. In the kidney tissues, CDKN1C, DLK1, GNAS, IGF2R, and PEG3 expression increased, while H19 and XIST expression decreased. The overall expression of imprinted genes was abnormal in different tissues of transgenic cloned goats, and the degree of abnormal genomic imprinting was more severe in the abortion group compared to the death and control groups. These results suggest that abnormal expression of imprinted genes may cause developmental defects in transgenic cloned goats. Moreover, abnormal epigenetic modifications may affect the reprogramming of transgenic donor cells.

Identification of transgenic cloned dairy goats harboring human lactoferrin and methylation status of the imprinted gene IGF2R in their lungs.

Dairy goat is a good model for production of transgenic proteins in milk using somatic cell nuclear transfer (SCNT). However, animals produced from SCNT are often associated with lung deficiencies. We recently produced six transgenic cloned dairy goats harboring the human lactoferrin gene, including three live transgenic clones and three deceased transgenic clones that died from respiratory failure during the perinatal period. Imprinted genes are important regulators of lung growth, and may be subjected to faulty reprogramming. In the present study, first, microsatellite analysis, PCR, and DNA sequence identification were conducted to confirm that these three dead kids were genetically identical to the transgenic donor cells. Second, the CpG island methylation profile of the imprinted insulin-like growth factor receptor (IGF2R) gene was assessed in the lungs of the three dead transgenic kids and the normally produced kids using bisulfite sequencing PCR. In addition, the relative mRNA level of IGF2R was also determined by real-time PCR. Results showed that the IGF2R gene in the lungs of the dead cloned kids showed abnormal hypermethylation and higher mRNA expression levels than the control, indicating that aberrant DNA methylation reprogramming is one of the important factors in the death of transgenic cloned animals.

Polymorphism analysis of IGFBP-5 gene exon 1 in Tibet Mini-pig and Junmu No. 1 White pig.

The genetic resources and the mechanism of miniaturization in the Tibet Mini-pig have not been comprehensively studied. Polymorphisms in genes related to the insulin-like growth factor (IGF) axis have been investigated for years, but few on the polymorphism of IGF-binding protein-5 (IGFBP-5) in the Tibetan pig. In this study, allele-specific polymerase chain reaction (AS-PCR) was used to analyze polymorphisms in exon 1 of the IGFBP-5 gene in two pig breeds, Tibet Mini-pigs and Junmu No. 1 White pigs. A BLAST analysis of the expressed sequence tags in the porcine IGFBP-5 gene revealed that exon 1 of this gene has two single nucleotide polymorphisms (SNPs), G188T and G503A. The AS-PCR results demonstrated that in both pig breeds examined, the TT, GT, and GG genotypes existed at the G188T locus, with GT as the most common genotype. At the G503A locus, GG, GA, and AA genotypes existed in Junmu No. 1 White pigs, with the GA genotype as the most frequently occurring. By contrast, at this locus, only the GA and AA genotypes were observed in the Tibetan pigs, and AA was more common than GA. There was a significant difference (P < 0.01) in allele distribution between the two breeds at the G503A locus but not the G188T locus, and there was a lower polymorphism information content for the two polymorphic loci in Tibet Mini-pigs than in Junmu No. 1 White pigs. The present study revealed SNPs in exon 1 of IGFBP-5 gene in the Tibet Mini-pig, possibly providing more understanding of the mechanism of miniaturization.

Oct4 and Sox2 overexpression improves the proliferation and differentiation of bone mesenchymal stem cells in Xiaomeishan porcine.

