Tissues Of Intact Animals Research Articles

Protein turnover: Measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.

Changes in fatty acid composition of thymus cells, liver, blood plasma, and muscle tissue in mice with solid Ehrlich carcinoma

The fatty acid composition of thymus cells, liver, blood plasma, muscle tissue, and tumor focus has been studied in mice with solid Ehrlich carcinoma. The tumor growth in the mice was associated with an increase in the total content of monounsaturated fatty acids in all organs and tissues studied and with a decrease in the total amount of polyunsaturated fatty acids in all tissues except blood plasma. The tumor tissue was characterized by increased levels of monounsaturated fatty acids in comparison with their levels in organs and tissues of intact animals. In the thymus of tumor-bearing mice, the contents of myristic and palmitic saturated fatty acids, which are associated with activation of the T-cell immunity, were increased. The most expressed and considerable changes in the fatty acid composition during tumor growth were observed in the muscle tissue of the animals. A possible role of changes in the fatty acid composition in the investigated organs and tissues of tumor-bearing mice in the organism's response to tumor growth is discussed.

Changes in Tissue Metalloproteinase Inhibitor-1 and Matrix Metalloproteinases during Tumor Development and Metastasizing in Mice

Tissue inhibitor of matrix metalloproteinases type 1, inhibiting the majority of matrix metalloproteinases, can both suppress and stimulate tumor growth. The concentrations and activities of tissue matrix metalloproteinase inhibitor-1 were measured in C57Bl/6 mice during progression and metastasizing of Lewis lung adenocarcinoma. Activities of matrix metalloproteinases in tumor tissue of mice were lower than in liver and lung tissues of intact animals. Serum concentration of tissue inhibitor increased significantly during the development of Lewis lung adenocarcinoma. Macrophage depression (injection of gadolinium chloride associated with a decrease in metastasis number) decreased serum concentration of tissue inhibitor, but it did not attain the control level observed in intact mice. These findings attest to a pleiotropic antitumor effect of tissue matrix metalloproteinase inhibitor-1 reflecting disorders in matrix metalloproteinase regulation during the progress of Lewis lung adenocarcinoma in mice.

Interrogating 7TM receptors: Does texture in the question yield greater texture in the answer?

The key to detecting and classifying drug effect at seven transmembrane (7TM) receptors is the pharmacological assay. Drug discovery had been rooted in testing of molecules on intact animal tissue until technology provided high-throughput binding assays for screening. While this allowed for the testing of large numbers of molecules, it also limited detection to molecules that interfere with the interaction of the receptor with a defined probe (i.e., radioligand). The ability to monitor functional changes in cells (recombinant or natural) provided a huge leap forward. Earlier functional assays were tied to specific signaling pathways (i.e., cyclic AMP and calcium) but now label-free assays in live cells provide the opportunity to detect more ligands and more fully characterize their efficacy. These ideas will be discussed in terms of harnessing the phenomenon of “functional selectivity” for therapeutic advantage.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.

Bioprobe‐assisted In Vivo dissection of molecular signaling by Rho GTPases in developing neurons

Knowing when and where a given signaling protein is normally activated within individual cells and tissues of intact animals permits elucidating its in vivo functions. However, tools for non‐invasive and quantitative visualization are lacking. Here, using genetically encoded A‐probe (activation bioprobe) designed for Cdc42 GTPase, I show that Drosophila embryos maintain Cdc42 activities at low levels until hour 15:00 of embryogenesis. Within the nervous system where Cdc42 activity first becomes upregulated, individual neurons display precise spatiotemporal patterns of activation restricted to specific ~10 µm compartments. At both organismal and cellular levels, Cdc42's endogenous activation patterns in wild type allow predictions of where loss‐of‐function phenotypes emerge in cdc42/cdc42 mutants. Genetic tests within neurons further support the importance of suppressing endogenous Cdc42 activities until needed in particular compartments. Thus, bioprobe‐assisted analyses uncover how potent, ubiquitously expressed signaling proteins control normal cellular events through intricate regulation of their activities within an intact animal.

