Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.     

Comparative patterns of synthesis of polyadenylated and nonpolyadenylated RNA in various brain regions following intraventricular administration of 3H-uridine into adult rats

Measurements of populations of unlabeled RNA indicate that the absolute concentrations and relative proportions of poly(A)-RNA and of nonpoly(A)-RNA, relative to total cellular RNA are similar in three brain regions. The incorporation of ³H-uridine into poly(A)-RNA and nonpoly(A)-RNA was measured in cerebrum, diencephalon, and midbrain-hindbrain from 15 min through 8.0 hr after intraventricular injection of the precursor into adult rat brains. Incorporation of ³H-uridine into poly(A)-RNA was very rapid and reached maximum levels of specific activity within 30 to 120 minutes, depending upon locus, after injection of the precursor. The specific activity of nonpoly(A)-RNA increased with time, but remained lower than that of poly(A)-RNA throughout the 8.0 hr period. Regionally differential synthesis occurred both in poly(A)-RNA and nonpoly(A)-RNA in the several brain regions. Establishment of the time kinetics of brain RNA synthesis should provide useful basis for selection of the conditions for labeling pulses for further studies of $\text{in vivo}$ RNA metabolism.     

Protein biosynthetic capacity in the endosperm tissue of ripening castor bean seeds

Endosperm tissue was excised from Ricinus communis plants at different stages during seed maturation. The various stages were characterized on the basis of total RNA, protein and lipid content. Polyadenylated RNA was recovered from the total RNA by affinity chromatography on oligo(dT)cellulose. With the exception of that isolated from dry seeds, this poly(A⁺) RNA actively programmed protein synthesis in cell-free systems containing either wheat germ S30 extracts or nuclease treated rabbit reticulocyte lysates at each developmental stage examined. Translational products were separated electrophoretically and were visualized by fluorography. The capacity to synthesize protein was also estimated during in vivo labelling studies. Developmental changes in the capacity of maturing endosperm tissue to synthesize a characteristic protein, R. communis agglutinin, were followed by immunoprecipitating this protein from the total in vitro products synthesized at various stages. Endoplasmic reticulum membranes were isolated from maturing endosperm tissue by sucrose density gradient centrifugation. The role of the endoplasmic reticulum (ER) in protein glycosylation was indicated by (a) localizing the enzymes catalysing the incorporation of N-acetylglucosamine and mannose into mono- and oligosaccharide lipid and into glycoprotein, (b) localizing particulate ³H-labelled glycoprotein amongst cellular fractions prepared from endosperm tissue which had been incubated with [³H]N-acetylglucosamine.Abbreviations polyCA⁺)RNA polyadenylated RNA - SDS sodiumdodecylsulphate - TCA trichloroacetic acid - RCA₁₂₀ Ricinus communis agglutinin, type I - NP40 Nonidet P40 - PMSF phenylmethylsulphonyl fluoride     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, ¹H and ¹³C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. ¹³C‐isotopomer analysis revealed decreased de novo synthesis of [4–¹³C]glutamate (30%p < 0.02) and [2–¹³C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–¹³C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].     

DIFFERENTIAL METABOLISM OF RNA IN NEURONAL-ENRICHED AND GLIAL-ENRICHED FRACTIONS OF RAT CEREBRUM

Cerebral tissue of rat, disrupted by passage through a custom-designed tissue press, was diluted to a 10% (w/v) cellular suspension in 10% (w/v) Ficoll and was then fractionated in the Spinco Model L ultracentrifuge into: (1) two enriched cellular layers (alpha and beta) in an isopycnic Ficoll gradient in the Spinco 40 angular rotor; or (2) into four layers (A, B, C, and D) in a discontinuous Ficoll gradient in the Spinco SW 39 swinging bucket rotor. The cellular composition of these layers was identified microscopically and enzymically as either glial-enriched (alpha and B layers) or neuronal-enriched (beta and C layers) fractions of cerebral cortex. Portions of the neuronal and glial fractions were used for determinations of total nitrogen, and for colorimetric determinations of carbonic anhydrase activity (a glial cell marker). These data established that glial contamination of the neuronal-enriched layer averaged 6·5 per cent. The data also indicated glial enrichment of Layer B, although no quantitative assessment of the amount of neuronal contamination was possible. The kinetics of metabolism of RNA in the glial-enriched and neuronal-enriched fractions were studied from 0·5 to 16 h after-intracisternal injection of either [³H]cytidine or [³H]orotic acid. In addition, the incorporation of [³H]cytidine into crude nuclear and cytoplasmic components of the layers was studied by the use of 1 h pulses. Our findings indicated greater incorporation of [³H]cytidine into nuclear fractions than into cytoplasmic fractions at 1 h and greater incorporation of both precursors into neuronal-enriched fractions than into glial-enriched fractions at all pulse times.     

