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PubMed2002

Early steps of metabolism evolution inferred by cladistic analysis of amino acid catabolic pathways.

Among abiotic molecules available in primitive environments, free amino acids are good candidates as the first source of energy and molecules for early protocells. Amino acid catabolic pathways are likely to be one of the very first metabolic pathways of life. Among them, which ones were the first to emerge? A cladistic analysis of catabolic pathways of the sixteen aliphatic amino acids and two portions of the Krebs cycle is performed using four criteria of homology. The cladogram shows that the earliest pathways to emerge are not portions of the Krebs cycle but catabolisms of aspartate, asparagine, glutamate, glutamine, proline, arginine. Earliest enzymatic catabolic functions were deaminations and transaminations. Later on appeared enzymatic decarboxylations. The consensus tree allows to propose four time spans for catabolism development and corroborates the views of Cordón in 1990 about the evolution of catabolism.

Amino Acids↗

PubMed2001

Sorting of internalized neurotrophins into an endocytic transcytosis pathway via the Golgi system: Ultrastructural analysis in retinal ganglion cells.

Subcellular pathways and accumulation of internalized radiolabeled neurotrophins NGF, BDNF, and NT-3 were examined in retinal ganglion cells (RGCs) of chick embryos by using quantitative electron microscopic autoradiography. All three neurotrophins accumulated in endosomes and multivesicular bodies. BDNF and NGF also concentrated at the plasma membrane, whereas NT-3 accumulated transiently in the Golgi system. The enhanced targeting of NT-3 to the Golgi system correlated with the anterograde axonal transport of this neurotrophin. Anterograde transport of NT-3, but not its internalization, was significantly attenuated by the tyrosine kinase (trk) inhibitor K252a. Abolishment of trk activity with K252a shifted NT-3 (and BDNF) away from the Golgi system and into a lysosomal pathway, indicating that trk activity regulated sorting of the ligand-receptor complex. Cross-linking of neurotrophins and immunoprecipitation with antibodies to the neurotrophin receptors p75, trkA, trkB, and trkC showed that the large majority of exogenous, receptor-bound NT-3 was bound to trkC in RGC somata, but during anterograde transport in the optic nerve most receptor-bound NT-3 was associated with p75, and after arrival and release in the optic tectum transferred to presumably postsynaptic trkC. These results reveal remarkable and unexpected differences in the intracellular pathways and fates of different neurotrophins within the same cell type. They provide first evidence for an endocytic pathway of internalized neurotrophic factors via the Golgi system before anterograde transport and transcytosis. The results challenge the belief that after internalization all neurotrophins are rapidly degraded in lysosomes.

Animals↗

PubMed2001

Genomic expression analysis implicates Wnt signaling pathway and extracellular matrix alterations in hepatic specification and differentiation of murine hepatic stem cells.

HBC-3 hepatic stem cells maintained in the undifferentiated state can be induced to differentiate along the hepatocyte lineage in response to DMSO (Rogler, 1997). In order to understand the complex transcriptional regulatory mechanisms associated with the differentiation of these somatic stem cells and to identify novel candidate stem cell and differentiation associated genes, we have begun to characterize the transcriptome of HBC-3 cells during a 7-day differentiation protocol. This analysis showed that differentiating HBC-3 cells undergo biphasic bursts of gene regulation peaking at 3 hours and 120 hours of DMSO treatment. In the undifferentiated state, HBC-3 cells express muscle, neuron, myeloid, and lymphoid specific genes that are rapidly downregulated during hepatocytic differentiation. Cluster analysis has revealed large groups of genes with different temporal regulation profiles demonstrating complex and widespread transcriptional changes. Specifically, we discovered a multifaceted downregulation of the Wnt/beta-catenin pathway accompanied by the repression of TCF target genes during HBC-3 differentiation. In addition, there is downregulation of cellular receptors for fibronectin and laminin and other extracellular matrix molecules indicative of widespread cell surface alterations. DMSO induces cell cycle arrest, and this is reflected in upregulation of growth inhibitory proteins such as cyclin I and p18 and downregulation of cyclins B1 and D. Genes needed for hepatocytic functions, such as apolipoprotein C-IV, phosphoenolpyruvate carboxykinase, alcohol dehydrogenase, and asialoglycoprotein receptor were upregulated. Finally, transcriptional regulators including Twist, Snail, HNF1a, and GATA6 were upregulated during differentiation of HBC-3 cells. The significance of these findings is that our genome-based approach has allowed the parallel identification of multiple regulatory pathways that is needed to begin to fully understand the complex differentiation process.

