Effect of aflatoxin B 1 on respiration and oxidative phosphorylation in the rabbit. I. Studies of the hepatic mitochondria

The effect of various concentrations of aflatoxin B1 (AB1) is studied "in vitro" on rabbit liver mitochondria. AB1 inhibits at concentrations from 1 to 2-4 x 10(-4) M, the respiratory rate from 20% a maximum 41-35% by glutamate and succinate as substrates, but it does not uncouple oxidative phosphorylation nor does it inhibit site III (ascorbate + TMPD). The inhibited site appears to be between cytochromes b and c (c1). AB1 seems to be easily transported across the mitochondrial membrane. The maximum degree of inhibition and the promoting AB1 concentration are too high to explain the liver cell necrosis in rabbit induced by AB1 "in vivo".     

Comparative immunochemical characteristics of polyclonal and monoclonal antibodies to aflatoxin B1

Four hybrid clones (MM-(AB1)-1, MM-(AB1)-2, MM-(AB1)-3, and MM-(AB1)-4) were obtained by hybridoma technology with immunization of BALB/c mice with a BSA conjugate of aflatoxin B1 carboxymethyloxime derivative. Antibodies produced by these clones varied in the ability to recognize the aflatoxin B1 analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively).     

Specific features of albumin interactions with hemiacetals of aflatoxins and sterigmatocystin

Aflatoxin B2a (AB2a), aflatoxin G2a (AG2a), and hemiacetal of sterigmatocystin have been shown to form immunoreactive conjugates with albumin. The conjugates were formed following incubation of solution mixtures at room temperature for 1 h, as demonstrated by spectrophotometry and enzyme immunoassay. Anti-AB2a antibodies reacted with AB2a, aflatoxin B1, and aflatoxin AB2 (100, 8.8, and 5.9%, respectively); a similar result was obtained for anti-AG2a antibodies reacting with AG2a, aflatoxin G1, and aflatoxin AG@2 (100, 2.5, and < 1.0%, respectively). Binding of anti-AB2a and anti-AG2a antibodies to solid-phase conjugates of AB2a or AG2a exhibited similar analytical characteristics.     

Activated and memory T lymphocytes in human milk.

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean +/- 1 SD of 4 +/- 3% of total HM leukocytes were lymphocytes and 96 +/- 3% were macrophages and granulocytes (N = 33 subjects). HM lymphocyte populations were further analyzed in five subjects. T cells (CD3+) represented 83 +/- 11% and B cells (CD19+) were 6 +/- 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40-1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P less than 0.02) and postpartum women (P less than 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P less than 0.002) and postpartum women (P less than 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 +/- 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 +/- 4%; P less than 0.001), and 15 +/- 6% of CD3+ cells in HM were IL-2R+ (controls, 6 +/- 2%; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

Acinetobacter sp. HM746599 isolated from leatherback turtle blood

A newly described bacterial isolate, Acinetobacter sp. HM746599, has been obtained from leatherback sea turtle hatchling blood. The implication is that the hatchling was infected during development in the egg, which is substantiated by other studies to be reported by us in the future. The 16S rRNA gene sequence of the bacterium (GenBank accession number: HM746599) showed the greatest similarity to the identified species, Acinetobacter beijerinckii (97.6-99.78%) and Acinetobacter venetianus (99.78%). Acinetobacter sp. HM746599 are gram-negative, rod-shaped coccobacilli and are hemolytic/cytotoxic to human and sea turtle red blood cells (RBCs). Hemolysis is not the result of any detectable soluble toxin. Acinetobacter beijerinckii and A. venetianus hemolyze sheep RBCs while Acinetobacter sp. HM746599 does not, and unlike A. venetianus, the growth of Acinetobacter sp. HM746599 and A. beijerinckii is not supported by l-arginine. Many Acinetobacter species, especially hemolytic ones, are pathogenic to immunologically compromised humans and it is possible that, in addition to sea turtles, this bacterium might also be a danger to susceptible humans who handle infected hatchlings. The bacteria are available from CCUG (Culture Collection, University Gothenburg, G?teborg, Sweden) and from NRRL (Agricultural Research Service Culture Collection, Peoria, IL).     