Comparative studies of zinc metabolism in cultured chinese hamster cells with differing metallothionein-induction capacities

Previous work in our laboratory led to the isolation of a cadmium (Cd)-resistant variant (Cd^r2C10) of the line CHO Chinese Hamster cell having a 10-fold greater resistance to the cytotoxic action of Cd²⁺ compared with the CHO cell. This resistance was attributed to an increased capacity of the Cd²⁺-resistant Cd^r2C10 subline to induce synthesis of the Cd²⁺- and Zn²⁺-binding protein(s), metallothionein(s) (MT). Evidence that Cd²⁺ behaves as an analog of the essential trace metal, Zn²⁺, especially as an inducer of MT synthesis, suggested that the Cd^r and CHO cell types could be employed to investigate cellular Zn²⁺ metabolism. In the present study, measurements were made to compare CHO and Cd^r cell types for (a) growth as a function of the level of ZnCl₂ added to the culture medium, (b) uptake and subcellular distribution of Zn²⁺, and (c) capacity to induce MT synthesis. The results of these measurements indicated that (a) both CHO and Cd^r cell types grew normally (T _d≊16–18 h) during exposures to Zn²⁺ at levels up to 100 μM added to the growth medium, but displayed abrupt growth inhibition at higher Zn²⁺ levels, (b) Cd^r cells incorporate fourfold more Zn²⁺ during a 24-h exposure to the maximal subtoxic level of Zn²⁺ and (c) the CHO cell lacks the capacity to induce MT synethesis while the Cd^r cell is proficient in this response during exposure to the maximal subtoxic Zn²⁺ level. These findings suggest that (a) the CHO and Cd^r cell systems will be useful in further studies of cellular Zn²⁺ metabolism, especially in comparisons of Zn²⁺ metabolism in the presence and absence of induction of the Zn²⁺-sequestering MT and (b) a relationship exists between cellular capacity to induce MT synthesis and capacity for cellular Zn²⁺ uptake.     

EFFECT OF DIMETHYL SULFOXIDE ON ZINC65 UPTAKE,RESPIRATION, AND RNA AND PROTEIN METABOLISM IN BEAN (PHASEOLUS VULGARIS) TISSUES

The effect of dimethyl sulfoxide (DMSO) on zinc⁶⁵ uptake, respiration, RNA, and protein metabolism in various tissues of two bean (Phaseolus vulgaris L.) cultivars showing differential growth responses to zinc has been studied. At a concentration of 1%, DMSO stimulated zinc uptake in excised roots, stem-callus tissue, leaf disks, and enzymically isolated leaf cells, but did not significantly alter the uptake and incorporation of C¹⁴-uracil into RNA and C¹⁴-methionine into protein, although a slight inhibition was discernible in some tissues. At a higher concentration (10%) DMSO increased Zn⁶⁵ uptake in excise roots incubated for 2 hr; however, at the same concentration, C¹⁴-uracil and C¹⁴-methionine uptake and incorporation were considerably inhibited in all the tissues. Oxygen uptake as measured with Warburg manometers was impaired, and the inhibition showed a time and concentration dependency. The fact that DMSO inhibited respiration and RNA and protein metabolism, while at the same concentration zinc uptake was increased, suggests that zinc uptake in beans is primarily a non-metabolic process. The possible mechanisms of DMSO action are discussed in the light of its reported effects on membrane permeability and cell metabolism.     