Two-photon Imaging of Glutathione Levels in Intact Brain Indicates Enhanced Redox Buffering in Developing Neurons and Cells at the Cerebrospinal Fluid and Blood-Brain Interface

Glutathione is the major cellular thiol present in mammalian cells and is critical for maintenance of redox homeostasis. However, current assay systems for glutathione lack application to intact animal tissues. To map the levels of glutathione in intact brain with cellular resolution (acute tissue slices and live animals), we have used two-photon imaging of monochlorobimane fluorescence, a selective enzyme-mediated marker for reduced glutathione. Previously, in vitro experiments using purified components and cultured glial cells attributed cellular monochlorobimane fluorescence to a glutathione S-transferase-dependent reaction with GSH. Our results indicate that cells at the cerebrospinal fluid or blood-brain interface, such as lateral ventricle ependymal cells (2.73 +/- 0.56 mm; glutathione), meningeal cells (1.45 +/- 0.09 mm), and astroglia (0.91 +/- 0.08 mm), contain high levels of glutathione. In comparison, layer II cortical neurons contained 20% (0.21 +/- 0.02 mm) the glutathione content of nearby astrocytes. Neuronal glutathione labeling increased 250% by the addition of the cell-permeable glutathione precursor N-acetylcysteine indicating that the monochlorobimane level or glutathione S-transferase activity within neurons was not limiting. Regional mapping showed that glutathione was highest in cells lining the lateral ventricles, specifically ependymal cells and the subventricular zone, suggesting a possible function for glutathione in oxidant homeostasis of developing neuronal progenitors. Consistently, developing neurons in the subgranular zone of dentate gyrus contained 3-fold more glutathione than older neurons found in the neighboring granular layer. In conclusion, mapping of glutathione levels in intact brain demonstrates a unique role for enhanced redox potential in developing neurons and cells at the cerebrospinal fluid and blood-brain interface.

5‐Hydroxytryptamine Receptor Assays

AbstractThis unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues. In total, nine in vitro bioassays are described for 5‐HT1A, 5‐HT1B, 5‐HT1D, 5‐HT2A, 5‐HT2B, 5‐HT2C, 5‐HT3, 5‐HT4, and 5‐HT7 receptors in isolated intact guinea pig, rat and rabbit tissues. The 5‐HT1A, 5‐HT3, and 5‐HT7 assays use ileum preparations, whereas, the 5‐HT1B, 5‐HT1D, 5‐HT2A and 5‐HT2C receptor assays are all based on vascular preparations. The 5‐HT2B receptor is best studied in stomach fundic muscle and the 5‐HT4 receptor in esophageal tissue. To date, no functional isolated organ bioassays are available for the 5‐ht1e, 5‐ht1f, 5‐ht5 or 5‐HT6 receptors. This unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues

Androgen ablation results in differential regulation of transforming growth factor-beta isoforms in rat male accessory sex organs and epididymis.

The male accessory sex organs and epididymis regress following androgen depletion, although the onset of apoptosis varies temporally depending upon the tissue type. Transforming growth factor-beta1 (TGF-beta1) is an androgen-repressed gene and believed to be an apoptotic agent in the regressing rat ventral prostate (VP). Hence, in order to investigate the status of TGF-beta isoforms following castration in androgen-dependent tissues other than VP, this study was undertaken. Northern blot analysis using total RNA from these tissues of intact animals showed higher levels of TGF-beta1 expression as compared with VP, indicating a function other than that of an apoptotic agent for this isoform. Following orchiectomy, TGF-beta1 was induced in all organs studied and the levels were highest at day 3 following castration in seminal vesicle (SV) and the epididymis and decreased by day 5 despite the absence of androgens. This observation implies that TGF-beta1 might not be a truly androgen-repressed gene in these tissues. TGF-beta2 was up-regulated in VP, SV, caput and corpus epididymis but was undetectable in the dorsolateral prostate and cauda epididymis. On the other hand, TGF-beta3 expression was refractory to the androgen status in corpus epididymis and SV but was up-regulated in the remaining tissues. The castration-induced induction of mRNAs was attenuated after exogenous androgen administration. Most importantly, all the isoforms differed significantly in the time and magnitude of induction following castration, suggesting that a single hormone, testosterone, modulates the expression of TGF-betas in an isoform- and tissue-specific manner.