Some Cultural Conditions That Control Biosynthesis of Lipid and Aflatoxin by Aspergillus parasiticus

Synthesis of total lipid and aflatoxin by Aspergillus parasiticus as affected by various concentrations of glucose and nitrogen in a defined medium and by different incubation temperatures was studied. Maximal yields of lipid and aflatoxin were obtained with 30% glucose, whereas mold growth, expressed as dry weight, was maximal when the medium contained 10% glucose. Maximal mold growth occurred when the medium contained 3% (NH(4))(2)SO(4); however, 1% (NH(4))(2)SO(4) favored maximum accumulation of lipid and aflatoxin. Growth of mold and synthesis of lipid and toxin also varied with the incubation temperature. Maximal mold growth occurred at 35 C, whereas most toxin appeared at 25 C. Maximal production of lipid occurred at 25 and 35 C but production was more rapid at 35 C. Essentially all glucose in the medium (5% initially) was utilized in 3 days at 25 and 35 C but not in 7 days at 15 and 45 C. Patterns for formation of lipid and aflatoxin were similar at 15 and 25 C when a complete growth medium was used and at 28 C when the substrate contained various concentrations of glucose or (NH(4))(2)SO(4). They were dissimilar when the mold grew at 35 or 45 C. At these temperatures lipid was produced preferentially and only small amounts of aflatoxin appeared.     

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.     

Monoclonal anti-idiotypic antibody mimicking the principal neutralization site in HIV-1 GP120 induces HIV-1 neutralizing antibodies in rabbits

A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.     

Production and characterization of monoclonal antibodies against aflatoxin M1.

By using an indirect enzyme-linked immunosorbent assay, four monoclonal antibodies were selected after fusion of mouse P3-NS1-Ag4-1 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with aflatoxin M1 (AFM1) conjugated to bovine serum albumin. Two of these antibodies were found to be specific for AFM1 and were designated AMW-1 and AMW-4. The specificities of AMW-1, which had higher affinity to AFM1, were determined by a competitive direct enzyme-linked immunosorbent assay with peroxidase-AFM1 as the marker. The relative cross-reactivity of each toxin (relative to AFM1) with AMW-1, as determined by the amount of aflatoxin necessary to cause 50% inhibition of enzyme activity, was 12, greater than 40, 12, and greater than 40 for B1, B2, G1, and G2, respectively.     

Efficacy of a Community-Based Physical Activity Program KM2H2 for Stroke and Heart Attack Prevention among Senior Hypertensive Patients: A Cluster Randomized Controlled Phase-II Trial

Objective

To evaluate the efficacy of the program Keep Moving toward Healthy Heart and Healthy Brain (KM2H²) in encouraging physical activities for the prevention of heart attack and stroke among hypertensive patients enrolled in the Community-Based Hypertension Control Program (CBHCP).

Design

Cluster randomized controlled trial with three waves of longitudinal assessments at baseline, 3 and 6 months post intervention.

Setting

Community-based and patient-centered self-care for behavioral intervention in urban settings of China.

Participants

A total of 450 participants diagnosed with hypertension from 12 community health centers in Wuhan, China were recruited, and were randomly assigned by center to receive either KM2H² plus standard CBHCP care (6 centers and 232 patients) or the standard care only (6 centers and 218 patients).

Intervention

KM2H² is a behavioral intervention guided by the Transtheoretical Model, the Model of Personalized Medicine and Social Capital Theory. It consists of six intervention sessions and two booster sessions engineered in a progressive manner. The purpose is to motivate and maintain physical activities for the prevention of heart attack and stroke.

Outcome Measures

Heart attack and stroke (clinically diagnosed, primary outcome), blood pressure (measured, secondary outcome), and physical activity (self-report, tertiary outcome) were assessed at the individual level during the baseline, 3- and 6-month post-intervention.

Results

Relative to the standard care, receiving KM2H² was associated with significant reductions in the incidence of heart attack (3.60% vs. 7.03%, p < .05) and stroke (5.11% vs. 9.90%, p<0.05), and moderate reduction in blood pressure (-3.72mmHg in DBP and -2.92 mmHg in DBP) at 6-month post-intervention; and significant increases in physical activity at 3- (d = 0.53, 95% CI: 0.21, 0.85) and 6-month (d = 0.45, 95% CI: 0.04, 0.85) post-intervention, respectively.

Conclusion

The program KM2H² is efficacious to reduce the risk of heart attack and stroke among senior patients who are on anti-hypertensive medication. Findings of this study provide solid data supporting a formal phase-III trial to establish the effectiveness of KM2H² for use in community settings for prevention.

Trial Registration

ISRCTN Register ISRCTN12608966     

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.     

The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.     