In vitro Methionine<Superscript>5</Superscript>-Enkephalin Degradation Kinetics by Human Brain Preparations

Incubation of [³H]-tyrosine methionine⁵-enkephalin (MET) with human brain preparations (100,000g supernatant; sections of the limbic system, thalamus, basal ganglia, cerebellum, and cortex) results in its rapid and complete degradation; over 95% of the initial labeled tyrosine is recovered as the free aminoacid within 10 min. Results show a considerable range in the peptide initial velocity (Iv) and half-life (t_1/2) degradation values obtained from different brain sections of individual brains, either from the same or from different main brain areas. This relatively wide range of values was scattered, failing to identify consistent differences between the various brains areas studied. Differences in brain tissue storage time or repeated sample freezing and thawing failed to alter significantly either of these kinetic parameters of MET metabolism. Peptide degradation rate (optimum pH and temperature of 7.4 and 37°C, respectively) was concentration-dependent inhibited by known aminopeptidase inhibitors (puromycin, bacitracin, and bestatin, and to a lesser extent by thioridazine). However, it was not significantly affected by either N-carboxymethyl phenyl leucine, captopril or thiorphan [dipeptidyl peptidase(s) or peptidyl dipeptidase(s) inhibitors, respectively]. A better understanding of the mechanisms regulating brain MET metabolism may contribute to the rational design of pharmacological strategies based in the modulation of its bioavailability.     

A MODIFIED PROCEDURE FOR ISOLATION OF ASTROCYTE- AND NEURON-ENRICHED FRACTIONS FROM RAT BRAIN

Our previously published method for isolation of neurons with extensive processes (Farooq et al., 1977) has been modified to permit the isolation of both astrocyte- and neuron-enriched fractions. Rat cerebral tissue is incubated with acetylated trypsin and disrupted. The cell suspension is separated first by differential centrifugation and then by gradient centrifugation on discontinuous Ficoll gradients. The method is reproducible and is applicable equally well to immature and adult animals. The yield of astrocytes of 57% particle purity, and higher weight purity, is 4–7 × 10⁶ cells/brain, amounting to 1.5–2.0 mg of protein. The astrocytes appear to be a mixture of fibrous and protoplasmic types. The yield of neurons of 90% particle purity is 10–14 × 10⁶ cells/brain, amounting to 2.4–3.0 mg of protein. A total yield of neurons of 28–37 × 10⁶ cells/brain can be obtained at 70% purity. These preparations have been characterized by light microscopy and protein, RNA and DNA content.     

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity ^1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection ³. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop ^4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.The principle and protocol of this methodology have been described in the literature ^7,8. Additionally, alternate methodologies to isolate primary microglia are well described ^9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia ¹². However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture ^9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.     

An enriched preparation of basal-lateral plasma membranes from gastric glandular cells

Summary A procedure is described for the preparation of a membrane fraction enriched in basal-lateral plasma membranes from gastric mucosa. Gastric glands isolated from rabbit were employed as starting material, greatly reducing contamination from nonglandular cell types. The distribution of cellular components during the fractionation procedure was monitored with specific marker enzymes. (Na⁺+K⁺)-ATPase, ouabain-sensitive K⁺-stimulatedp-nitrophenyl-phosphatase and histamine-stimulated adenylate cyclase were used as markers for basal-lateral membranes. These three markers were similarly distributed during both differential and equilibrium density gradient centrifugation. The enriched membrane fraction contained more than 30% of the total initial activities of the three basal-lateral membrane markers which were purified better than 11-fold with respect to protein. (Na⁺+K⁺)-ATPase activity was resolved from the activities of acid phosphatase, pepsin, Mg²⁺-ATPase, cytochromec oxidase, NADPH-cytochromec reductase, glucose-6-phosphatase, (K⁺+H⁺)-ATPase, DNA and RNA.     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