Animals↗

PubMed2004

Analysis of Ah receptor pathway activation by brominated flame retardants.

Brominated flame-retardants (BFRs) are used as additives in plastics to decrease the rate of combustion of these materials, leading to greater consumer safety. As the use of plastics has increased, the production and use of flame-retardants has also grown. Many BFRs are persistent and have been detected in environmental samples, raising concerns about the biological/toxicological risk associated with their use. Most BFRs appear to be non-toxic, however there is still some concern that these compounds, or possible contaminants in BFRs mixtures could interact with cellular receptors. In this study we have examined the interaction of decabromodiphenyl ether, Firemaster BP4A (tetrabromobisphenol A), Firemaster PHT4 (tetrabromophthalic anhydride), hexabromobenzene, pentabromotoluene, decabromobiphenyl, Firemaster BP-6 (2,2',4,4',5,5'-hexabromobiphenyl) and possible contaminants of BFR mixtures with the Ah receptor. Receptor binding and activation was examined using the Gel Retardation Assay and increased expression of dioxin responsive genes was detected using the reporter gene based CALUX assay. The results demonstrate the ability of BFRs to activate the AhR signal transduction pathway at moderate to high concentrations as assessed using both assays. AhR-dependent activation by BFRs may be due in part to contaminants present in commercial/technical mixtures. This was suggested by our comparative analysis of Firemaster BP-6 versus its primary component 2,2',4,4',5,5'-hexabromobiphenyl. Some technical mixtures of brominated flame-retardants contain brominated biphenyls, dioxins or dibenzofurans as contaminants. When tested in the CALUX assay these compounds were found to be equivalent to, or more active than their chlorinated analogues. Relative effective potency values were determined from dose response curves for these brominated HAHs.

Animals↗

PubMed2005

Lhermitte-Duclos disease: a report of 31 cases with immunohistochemical analysis of the PTEN/AKT/mTOR pathway.

Lhermitte-Duclos disease (LDD) is a rare cerebellar tumor associated with Cowden disease (CD) and germline mutations in the PTEN gene. To further define these relationships, we reviewed clinical and pathologic findings in 31 LDD cases and analyzed the status of the PTEN pathway in 11 of them. We hypothesized that the granule cell hypertrophy in LDD is secondary to activation of mammalian target of rapamycin (mTOR), a downstream effector in the PTEN/AKT pathway and a major regulator of cell growth. Histopathologically, in addition to the classical findings of LDD, we observed prominent vascular proliferation and vacuolization of the white matter in many of the lesions. Four patients met diagnostic criteria for CD, and many of the remaining patients had some clinical features of CD. Immunohistochemical analysis showed high levels of phospho-AKT and phospho-S6 in the large ganglionic cells forming the lesions, indicating activation of the PTEN/AKT/mTOR pathway and suggesting a central role for mTOR in the pathogenesis of LDD. These data support recommendations for genetic testing and screening for CD in patients with LDD and suggest a novel therapy for LDD through pharmacologic inhibition of mTOR.

Adolescent↗

PubMed1992

The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding.

The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter phi. There are two extreme values of phi that are often found in practice and may be interpreted in a simple manner. A value of phi = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, phi = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of phi are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of phi found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of phi = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the double-mutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.

