Multiple actins in drosophila melanogaster

The tissue and developmental specificities of the three Drosophila isoactins, originally identified in primary myogenic cultures and in the permanent Schneider L-2 cell line, have been investigated. Of these three isoactins (I, II, and III), actins I and II are stable and actin III is unstable. Two-dimensional polyacrylamide gel electrophoretic analyses of total cellular extracts after 1-h [(35)S]methionine pulses were performed on a large variety of embryonic, larval, and adult muscle and nonmuscle tissues. The results suggest that isoactins II and III are generalized cellular actins found in all drosophila cell types. Actin I, on the other hand, is muscle-associated and is found exclusively in supercontractile muscle (such as larval body wall and larval and adult viscera) including primary myogenic cell cultures. Although actin I synthesis is not detectable during very early embryogenesis, it is detectable by 25 h and actin I is a major stable actin in all larval muscle tissues. Actin I is synthesized in reduced amounts relative to the other actins in late third instar larvae but is again a major product of actin synthesis in the adult abdomen. A stable actin species with the same pI as actin III has been identified in the adult thorax and appears to be unique to flight muscle tissue. This new stable form of thoracic actin may be the result of a stabilization of the actin III found in other tissues or may be an entirely separate gene product.     

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.     

Effect of fruit maturity,seed weight and storage time on the viability and germination of the seed of candelilla (<i>Euphorbia antisiphylitica</i> Zucc.)

Candelilla (Euphorbia antisiphylitica Zucc.) is a very important plant resource in the arid lands of Northern Mexico. This is because the wax content coating the stem has unique properties which have been useful for multiple applications in the food industry, electronics, cosmetics, etc. However, the intensive exploitation of this resource has caused a great decrease in the populations of this species making necessary to consider strategies for their conservation and sustainable use. One of the primary needs with regeneration purposes is to know their reproductive processes, particularly the biotic and/or abiotic factors that determine the viability and germination of seeds. The present study evaluated the (1) germination and seed viability in relation to the ripeness degree of the fruit at the time of collection, (2) weight of the seed (low, medium and high), and (3) storage time (1, 3, and 5 months). Fruits from four locations, two in the State of Coahuila (Las Coloradas and Candela) and two in the State of Nuevo Leon (Icamole 1 and Icamole 2), were collected. Three germination assays were carried out corresponding to each month of storage. Seed viability was determined by the tetrazolium test. The average weight of the candelilla seeds was 0.0029 ± 0.0010 g, with extreme average values of 0.0018 ± 0.0006 g at Las Coloradas and 0.0036 ± 0.0010 g in Icamole 2. Those seeds with heavier weight obtained from red fruits and with 1 month of storage showed the highest average percentage of viability (66.87 ± 24.19%). At the same time, seeds with around average weight, obtained from red fruits and five months of storage, showed the highest average germination percentage (50.00 ± 9.42%).     

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.     

Fat malabsorption in essential fatty acid-deficient mice is not due to impaired bile formation

Essential fatty acid (EFA) deficiency induces fat malabsorption, but the pathophysiological mechanism is unknown. Bile salts (BS) and EFA-rich biliary phospholipids affect dietary fat solubilization and chylomicron formation, respectively. We investigated whether altered biliary BS and/or phospholipid secretion mediate EFA deficiency-induced fat malabsorption in mice. Free virus breed (FVB) mice received EFA-containing (EFA(+)) or EFA-deficient (EFA(-)) chow for 8 wk. Subsequently, fat absorption, bile flow, and bile composition were determined. Identical dietary experiments were performed in multidrug resistance gene-2-deficient [Mdr2((-/-))] mice, secreting phospholipid-free bile. After 8 wk, EFA(-)-fed wild-type [Mdr2((+/+))] and Mdr2((-/-)) mice were markedly EFA deficient [plasma triene (20:3n-9)-to-tetraene (20:4n-6) ratio >0.2]. Fat absorption decreased (70.1 +/- 4.2 vs. 99.1 +/- 0.3%, P < 0.001), but bile flow and biliary BS secretion increased in EFA(-) mice compared with EFA(+) controls (4.87 +/- 0.36 vs. 2.87 +/- 0.29 microl x min(-1) x 100 g body wt(-1), P < 0.001, and 252 +/- 30 vs. 145 +/- 20 nmol x min(-1) x 100 g body wt(-1), P < 0.001, respectively). BS composition was similar in EFA(+)- and EFA(-)-fed mice. Similar to EFA(-) Mdr2((+/+)) mice, EFA(-) Mdr2((-/-)) mice developed fat malabsorption associated with twofold increase in bile flow and BS secretion. Fat malabsorption in EFA(-) mice is not due to impaired biliary BS or phospholipid secretion. We hypothesize that EFA deficiency affects intracellular processing of dietary fat by enterocytes.     

Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons,astrocytes, oligodendrocytes and microglial cells

NADP+-dependent isocitrate dehydrogenases (ICDHs) are enzymes that reduce NADP+ to NADPH using isocitrate as electron donor. Cytosolic and mitochondrial isoforms of ICDH have been described. Little is known on the expression of ICDHs in brain cells. We have cloned the rat mitochondrial ICDH (mICDH) in order to obtain the sequence information necessary to study the expression of ICDHs in brain cells by RT-PCR. The cDNA sequence of rat mICDH was highly homologous to that of mICDH cDNAs from other species. By RT-PCR the presence of mRNAs for both the cytosolic and the mitochondrial ICDHs was demonstrated for cultured rat neurons, astrocytes, oligodendrocytes and microglia. The expression of both ICDH isoenzymes was confirmed by western blot analysis using ICDH-isoenzyme specific antibodies as well as by determination of ICDH activities in cytosolic and mitochondrial fractions of the neural cell cultures. In astroglial and microglial cultures, the total ICDH activity was almost equally distributed between cytosolic and mitochondrial fractions. In contrast, in cultures of neurons and oligodendrocytes about 75% of total ICDH activity was present in the cytosolic fractions. Putative functions of ICDHs in cytosol and mitochondria of brain cells are discussed.     

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.     

Improved Multiple Displacement Amplification (iMDA) and Ultraclean Reagents

Background

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).

Results

A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.

Conclusions

The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.     

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.