Colchicine inhibition of microtubule assembly via copolymer formation.

Colchicine.tubulin complex (CD) inhibits microtubule assembly. We examined this inhibition under conditions where spontaneous nucleation was suppressed and assembly was restricted to an elongation polymerization. We found that CD inhibited assembly by a mechanism which preserved the ability of microtubule ends to add tubulin. This observation is inconsistent with the end-poisoning model which recently was proposed as a general mechanism for assembly inhibition by CD. Our data are consistent with the following model: (a) microtubules formed in the presence of CD are CD-tubulin copolymers; (b) these copolymers can have appreciable numbers of incorporated CDs which are, most likely, randomly distributed in the copolymers; (c) CD-tubulin copolymers have assembly-competent ends with association and dissociation rate constants which decrease as the CD/tubulin ratio in the copolymers, (CD/T)MT, increases; and (d) the critical tubulin concentrations required for microtubule assembly increase in the presence of CD, indicating that copolymer affinity for tubulin decreases as (CD/T)MT increases.     

Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.     

Correlation between thyroid hormone status and hepatic hyperplasia and hypertrophy caused by the peroxisome proliferator-activated receptor alpha agonist Wy-14,643

BACKGROUND: The metabolic inhibitor rotenone inhibits hepatocellular proliferation and the incidence of liver cancer resulting from exposure to the PPARalpha agonist Wy-14,643, via unknown mechanisms. Since the absence of thyroid hormones diminishes hepatomegaly, an early biomarker for the hepatocarcinogenicity induced by PPARalpha agonists, this study was undertaken to investigate whether rotenone might interference with the ability of Wy-14,643 to alter the animal thyroid status. METHODS: Male B6C3F1 mice were given Wy-14,643 (100 ppm), rotenone (600 ppm) or a mixture of both, in the feed for 7 days. Bromodeoxyuridine (BrDU), marker of cell replication, was delivered through subcutaneously implanted osmotic mini-pumps. At the end of the experiment, sera were collected and corticosterone and thyroid hormone levels were measured by solid-phase radioimmunoassay kits. In addition, liver tissue samples were stained immunohistochemically for BrDU to determine percentages of labeled cells. Further, cell surface area was determined from images generated by a Zeiss Axioplan microscope equipped with a plan Neofluar x40 0.75 na objective. Tracings of individual hepatocyte perimeters were then analyzed and cell-surface areas were calculated using MicroMeasure FL-4000. RESULTS: Wy-14,643 caused a significant increase in liver weights, hepatocyte BrDU labeling index (LI), and hepatocyte surface area. In animals which received both Wy-14,643 and rotenone simultaneously, all of these effects were significantly less pronounced compared with mice that received Wy-14,643 alone. Rotenone alone decreased liver weights, LI and surface area. The Free Thyroid Index (FTI), which provides an accurate reflection of the animal's thyroid status, was 5.0 +/- 0.3 in control mice. In animals exposed to rotenone, these values decreased to 2.0 +/- 0.9, but in animals which received Wy-14,643, levels increased significantly to 7.7 +/- 0.9. FTI values decreased to 3.4 +/- 0.8 in mice receiving both rotenone and Wy-14,643. CONCLUSION: A strong correlation was observed between the animal thyroid status and both, hepatocyte proliferation (r2 = 0.62), and hepatocyte surface area (r2 = 0.83). These results support the hypothesis that the thyroid status of the animal plays a role in PPARalpha-induced hepatocellular proliferation and liver cell enlargement. Both these events are known to contribute to the expression of liver cancer in response to the activation of PPARalpha.     

Beta-adrenergic receptors are not responsible for exercise stimulation of glucose transport