Mesenchymal stem cells derived from bone marrow (BMSCs) are a population of self-renewing multipotent cells that are capable of differentiating into various cellular lineages, and are widely employed in tissue engineering and cell therapy. Recently, clinical research involving BMSCs has become increasingly popular. In order to conduct appropriate research, it is first necessary to amplify large amounts of functional BMSCs in vitro. However, after several passages of expanding in vitro, the proliferation and differentiation potential of BMSCs gradually decline. To determine whether overexpression of Oct4 or Sox2 might prevent this decline, we transfected Oct4 or Sox2, which are essential for the pluripotency and self-renewal of embryonic stem cells, into BMSCs of Xiaomeishan porcine by a lentivirus. The results showed that overexpression of Sox2 or Oct4 BMSCs in culture media containing a basic fibroblast growth factor resulted in higher proliferation and differentiation compared to controls, suggesting that genetic modification of stemness-related genes is an efficient way to maintain the proliferation and differentiation potential of BMSCs.

Antibody-based detection of alkaline phosphatase in lepidopteran insects (Lepidoptera: Noctuidae).

Alkaline phosphatase (ALP) of Helicoverpa armigera Hub. (Lepidoptera; Noctuidae) (GenBank accession No. EU729322) was cloned and expressed. The target gene H.a-ALP, having an open reading frame of 1608 bp, was reverse-transcribed from cDNA by the polymerase chain reaction. The open reading frame of the target gene was cloned into the pET-32a expression vector to obtain recombinant protein in Escherichia coli DE-3 cells for the subsequent production of polyclonal antibody. New Zealand white rabbits were used for production of anti-pET-32a-H.a-ALP. The production of antibody was also optimized by employing ELISA for titer determination. The produced antiserum was processed and used as an antibody. Western blot results showed that the polyclonal antibody produced was capable of effectively binding target protein not only from H. armigera but also from other lepidopterans such as Mythimna separata and Plutella xylostella. This antibody was also used to detect levels of ALP within different instars of H. armigera. Thus, it is concluded that this antibody-based assay is very useful for the effective detection of gene-specific expression. Furthermore, it may also be used to detect the expression levels and tissue localization of ALP, as well as in other physiological studies involving this enzyme.

Molecular and biochemical characterization of the effects of insecticidal toxin from Meloidae beetles on Helicoverpa armigera (Hub.) (Lepidoptera: Noctuidae).

The molecular and biochemical effects of an insecticidal toxin extracted from Meloidae beetles were investigated on Helicoverpa armigera. The toxin was identified as cantharidin, a well-known natural compound produced by beetles of family Meloidae and Oedemeridae. Furthermore, the effect of the toxin on the metabolic enzymes alkaline phosphatase (ALP) and glutathione S-transferase (GST), responsible for the metabolism of insecticides, was also investigated. Results of a diet incorporation bioassay performed under laboratory conditions showed that the LC50 value of cantharidin was 0.068 mg/g. The body weight of the insect was also significantly reduced by cantharidin treatment. The LC10 concentration of cantharidin, 0.01 mg/g, was also tested to determine its effect on ALP and GST. Our results showed that cantharidin significantly inhibited ALP activity after 48 h, whereas GST activity was significantly inhibited after 24 h. The decline of ALP and GST transcript levels was also validated by semiquantitative RT-PCR analysis. It may be concluded from the results that ALPs and GSTs may be targets of the cantharidin intoxication mechanism. Moreover, the inability of ALP and GST to metabolize cantharidin shows that the mechanism of detoxification for cantharidin is different from that for conventional insecticides. On the basis of our investigations, the chemical structure of insecticides may be modified using a model structure of cantharidin, to avoid metabolism by metabolic enzymes.

Differences of ventricular late potential between acute STEMI and NSTEMI patients / Diferencias del potencial tardío ventricular entre pacientes con infartos agudos STEMI y NSTEMI

OBJECTIVE: To discuss the positive rate of ventricular late potential (VLP) between patients with acute ST-segment elevation myocardial infarction (STEMI) and patients with acute non NSTEMI. METHODS: One hundred and sixty-three cases of acute myocardial infarction (90 patients with STEMI and 73 with NSTEMI), admitted to the first hospital of China Medical University between June 2011 and August 2011, underwent VLP examination. RESULTS: The VLP positive rate of the STEMI group was 54.4%, while that of the NSTEMI group was 38.4%, and the differences have statistical meaning (χ2 = 4.186, p < 0.05). The occurrence rate of ventricular arrhythmia in VLP positive patients was 11.7%, while in VLP negative patients it was 3.5% (χ2 = 4.005, p < 0.05). CONCLUSION: The VLP positive rate of the STEMI group is higher than that of the NSTEMI group.