Chromosomal organization of Drosophila tumours

In otu mutants of Drosophila melanogaster ovarian tumours develop because of the high mitotic activity of the mutant cystocytes; the latter are normally endopolyploid. In certain alleles of otu, however, a varying proportion of the mutant ovarian cystocytes undergo polyteny. Mutant cystocytes with polytene chromosomes are termed pseudonurse cells (PNC). Polytene chromosome morphology and banding patterns in PNC of otu ¹/otu³ flies were cytologically analysed. Extensive variability was noted in the quality of the banding pattern of the PNC chromosomes which ranged from highly condensed (condensed PNC chromosomes) to those with a banding pattern (banded PNC chromosomes) similar to that in larval salivary gland cells (SGC). Both the condensed and banded PNC chromosomes frequently enter into a diffuse state characterised by weakened synapsis of the polytene chromatids and alterations in their banding pattern (diffuse PNC chromosomes). Analysis of DNA synthesis patterns in the various morphological forms of PNC polytene chromosomes by ³H-thymidine autoradiography revealed a basic similarity to the pattern seen in polytene nuclei of larval SGC. Independently replicating sites, however, could be unambiguously identified only in banded PNC chromosomes. Comparison of late replicating sites in such PNC chromosomes with those of larval SGC showed a remarkable similarity in the two cell types. These results suggest a close correlation between the polytene chromosome banding pattern and its replicative organization.     

Gene expression analysis of in vivo fluorescent cells

Background

The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.

Methodology/Principal Findings

We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).

Conclusions/Significance

Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.     

Turnover of O-Glucosides of Dihydrozeatin and Dihydrozeatin-9-riboside During the Cell Growth Cycle of Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum

The cellular levels of O-glucosides of ³H-(diH)Z and ³H-(diH)[9R]Z, the major short-term metabolites of ³H-(diH)Z having been exogenously supplied to photoautotrophically growing suspension cell cultures of Chenopodium rubrum, decreased significantly during further culture, irrespective of whether the cells were maintained in the stationary phase or were transferred to conditions restoring cell divison. Metabolism of both compounds was more pronounced during the active growth phase than during the stationary phase. The O-glucosides were converted preferentially to polar compounds of as yet unknown nature, which were partly excreted into the medium. The cellular pools of both glycosides remained compartmented within the vacuole. In contrast to the O-glycosides, the small cellular pools of the aglycones ³H-(diH)Z and ³H-(diH)[9R]Z maintained their level during the experimental period of 30 days. Small amounts of the glucosides, as well as of the aglycones, were recovered from the medium and could have resulted from the lysis of a few cells. The results demonstrate, for the first time, that O-glucosides of cytokinins are not irreversibly deposited within the vacuole of plant cells but may serve to maintain a small, but more or less constant pool of extra-vacuolar, presumably cytosolic, aglycones. (DiH)Z and its derivatives could be demonstrated to be endogenous cytokinins of Chenopodium rubrum suspension cultured cells occurring along with those of the isopentenyladenine and zeatin types.     

Morphogenesis of albino rat lung: an autoradiographic analysis of the embryological origin of the type I and II pulmonary epithelial cells

A correlation of autoradiographic and histochemical data indicates that the type I and II pulmonary epithelial cells are endodermally-derived; and, that the interstitial pulmonary cells are mesodermally-derived. Tritiated thymidine (T-H³) was found to be an excellent cell marker for in vivo developmental studies of mammalian (rat) lung. At a dose of 3 μc per gm (specific activity, 15.6–16.9 c per mM) maternal body weight, T-H³ crosses the placenta in amounts sufficient to effect heavy labeling of dividing cells. A partial placental barrier to T-H³ was found in late stages of development. Following an injection of T-H³ on day 16 of gestation, a higher rate of endodermal cell division was reflected by higher labeling indices and a steeper slope of the endodermal dilution curve as opposed to the mesoderm. This differential in labeling was maintained through the third postnatal day. Neonatal labeling patterns of the definitive cell types (type I and II pulmonary epithelial cells, interstitial pulmonary cells) reflected those of their germ layer precursors. Histochemical analysis of the developing rat lung demonstrated large accumulations of cytoplasmic glycogen in areas of rapid cell division (endodermal cells). As the mitotic rate decreased and cellular differentiation progressed, glycogen decreased; postnatally it is not a feature of mature pulmonary cell types.