Bacterial Proteins↗

PubMed2007

Psychophysical evidence for long-term potentiation of C-fiber and Adelta-fiber pathways in humans by analysis of pain descriptors.

Long-term potentiation of human pain perception (nociceptive LTP) to single electrical test stimuli was induced by high-frequency stimulation (HFS) of cutaneous nociceptive afferents. Numerical pain ratings and a list of sensory pain descriptors disclosed the same magnitude of nociceptive LTP (23% increase for >60 min, P < 0.001), whereas affective pain descriptors were not significantly enhanced. Factor analysis of the sensory pain descriptors showed that facilitation was restricted to two factors characterized by hot and burning (+41%) and piercing and stinging (+21%, both P < 0.01), whereas a factor represented by throbbing and beating was not significantly increased (+9%, P = 0.47). The increased perception of the burning pain quality for >1 h after HFS is interpreted as a LTP-like facilitation of the conditioned cutaneous C-fiber pathway. Additionally, the increase of the stinging pain quality supplied evidence for facilitation of a sharpness-sensitive Adelta-fiber pathway.

Adult↗

PubMed2003

Identification and characterization of the Xlsirt cis-acting RNA localization element.

There are many RNAs that are localized to the vegetal cortex of Xenopus laevis oocytes. One family of localized transcripts, Xlsirts (Xenopus laevis short interspersed repeat transcripts), are defined by the presence of non-coding repeat units 79-81 nucleotides long. Endogenous Xlsirt RNAs are localized through the METRO (message transport organizer) pathway that localizes RNAs during stages I and II of oogenesis. Interestingly, exogenous Xlsirt RNAs that are injected into oocytes can utilize both the METRO pathway as well as the Late pathway, which localizes RNAs during the late stages of oogenesis (stages III-VI). In all cases thus far analyzed, the localization process relies on the presence of cis-acting elements on the transcripts that are responsible for directing localization. To better understand the mechanism responsible for the use of the METRO and Late localization pathways, we sought to identify pathway-specific cis-acting localization elements contained in Xlsirts. The results showed that an intact 137 nucleotide element was necessary and sufficient to localize RNAs through the METRO and Late pathways. Further analysis of this element identified putative METRO and Late pathway localization sub-elements. Computer analysis relates the secondary structure of the 137 nt element to its ability to function as a localization element.

Amino Acid Sequence↗

PubMed2002

A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries.

The sequence of much of the human genome is now available and the next goal is to identify functional genes and to clarify their roles. We have recently developed a novel system for isolation of genes in the Fas- and TNF-alpha-mediated pathways to apoptosis using poly(A)-connected hammerhead ribozyme libraries with randomized substrate-binding arms at both the 5' and 3' ends of ribozymes. The transcripts of these hybrid ribozymes have a poly(A) motif that can recruit RNA helicases and, thus, they can effectively attack target sites. In the previous studies, hybrid ribozymes were stably expressed. In order to save selection times, in this study we adopted transiently expressed hybrid ribozymes. In the case of Fas-mediated apoptosis, when we transiently introduced these hybrid-ribozyme libraries into Fas-expressing HeLa cells, we were able to isolate surviving clones that were resistant to or exhibited a delay in Fas-mediated apoptosis. We identified many pro-apoptotic genes and novel genes using this strategy with these transiently expressed hybrid-ribozyme libraries. In contrast, we identified significantly smaller numbers of candidate genes using conventional ribozyme libraries that were expressed transiently. Thus, when changes of a particular phenotype occur within a short period of time, our gene discovery system based on transiently expressed hybrid-ribozyme libraries should also be useful for the rapid identification of functional genes in the post-genome era.

Apoptosis↗

PubMed1989

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration.

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.

Animals↗

PubMed2004

Ubiquitin-mediated proteolysis: an ideal pathway for systems biology analysis.