OBJETIVO: Analizar la tasa positiva del potencial tardío ventricular (PTV) entre pacientes con infarto agudo del miocardio sin elevación del segmento ST (NSTEMI por sus siglas en inglés) y el infarto agudo del miocardio con elevación del segmento ST (STEMI por sus siglas en inglés). MÉTODOS: Ciento sesenta y tres casos de infarto agudo de miocardio (90pacientes con STEMI) y 73 con NSTEMI, ingresados en la Universidad primer hospital de Medicina China entre junio y agosto de 2011, fueron sometidos a examen de PTV. RESULTADOS: La tasa positiva PVT del grupo STEMI fue 54.4%, mientras que la del grupo NSTEMI fue 38.4%, y las diferencias tienen significado estadístico (χ² = 4.186, p < 0.05). La tasa de ocurrencia de arritmia ventricular en pacientes PVTpositivos fue 11.7%, mientras que en los pacientes PVT negativos fue 3.5% (χ² = 4.005, p < 0.05). CONCLUSIÓN: La tasa PTV positiva del grupo STEMI es mayor que la del grupo NSTEMI.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae).

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

Significant association of APOA5 and APOC3 gene polymorphisms with meat quality traits in Kele pigs.

Apolipoprotein A5 (APOA5) and C3 (APOC3) genes are involved in the PPAR lipid metabolism pathway and thus associated with elevated triglyceride levels. However, whether APOA5 and APOC3 genetic polymorphisms affect intramuscular fat deposition and other meat quality traits remains unknown in pigs. One hundred and seventy-one Kele pigs were sampled to investigate genetic variants in the APOA5 and APOC3 genes and their association with seven pork quality traits. We identified 5 single nucleotide polymorphisms (SNPs) in the promoter region of the APOA5 gene and 17 SNPs in the APOC3 gene. Linkage disequilibrium analysis revealed 5 complete linkage disequilibria among these 22 SNPs. We found that 10 SNPs were significantly correlated with meat quality traits, including the mutation A5/-769 in the APOA5 gene, which was significantly associated with cooked weight percentage, and 9 SNPs in the APOC3 gene that were significantly associated with drip loss rate, meat color value of longissimus dorsi muscle and shear force. Therefore, these SNP markers will be useful for marker-assisted selection for improved pork quality.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

Paeonia (Paeoniaceae) expressed sequence tag-derived microsatellite markers transferred to Paeonia delavayi.

Paeonia Franchet contains approximately 30 species of perennial herbs and is the only member of the family Paeoniaceae. Paeonia delavayi is an endangered ornamental plant that has a compound flower color and is endemic to southwest China. Its optimal habitats have been fragmented and its population size has greatly decreased as a result of human activities. Using a modified biotin-streptavidin capture method, 51 primer sets were identified in 3 wild populations of P. delavayi in this study. Ten primers displayed polymorphisms and 41 amplified no products, gave weak or indistinct bands, or gave larger sizes than expected. The number of alleles per locus ranged from 1 to 3, and the values for observed and expected heterozygosities ranged from 0.000 to 0.571 and from 0.000 to 0.509, with averages of 0.113 and 0.295, respectively. These markers will be useful for further investigations in resource conservation, selection of parental types in cross-breeding, the molecular evolution of this species, and for related research in Paeonia species.

Comparison of the neurobiological effects of attribution retraining group therapy with those of selective serotonin reuptake inhibitors.