This study was designed to examine whether the increased glucose transport after acute exercise in rat skeletal muscle is mediated via beta-adrenergic receptor stimulation. Sarcolemmal (SL) membranes were isolated from three groups: control (C), acute exercise (E), and exercise + propranolol (E+P). The acute exercise bout was performed on a treadmill and consisted of a 45-min run until near exhaustion. E+P received an intravenous propranolol injection (0.8 mg/kg) 10 min before the exercise session. Michaelis-Menten kinetics at 1.5 s indicated that the Vmax for glucose transport was increased after each perturbation compared with C but were not different from each other (E, 4,334 +/- 377; E+P, 4,824 +/- 357; and C, 1,366 +/- 124 pmol.mg protein-1.s-1). The apparent Km's were similar in all groups. Scatchard plots for the D-glucose inhibitable class of cytochalasin B binding sites indicated no differences in either the total number of binding sites in the SL vesicles (C, 5.5 +/- 0.3; E, 5.1 +/- 0.2, and E+P, 5.1 +/- 0.3 pmol/mg protein) or in their dissociation constant (Kd) (C, 46 +/- 3; E, 48 +/- 3; and E+P, 49 +/- 2 nM). The increase in Vmax for transport was similar between E and E+P, indicating that exercise does not stimulate glucose transport via the beta-adrenergic receptor.     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.     

Subapical Wall Synthesis and Wall Thickening Induced by Cycloheximide in Hyphae of Aspergillus nidulans

Hyphae of Aspergillus nidulans continued to synthesize all the major polysaccharide components of the cell wall when cycloheximide was added to cultures. In the presence of cycloheximide, hyphae did not elongate, but electron microscopy showed that the walls became thicker around the cell. The conclusion that cycloheximide changed wall synthesis from extension at the apex to subapical thickening was supported by grain distributions on radioautograms of mutant hyphae labeled with galactose. These findings are discussed in relation to the control of hyphal wall synthesis and are compared with the effects of protein synthesis inhibitors on wall formation in gram-positive bacteria.     

Nifedipine loaded chitosan microspheres: characterization of internal structure

In the present investigation, a simple technique was employed to obtain cross-sections of unloaded and nifedipine loaded chitosan microspheres. Microspheres, adhering to a polymerized resin block, were cut with an ultramicrotome and viewed with a scanning electron microscope. Unloaded microspheres exhibited a uniform dense matrix structure while crystals of nifedipine were clearly visible in the drug-loaded microspheres. At 2% drug loading, however, no crystals could be seen in the microspheres indicating that either the drug was molecularly dispersed or dissolved in the matrix at this concentration. This was confirmed by powder X-ray diffractometry studies where no peak due to crystalline nifedipine was observed. At high Span 85 concentration (1.5% w/v), the external surface of the microspheres collapsed, but the internal structure remained dense. When the drug was dispersed in the chitosan solution with stirring during preparation, the entrapment was good and the shape of the crystals was changed. The internal structure of the microspheres following dissolution exhibited the presence of pores.     

土壤低剂量芘污染对蚯蚓若干生化指标的影响

通过人工污染土壤的方法,设计芘的暴露浓度为0、60、120、240、480、960μg.kg-1.暴露实验进行1、3、7和14d后,分别检测蚯蚓内脏中细胞色素P450含量、谷胱甘肽转移酶(GST)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量.结果表明,在供试浓度范围内,蚯蚓内脏中各生化指标对污染物暴露指示的敏感性存在差异:其中P450含量、GST和SOD活性最为敏感;POD和CAT活性次之;而MDA含量未对低剂量的芘暴露起到明显的指示作用.研究同时发现,低剂量污染物暴露的时间效应要强于剂量效应的影响.因而,在进行生态毒性诊断时,采用多指标和多时段的检测对增强指示的灵敏性和有效性尤为重要.     

Loss of meiosis in Aspergillus

If strictly mitotic asexual fungi lack recombination, the conventional view predicts that they are recent derivatives from older meiotic lineages. We tested this by inferring phylogenetic relationships among closely related meiotic and strictly mitotic taxa with Aspergillus conidial (mitotic) states. Phylogenies were constructed by using DNA sequences from the mitochondrial small ribosomal subunit, the nuclear ribosomal internal transcribed spacers, and the nuclear 5.8S ribosomal gene. Over 920 bp of sequence was analyzed for each taxon. Phylogenetic analysis of both the mitochondrial and nuclear data sets showed at least four clades that possess both meiotic and strictly mitotic taxa. These results support the hypothesis that strictly mitotic lineages arise frequently from more ancient meiotic lineages with Aspergillus conidial states. Many of the strictly mitotic species examined retained characters that may be vestiges of a meiotic state, including the production of sclerotia, sclerotium-like structures, and hulle cells.