Liposome-induced release of cell membrane proteins from intact tissue epithelium.

During extraction and purification, membrane proteins very often undergo denaturation and deactivation. To overcome this problem, the authors have tried to establish a better methodology to make the study at in vivo tissue level, not at the isolated cellular level, possible and easier. This is in vivo direct exposure of animal tissue to the liposome that contains an artificial boundary lipid (D14DPC, 1,2-dimyristamido-1,2,-deoxyphosphatidylcholine). Bullfrog and rat tongues were used. To confirm the reasonableness of this methodology, several different techniques were adopted; the nerve response study, gel electrophoretic analysis, quartz crystal microbalance (QCM) measurement and the affinity gelchromatography. When the tongue was exposed to the D14DPC-containing DMPC liposome, a significant amount of membrane protein was found in the recovered liposome (this was the production of proteoliposome). The nerve response in the neurophysiological measurement to several taste stimuli, such as L-alanine, L-leucine, sucrose and quinine hydrochloride significantly decreased when the tongue was exposed to the same liposome. These phenomena were common to both bullfrog and rat tongues. The nerve response to the stimulation with L-alanine was the most remarkably affected in the liposomal treatment. Therefore, the L-alanine-binding protein was focused upon to confirm the reasonableness of the QCM measurement and the affinity gelchromatography. The D14DPC-containing proteoliposome always showed significant binding to both the L-alanine affinity gel and the L-alanine-conjugated QCM. The results revealed that membrane proteins can be directly and effectively released, even from intact animal tissue epithelium, using the artificial boundary lipid-containing liposome.

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Uptake of L‐ and D‐alanine by a brackish‐water Bivalve, Corbicula japonica, with special reference to their transport pathways and the salinity effect

AbstractThe uptake of L‐ and D‐alanine by intact animals or isolated gills of a brackish‐water bivalve, Corbicula japonica, was investigated in relation to their transport pathways and the possible physiological roles. The animals took up L‐alanine at concentrations as low as 10 μM. Both the net transport and the influx were strongly affected by external salinity; animals acclimated to fresh water showed much lower rates of net transport and influx than did animals acclimated to 250 mOsm. Patterns of accumulation of 14C‐L‐alanine in different tissues of intact animals revealed that the gill was the principal site for the transport of the amino acid. Influx of both 14C‐L‐ and 14C‐D‐alanine into isolated gills was inhibited by α‐neutral amino acids, but not by α‐acidic or β‐amino acids; all α‐neutral amino acids examined (glycine, L‐glutamine, L‐leucine, L‐valine, and even D‐ and L‐alanine) inhibited the influx of D‐alanine more strongly than the influx of L‐alanine. Kinetic analysis of the influx indicated that D‐ and L‐alanine were transported via the same carrier(s), but that the carrier(s) had a higher affinity for L‐alanine than for D‐alanine. The transport mechanism appears to play a significant role in the net uptake of D‐ and L‐alanine or in the re‐uptake of the amino acids diffused from the integument according to the ambient concentrations of the substrates. © 1992 Wiley‐Liss, Inc.

Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells.

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.