The aim of this study was to compare the effectiveness of attribution retraining group therapy (ARGT) with selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depressive disorder (MDD), generalized anxiety disorder (GAD), and obsessive-compulsive disorder (OCD). Subjects were sequentially recruited and randomized into two groups, one receiving ARGT (n = 63) and the other SSRIs (n = 66) for 8 weeks. Fifty-four ARGT outpatients with MDD (n = 19), GAD (n = 19), and OCD (n = 16) and 55 SSRI outpatients with MDD (n = 19), GAD (n = 19), and OCD (n = 17) completed the study. All subjects were assessed using the Hamilton Depression Scale and Hamilton Anxiety Scale before and after treatment. The 10-item Yale-Brown Obsessive Compulsive Scale was employed only for OCD subjects. Plasma levels of serotonin, norepinephrine, cortisol, and adrenocorticotropic hormone were also measured at baseline and 8 weeks after completion of treatment. Symptom scores were significantly reduced (P < 0.001) in both the ARGT and SSRI groups at the end of treatment. However, MDD, GAD and OCD patients in the ARGT group had significantly lower plasma cortisol concentrations compared to baseline (P < 0.05), whereas MDD and OCD patients receiving SSRIs showed significantly increased plasma levels of serotonin (P < 0.05). These findings suggest that ARGT may modulate plasma cortisol levels and affect the hypothalamus-pituitary-adrenal axis as opposed to SSRIs, which may up-regulate plasma serotonin levels via a different pathway to produce an overall improvement in the clinical condition of the patients.

Comparison of the neurobiological effects of attribution retraining group therapy with those of selective serotonin reuptake inhibitors

The aim of this study was to compare the effectiveness of attribution retraining group therapy (ARGT) with selective serotonin reuptake inhibitors (SSRIs) in the treatment of major depressive disorder (MDD), generalized anxiety disorder (GAD), and obsessive-compulsive disorder (OCD). Subjects were sequentially recruited and randomized into two groups, one receiving ARGT (n = 63) and the other SSRIs (n = 66) for 8 weeks. Fifty-four ARGT outpatients with MDD (n = 19), GAD (n = 19), and OCD (n = 16) and 55 SSRI outpatients with MDD (n = 19), GAD (n = 19), and OCD (n = 17) completed the study. All subjects were assessed using the Hamilton Depression Scale and Hamilton Anxiety Scale before and after treatment. The 10-item Yale-Brown Obsessive Compulsive Scale was employed only for OCD subjects. Plasma levels of serotonin, norepinephrine, cortisol, and adrenocorticotropic hormone were also measured at baseline and 8 weeks after completion of treatment. Symptom scores were significantly reduced (P < 0.001) in both the ARGT and SSRI groups at the end of treatment. However, MDD, GAD and OCD patients in the ARGT group had significantly lower plasma cortisol concentrations compared to baseline (P < 0.05), whereas MDD and OCD patients receiving SSRIs showed significantly increased plasma levels of serotonin (P < 0.05). These findings suggest that ARGT may modulate plasma cortisol levels and affect the hypothalamus-pituitary-adrenal axis as opposed to SSRIs, which may up-regulate plasma serotonin levels via a different pathway to produce an overall improvement in the clinical condition of the patients.

Gene expression patterns of invertase gene families and modulation of the inhibitor gene in tomato sucrose metabolism.

Patterns of gene expression in the different types of sucrose metabolism in the tomato are highly variable and heritable. This genetic variation causes considerable functional differences. We examined the patterns of expression of invertase (Inv) gene families and an invertase inhibitor (INH) gene involved in elongating roots, hypocotyls, and fruit of the tomato (Lycopersicon esculentum cv. Micro-Tom and L. chmielewskii) through a real-time quantitative PCR analysis. We found that the Lin6 gene plays an important role in the vegetative growth stage. Lin5 and Lin7 did not express in Micro-Tom, but did express in L. chmielewskii. Overall relative expression levels of sucrose Inv gene families were significantly lower in L. chmielewskii during the reproductive growth stage than in Micro-Tom, being up to hundreds of times lower. It was not expressed in the dissepiment in L. chmielewskii. We suggest that differences in sucrose accumulation in tomato fruit is mainly due to differentially expressed invertase gene families at the later fruit growth stages.