Ubiquitin is a small, evolutionarily conserved eukaryotic protein that can be attached to a wide variety of intracellular proteins including itself. Covalent attachment of ubiquitin to other proteins serves various functions, but its major role is to target cellular proteins for destruction. Cellular components that activate, transfer, remove, or simply recognize ubiquitin number in the hundreds, perhaps even in the thousands. In light of this complexity the ubiquitin pathway is ideal for a systems biology approach.

Cysteine Endopeptidases↗

PubMed1998

C5 convertase of the alternative pathway of complement. Kinetic analysis of the free and surface-bound forms of the enzyme.

Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (kcat). However, the surface-bound enzyme, ZymC3b,Bb had a Km (1.4 microM) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (Km = 24 microM). The kcat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s-1 and that for soluble C3b,Bb was 0.0110 s-1. Both forms of the enzyme had a low turnover number at Vmax (0.23 to 0.68 C5/min/enzyme). Substituting Mg2+ for Ni2+ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the Km determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.

Cell Membrane↗

PubMed2003

Apoptosome-independent pathway for apoptosis. Biochemical analysis of APAF-1 defects and biological outcomes.

Induction and execution of apoptosis programs are generally believed to be mediated through a hierarchy of caspase activation. By using two cellular variants obtained from the L1210 cell line (L1210/S and L1210/0), we have shown previously that staurosporine induces apoptotic cell death through both caspase-dependent and caspase-independent pathways. Both pathways normally coexisted in L1210/S cells, whereas L1210/0 cells lacked the ability to activate caspases despite the confirmed presence of both procaspase-3 and -9. Here we show that this defect in caspase activation is not due to mechanisms such as an absence of cytochrome c release, the expression of non-functional caspases, or the presence of an endogenous inhibitor but results from the loss of apoptosis protease activator protein-1 (APAF-1) expression. This absence of APAF-1 protein results from multiple alterations at both genomic and transcriptional levels. However, although this lack of APAF-1 delays the apoptotic program, it does not hamper its execution. Importantly, in these cells, apoptosis develops not only in an APAF-1-independent way but also in the absence of caspase-3 and -9 activation. Altogether these findings provide evidence that apoptosis may occur through alternative signaling pathways independent of APAF-1 expression and totally dissociated from any caspase processing. Therefore, the L1210/0 variant sub-line provides a valuable tool for the elucidation of these pathways.

Animals↗

PubMed2006

Multiplex analysis of inflammatory signaling pathways using a high-content imaging system.

This chapter describes a robust high-content cellular screening assay to simultaneously analyze the spatiotemporal activation of three different kinase-associated signaling pathways involving NF-kappaB, JNK, and p38, all of which are closely implicated in proliferative and proinflammatory responses. Signal transduction is dependent on the translocation of NF-kappaB p65 and phosphorylated c-Jun and p38 from the cytosol to the nucleus, and fluorescent immunolabeling was used to monitor changes in their cellular distribution. Cellular screening, data acquisition, and data interpretation were conducted on the ArrayScan HCS Reader (Cellomics Inc., Pittsburgh, PA). Assay adaptation to various cellular systems is feasible when sufficient separation of the nuclear and cytosolic compartment can be achieved and if cell adhesion properties permit proper attachment to the culture plates. Substitution of NF-kappaB p65 and phosphorylated forms of c-Jun and p38 as targets to analyze other translocating components is possible and is limited primarily by antibody specificity and the risk of fluorescent bleed-through between emission channels. Because assay validity is particularly confounded by inadequate spectral separation of the detection dyes in multicolor labeling assays, means of eliminating or counterbalancing staining artifacts are illustrated. Also, protocol parameter settings important for imaging and image processing are described, including object identification, image exposure settings, separation of cytosolic and nuclear regions, number of cells sufficient for analysis, and the use of gating thresholds critical for cell sorting and subpopulation analysis. This assay is a useful tool to investigate the interplay between signaling pathways and the mode of action, potency, and selectivity of compound inhibition of specific target molecules in a cellular context.