Measurement of instant rates of protein degradation in the livers of intact mice by the accumulation of bestatin-induced peptides

Bestatin induces the accumulation of di- and tripeptide intermediates in cellular protein breakdown. In liver, a single set of bestatin-sensitive cytosolic peptidases are involved in the degradation to amino acids of the major classes of cellular proteins. Accumulation of bestatin-induced peptides, in isolated hepatocytes, is proportional to the rate of protein degradation (Botbol, V., and Scornik, O. A. (1989) J. Biol. Chem. 264, 13504-13509). Injection of 1 mg of bestatin into mice results in detectable amounts of hepatic intermediates in 15 min. We propose to use the accumulation of these peptides as a relative measurement of liver protein degradation. There is at present no other way to determine transient changes in protein breakdown in the tissues of intact animals. As an example of the applications of this procedure, we present the effects of a single meal on hepatic protein metabolism. Protein synthesis was estimated by the incorporation into liver protein of a massive dose of radioactive leucine (Scornik, O. A. (1974) J. Biol. Chem. 249, 3876-3883) and degradation of long-lived or short-lived proteins by the accumulation of bestatin-induced peptides, labeled in carboxy-C of their Leu or Arg moieties, 1 day or 1 h beforehand. A single meal resulted in an 18% increase in liver protein in 8 h, a 45% increase in the rate of hepatic protein synthesis, and a 3-fold decrease in the rate of breakdown of long-lived proteins. Short-lived proteins were not affected. To establish the efficiency with which bestatin-induced peptides accumulate in the livers of fasting mice, we compared them with the disappearance, in 1 day, of protein-bound 14C-guanidino-Arg residues, labeled by previous injection of 14C-bicarbonate (Swick, R. W., and Ip, M. M. (1974) J. Biol. Chem. 249, 6836-6841). From this comparison, we estimated that bestatin-induced Leu-labeled intermediates, accumulating in 15 min, represented 39% of the hepatic proteins degraded in that interval. For Arg-labeled intermediates the value was 55%. Correcting for these efficiencies, we estimate that in 4 h a meal decreased the rate of degradation of long-lived Arg-labeled proteins from 2.02 to 0.73%/h. For Leu-labeled proteins the estimated rates were 1.76 and 0.66%/h, respectively. Although a transient slowdown of liver protein degradation after a single meal had been suggested before, this is the first time that acute changes such as this can be determined directly in intact animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Transfer of human membrane surface components by incorporating human cells into intact animal tissue by cell-tissue electrofusion in vivo

Current animal models employed for the study of the obligate human pathogen Neisseria gonorrhoeae fail to utilize specific human gonococcal attachment receptors required to initiate pathogenesis in a clinically meaningful way. This communication presents evidence that suggests that cell-tissue electrofusion may be employed to create an animal model for this human specific pathogen. This new biotechnology was used to incorporate human membrane gonococcal receptors directly into epithelium of laboratory animals and subsequently infecting the histologically modified tissue with N. gonorrhoeae strain Pgh 3-2.

The role of the low density lipoprotein receptor for alpha-tocopherol delivery to tissues.

To study the role of the LDL receptor pathway for the maintenance of alpha-tocopherol concentrations in tissues of intact animals, we have compared vitamin E levels in plasma and tissues of normal rabbits and WHHL rabbits. WHHL rabbits are deficient in LDL receptor activity. For WHHL rabbits, alpha-tocopherol plasma concentrations were elevated to 10 times normal levels. When plasma lipoprotein profiles were analyzed by density gradient centrifugation, concentrations of VLDL, IDL, and LDL were increased in WHHL rabbits, and plasma alpha-tocopherol was only recovered in these fractions. In normal rabbits, plasma alpha-tocopherol was confined mainly to the HDL fraction; levels associated with LDL were markedly lower. Despite LDL receptor deficiency, alpha-tocopherol concentrations in various tissues of WHHL rabbits were not found to be reduced, with the exception of the adrenal. Vitamin E levels in muscle and kidney of WHHL rabbits exceeded those of normal animals. Our results demonstrate the importance of the LDL receptor pathway for vitamin E clearance in the normal rabbit, although at high LDL concentrations, alternative mechanisms may become more efficient for the delivery of vitamin E to tissues.