Differences of Ventricular Late Potential between Acute STEMI and NSTEMI Patients.

OBJECTIVE: To discuss the positive rate of ventricular late potential (VLP) between patients with acute ST-segment elevation myocardial infarction (STEMI) and patients with acute non NSTEMI. METHODS: One hundred and sixty-three cases of acute myocardial infarction (90 patients with STEMI and 73 with NSTEMI), admitted to the First Hospital of China Medical University between June 2011 and August 2011, underwent VLP examination. RESULTS: The VLP positive rate of the STEMI group was 54.4%, while that of the NSTEMI group was 38.4%, and the differences have statistical meaning (χ2 = 4.186, p < 0.05). The occurrence rate of ventricular arrhythmia in VLP positive patients was 11.7%, while in VLP negative patients it was 3.5% (χ2 = 4.005, p < 0.05). CONCLUSION: The VLP positive rate of the STEMI group is higher than that of the NSTEMI group.

Optimization of factors affecting Agrobacterium-mediated transformation of Micro-Tom tomatoes.

Micro-Tom is the smallest known variety of tomatoes. An orthogonal experimental design L(16) (4(5)) was used to optimize Agrobacterium-mediated transformation of cotyledon explants of Lycopersicon esculentum cv. Micro-Tom. Four parameters were investigated to determine their effect on transformation frequency: the concentration of bacterial suspension, time of dip in bacterial suspension, co-cultivation time, and concentration of carbenicillin. We also examined the effect of these parameters on contamination rate, necrosis rate, mortality, cut-surface browning rate, and undamaged explant rate. Both the bacterial and carbenicillin concentrations had a significant influence on the rate of infected explants. The time of co-cultivation also had a significant influence on the transformation parameters. The optimal transformation protocol consisted of an Agrobacterium suspension of 0.5 × 10(8) cells/mL (OD(600) = 0.5) and an infection time of 5 min, one day of co-cultivation and 500 mg/L carbenicillin. Under these conditions, the transformation efficiency of the shoots reached 5.1%; the mean transformation frequency was 3.9% (N = 838).

Clinical, metabolic, and antibody responses of adult volunteers to an investigational vaccine composed of pertussis toxin inactivated by hydrogen peroxide.

A toxoid vaccine, composed of purified pertussis toxin inactivated with H2O2 (NICHD-Ptxd), was developed on the basis of evidence that serum neutralizing antibodies (antitoxin) would confer immunity to pertussis. In vivo and in vitro assays of NICHD-Ptxd showed only trace or nondetectable levels of pyrogenic, adenosine diphosphate-ribosyltransferase, binding and pharmacologic activities. Nevertheless, about 40% of the antigenicity of pertussis toxin was retained. Adult volunteers were injected, two times 6 weeks apart, with either 10 (n = 21), 50 (n = 25), or 75 (n = 30) micrograms/dose of one lot, Ptx-06, adsorbed onto AI(OH)3. Neither fever nor changes in the levels of leukocytes, lymphocytes, fasting blood glucose, or insulin were observed in the volunteers. The optimal immunizing dose, 50 micrograms, induced levels of antitoxin (geometric mean (GM) 302 U) comparable to those found in eight adults convalescent from pertussis (GM 269 U) and greater than those found in 18-month-old children after their fourth dose of diphtheria and tetanus toxoids and pertussis vaccine (GM 20.0 U, p less than 0.001). These data indicate that NICHD-Ptxd is safe and immunogenic in adults, and they justify its evaluation in infants and children.