Chromosomal aberrations and sister-chromatid exchanges in swine

Frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) in peripheral blood lymphocytes were investigated in 56 swine from 3 herds (I, breeding sows; II and III, fattening pigs). The mean frequencies of aberrant cells (AB.C.) were 3.58 +/- 1.59%, 2.10 +/- 1.52% and 6.20 +/- 3.21%, respectively. The mean numbers of SCEs per cell were 7.73 +/- 0.86, 6.51 +/- 0.89 and 7.06 +/- 1.47, respectively. A significant difference was found between the herds under study with regard to the number of aberrant cells but not the SCE frequency. In a parallel study, the presence of aflatoxin B1, polychlorinated biphenyls (PCB), dichlorodiphenyltrichloromethylmethane (DDT), lindane, mercury, lead and cadmium in the environment of fattening pigs was investigated. The total exposure to mutagens of pigs from herd III with a mean frequency of 6.2% AB.C., was markedly higher than that of herd II with the mean frequency of 2.1% AB.C.     

Comparison of Anterior Segment Biometric Measurements between Pentacam HR and IOLMaster in Normal and High Myopic Eyes

Purpose

To compare the anterior chamber depth (ACD), keratometry (K) and astigmatism measurements taken by IOLMaster and Pentacam HR in normal and high myopic (HM) eyes.

Design

A prospective observational case series.

Methods

Sixty-six normal eyes and 59 HM eyes underwent ACD, keratometry and astigmatism measurements with both devices. Axial length (AL) was measured on IOLMaster. The interdevice agreement was evaluated using the Bland-Altman analysis and paired t-test. The correlations between age and AL & ACD were analyzed. Vector analysis was used to compare astigmatism measurements.

Results

The ACD from IOLMaster and Pentacam HR was different for the normal group (P = 0.003) but not for the HM group (P = 0.280). IOLMaster demonstrated higher steep K and mean K values than Pentacam HR for both normal and HM groups (P<0.001 for all). IOLMaster also have higher flat K values for the HM groups (P<0.001) but were statistically equivalent with Pentacam HR for the normal group (P = 0.119) IOLMaster and Pentacam HR were different in astigmatism measurements for the normal group but were statistically equivalent for the HM group. For the normal group, age was negatively correlated with AL, IOLMaster ACD and Pentacam HR ACD (r = -0.395, P = 0.001; r = -0.715, P < 0.001; r = -0.643, P < 0.001). For the HM group, age was positively correlated with AL but negatively correlated with IOLMaster ACD and Pentacam HR ACD (r = 0.377, P = 0.003; r = -0.392, P = 0.002; r = -0.616, P < 0.001).

Conclusions

The IOLMaster and Pentacam HR have significant difference in corneal power measurements for both normal and HM groups. The two instruments also differ in ACD and astigmatism measurement for the normal group. Therefore, a single instrument is recommended for studying longitudinal changes in anterior segment biometric measurements. Age should be considered as an influencing factor for both AL and ACD values in the normal and HM group.     

Model investigations on the structure of the purple dye complex of Giemsa staining

Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18,100 cm-1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure. In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO4- and COO- -binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18,000 cm-1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18,100 cm-1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.     

Histone deacetylase inhibitor valproic acid inhibits proliferation and induces apoptosis in KM3 cells via downregulating VEGF receptor

The expression of vascular endothelial growth factor receptor 1(VEGFR-1) in human multiple myeloma KM3 cells in vitro, effects of valproic acid (VPA), as a histone deacetylase inhibitor, on cell proliferation and apoptosis and the underlying molecular mechanism were investigated. The effects of VPA on the growth of KM3 cells were studied by MTT assay. The apoptosis rate was determined with flow cytometry. The mRNA level of VEGFR was determined by RT-PCR; and immunocytochemistry was used to detect the protein level of ac-H4 and VEGFR. VPA inhibited proliferation of KM3 cells in a time- and dose-dependent manner. Treatment with VPA (4, 2, 1 and 0.5 mmol/L) for 48h, the apoptosis rates of KM3 cells were (13.27+/-3.54)%, (22.13+/-1.20)%, (24.41+/-2.23)% and(40.62+/-4.28)% respectively. The expression of VEGFR-1 in KM3 cells were decreased in VPA-treated group by the immunochemistry and RT-PCR, whereas the acetylated histone H4(ac-H4) accumulated. It suggested VPA could decrease the expression of VEGFR-1 in KM3 cells, and it might play an important role in regulating the proliferation and apoptosis of multiple myeloma cell line KM3 cells. These results provide the framework for clinical trials.