Application of ESR spectroscopy in toxicology.

Spin trapping in vivo was first achieved in the author's laboratory and is shown to be a feasible method for demonstrating that highly reactive free radical intermediates are generated in the tissues of intact animals as a result of the exposure to certain toxic compounds and to ionizing radiation. The method is based on the property of spin trapping agents (nitrones) to react readily with reactive free radicals to produce stable radical adducts at the site of their origin in target organs. The radical adducts can then be detected by electron spin resonance spectroscopy to determine the intensity of radical production (i.e., number of radicals which were trapped), and, in most cases, identify the nature of the radical that was produced. The type of spin trapping agent employed determines the type of radicals which can be trapped and, at this stage of development of the technique, the number of useful in vivo trapping agents is rather limited.

Inositol bis-, tris-, and tetrakis(phosphate)s: analysis in tissues by HPLC.

The concentration of isomers of inositol tris(phosphate) (InsP3) was measured in tissues of intact animals. The method employed involved anion-exchange HPLC with on-line enzymatic hydrolysis of the phosphate esters and detection of the inorganic phosphate formed. All seven organs tested from rats killed by decapitation contained Ins(1,4,5)P3 in concentrations of 13-40 nmol/g; a distribution that bears no resemblance to that reported for its precursor [phosphatidylinositol bis(phosphate)]. A second InsP3 isomer [probably Ins(1,3,4)P3] was also detectable in brain and salivary gland. The content of Ins(1,4,5)P3 in brain and salivary gland from rats killed by decapitation was 10-60 times greater than that from rats killed by focused microwave irradiation to block postmortem metabolism. Inositol bis(phosphate) concentrations also changed dramatically postmortem. A much smaller postmortem change was seen in the content of Ins(1,3,4)P3. Receptor stimulation by muscarinic cholinergic agonists increased the content not only of Ins(1,3,4)P3, but also its recently discovered probable precursor, inositol tetrakis-(phosphate).

Influence of polyethylene glycol insulin on lipid tissues of experimental animals.

Earlier investigations have revealed that polyethylene glycol-B1-insulin (PEG insulin) causes lower utilization of glucose in fat cells than does the unaltered hormone, even though the blood glucose-lowering activity of both preparations in intact animals is identical. The present study was aimed to establish whether or not PEG insulin in regard to adipose tissues of intact animals has similar functions. Radioactive glucose was used to examine the influence of both native pork insulin and its PEG derivative on the incorporation of tracers into lipids. Male Wistar rats and male domestic rabbits received 14C- and/or 3H-labeled glucose intravenously, while the insulin preparations were administered by either the subcutaneous (s.c.) or the intravenous (i.v.) route. Under the influence of PEG insulin, diminished incorporation of 14C and/or 3H into adipose tissues was observed in all cases, yet both natural insulin and the derivative lowered the blood glucose to the same extent. These observations allow the assumption that, by using certain modified insulins, it may also be possible to manipulate the extent of glucose metabolism by lipid tissues in diabetic patients.

Lipid Absorption and Metabolism

Metabolic processes occurring within the mucosal cell are critical in determining results of interactions between environmental agents and the alimentary tract. The absorption, metabolism, and transport of lipids affects most those agents which are lipid soluble. The understanding of the process involved in lipid absorption and transport is therefore important for both appreciation of the mechanism of uptake of these toxins and for an effective interference with it. Most of the detailed mechanisms of lipid absorption and transport have been proposed from in vitro studies with soluble cell-free systems. The present review integrates these results with recent in vivo and in vitro findings with intact animal tissues and isolated mucosal cells. While there is much general agreement occasional startling differences are also observed, which may have a bearing on the mechanism of normal fat absorption and on the understanding of the transport of the fat-soluble toxins across the mucosal villus cell.