Adsorption of mutagens to cotton bearing covalently bound trisulfo-copper-phthalocyanine

A method for separating nonpolar mutagens from their dilute aqueous solutions is described. It utilizes the affinity of the mutagens to a phthalocyanine derivative attached to cotton through a covalent bond. For mutagens having 3 or more fused aromatic rings in their structures, efficient adsorption took place on soaking the cotton in their solutions. The mutagens adsorbed can be recovered by elution with ammoniacal methanol. Mutagenicity in smoker's urine, cooked beef, and river water was detected by use of this method.     

Establishing a new genus,Chiharadinium gen. nov. (Peridiniales,Dinophyceae) for a tidal pool dinoflagellate formerly known as Scrippsiella hexapraecingula

To determine its accurate taxonomic position, a tidal pool bloom-forming dinoflagellate, Scrippsiella hexapraecingula was re-investigated using light, scanning and transmission electron microscopy together with a phylogenetic analysis based on concatenated ribosomal DNA sequences. The culture strains used in this study were established from intertidal rock pool samples taken from Jogashima, Kanagawa prefecture and Heisaura, Chiba prefecture, Japan and were identified as S. hexapraecingula originally described by Horiguchi and Chihara from a tidal pool in Hachijo Island, Tokyo, Japan in 1983. The thecal plate arrangement was determined as Po, X, 4′, 3a, 6″, 6c, 5s, 5″′, 2″″. The internal structure was investigated for the first time. The organism has typical dinoflagellate cellular organelles such as a dinokaryotic nucleus, mitochondria with tubular cristae, trichocysts and pusule. The chloroplast was single and connected to the central pyrenoid (stalked type). The eyespot found in the sulcus is of the B type with two rows of superficial intraplastidal lipid globules directly overlain by an extraplastidal single layer of crystalline bricks enveloped by a common membrane. The apical pore is plugged by a double-layered stub-like structure. Stalk building material for attachment covered the apical pore. Phylogenetic analysis indicated that S. hexapraecingula was most closely related to a freshwater dinoflagellate, Peridiniopsis borgei, the type species of the genus Peridiniopsis. However, clear differences exist between these two organisms, including their thecal plate arrangement, habitat and habit. As a result, a new genus, Chiharadinium Dawut & T. Horiguchi gen. nov. has been proposed rather than attempting to accommodate S. hexapraecingula in the genus Peridiniopsis. The new combination, Chiharadinium hexapraecingulum (T. Horiguchi & Chihara) Dawut & T. Horiguchi comb. nov. has been proposed.     

The micronucleus test using peripheral blood reticulocytes from methotrexate-treated mice.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.     

The Epidemiology of HIV and HSV-2 Infections among Women Participating in Microbicide and Vaccine Feasibility Studies in Northern Tanzania

Objectives

To prepare for future HIV prevention trials, we conducted prospective cohort studies among women working in food and recreational facilities in northern Tanzania. We examined the prevalence and incidence of HIV and HSV-2, and associated risk factors.

Methods

Women aged 18–44 years working in food and recreational facilities were screened to determine their eligibility for the studies. Between 2008–2010, HIV-negative women were enrolled and followed for 12 months. At enrolment and 3-monthly, we collected socio-demographic and behavioural data, and performed clinical examinations for collection of biological specimens that were tested for reproductive tract infections. Risk factors for HIV and HSV-2 incidence were investigated using Poisson regression models.

Results

We screened 2,229 and enrolled 1,378 women. The median age was 27 years (interquartile range, IQR 22, 33), and median duration working at current facility was 2 years. The prevalences of HIV at screening and HSV-2 at enrolment were 16% and 67%, respectively. Attendance at the 12-month visit was 86%. HIV and HSV-2 incidence rates were 3.7 (95% confidence interval, CI: 2.8,5.1) and 28.6 (95% CI: 23.5,35.0)/100 person-years, respectively. Women who were separated, divorced, or widowed were at increased risk of HIV (adjusted incidence rate ratio, aRR = 6.63; 95% CI: 1.97,22.2) and HSV-2 (aRR = 2.00; 95% CI: 1.15,3.47) compared with married women. Women reporting ≥3 partners in the past 3 months were at higher HIV risk compared with women with 0–1 partner (aRR = 4.75; 95% CI: 2.10,10.8), while those who had reached secondary education or above were at lower risk of HSV-2 compared with women with incomplete primary education (aRR = 0.42; 95% CI: 0.22,0.82).

Conclusions

HIV and HSV-2 rates remain substantially higher in this cohort than in the general population, indicating urgent need for effective interventions. These studies demonstrate the feasibility of conducting trials to test new interventions in this highly-mobile population.     

Abstracts of papers read at the Winter Meeting, 1961

Herdmania litoralis is a heterotrophic, sand-dwelling dinoflagellate with morphological characters that do not provide clear evidence for its systematic position in any existing family of dinoflagellates. Protoperidinium minutum is a heterotrophic, planktonic species that has a typical tabulation for the genus Protoperidinium. In order to infer the phylogenetic positions of these two species more confidently, we characterized the thecal plate patterns and determined small-subunit and large-subunit ribosomal DNA sequences (SSU rDNA and LSU rDNA, respectively) from both species. Intraindividual and intraspecific diversity of SSU and LSU rDNA data were characterized in H. litoralis using a combination of single-cell PCR approaches and analyses of PCR clones derived from multi-cell DNA extractions. The results of the molecular phylogenetic analyses demonstrated a novel, well-supported clade comprising both sand-dwelling species (H. litoralis and Thecadinium dragescoi) and planktonic species (P. minutum). Because the establishment of this clade also demonstrated that P. minutum is not a member of Protoperidinium, we reinstated and emended the genus Archaeperidinium Jörgensen 1912 Jörgensen, E. 1912. Bericht über die von der schwedischen hydrographisch-biologischen Kommission in den schwedischen Gewässern in den Jahren 1909–10 eingesammelten Planktonproben. Svenska Hydrograph.-Biol. Komm. Skr., 4: 1–20.  [Google Scholar].     

Bisorbicillinol inhibits Lyn tyrosine kinase for allergic response on RBL-2H3 cells

Bisorbicillinol, which is isolated from Trichoderma sp. USF2690, is an inhibitor of β-hexosaminidase release and tumor necrosis factor (TNF)-α, and Interleukin (IL)-4 secretion from rat basophilic leukemia (RBL-2H3) cells, with IC₅₀ values of 2.8?μM, 2.9?μM and 2.8?μM respectively. We showed that the inhibitory mechanism of β-hexosaminidase release and TNF-α secretion involved inhibition of Lyn, a tyrosine kinase. The inhibitory activities of bisorbicillinol indicate that this compound is a new candidate anti-allergic agent.     

Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Report from the in vitro micronucleus assay working group

At the Washington "2nd International Workshop on Genotoxicity Testing" (25-26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth "3rd International Workshop on Genotoxicity Testing" (28-29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern: 1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test. 2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information. 3. Treatment schedules for cell lines and lymphocytes. 4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly). 5. Duplicate cultures and number of cells to be scored. 6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative. 7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.