Cell Nucleus↗

PubMed1995

Electron tunneling in substrate-reduced trimethylamine dehydrogenase: kinetics of electron transfer and analysis of the tunneling pathway.

The reoxidation of substrate-reduced trimethylamine dehydrogenase by the artificial electron acceptor ferricenium hexafluorophosphate was studied by stopped-flow spectroscopy. The rate constants for the two sequential one-electron transfers from the reduced 4Fe-4S center to ferricenium ions were measured, the first (ka = 49 s-1) being about 7 times greater than the second (kb = 7.3 s-1) at 20 degrees C and neutral pH. The temperature dependence of the second electron transfer was studied over the range 10-40 degrees C, and the rate constant ranged from 5.7 to 19.2 s-1. Analysis of the temperature perturbation of kb by Marcus theory yielded values for the reorganizational energy of 1.95 eV and the electronic coupling matrix element of 0.26 cm-1. An electron tunneling pathway distance of 13 +/- 0.7 A was calculated which correlates with the shortest pathway measured from the 4Fe-4S center to the protein surface using the crystallographic coordinates of trimethylamine dehydrogenase. Tyr-442 is implicated in facilitating electron transfer from the enzyme to ferricenium ions. The data suggest a location for the docking site on the surface of trimethylamine dehydrogenase for the physiological electron acceptor (ETF).

Electron Transport↗

PubMed2002

Mutational and expression analysis of the reelin pathway components CDK5 and doublecortin in gangliogliomas.

Gangliogliomas represent highly differentiated glioneuronal tumors frequently occurring in young patients with focal epilepsies. Dysplastic neurons are a neuropathological hallmark of this neoplasm. Here, we have analyzed two major components of the reelin pathway associated with neuronal migration and cortical cytoarchitecture in gangliogliomas, i.e., cyclin-dependent kinase 5 (CDK5) and doublecortin (DCX). The genomic structure of human CDK5 was identified by an " in silico" cloning approach using the "high throughput genomic sequencing" (htgs) databank, NCBI BLAST 2.1. DNA sequence analysis of CDK5 and DCX was carried out in tissue samples obtained from 23 patients and compared with control DNA from non-affected individuals ( n=100). For gene expression analysis of CDK5 and DCX, a quantitative real time reverse transcription-PCR TaqMan assay was used with mRNA from gangliogliomas ( n=22) and non-lesional central nervous tissue control tissue ( n=7). The human CDK5 gene is located on chromosome 7q36 and contains 12 exons. Its coding sequence reveals 90.1% homology to the mouse counterpart. A novel pseudogene of CDK5 was found on chromosome 8. While the mutational analysis of CDK5 and DCX did not reveal any sequence alterations in gangliogliomas, a lower expression was observed for both genes in tumor compared to control tissue samples. The present data indicate that mutations of CDK5 and DCX genes are not involved in the development of gangliogliomas. A novel pseudogene on chromosome 8 has to be taken into account for future studies on CDK5.

Animals↗

PubMed2004

Mutation analysis of the Ras pathway genes NRAS, HRAS, KRAS and BRAF in glioblastomas.

Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.

Adolescent↗

PubMed1981

High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary.

The secretory process in pituitary mammotrophs was analyzed by quantitative electron microscope autoradiography. Dispersed pituitary cells from estrogen-treated female rats were subjected to pulse-labeling with [3H]leucine (5 min) followed by a chase incubation of up to 4 h. Autoradiograms were prepared using fine-grained emulsion (Kodak 129-01), and analyzed using a three-step "mask analysis' procedure: (a) the distribution of autoradiographic grains is determined as in a simple grain density analysis; (b) masks (transparent overlays) are used to generate expected grains from assumed sources; and (c) a computer program compares these two distributions and varies the expected distribution to match the observed distribution, thereby identifying the radioactive sources in the tissue. The overall route of intracellular transport of prolactin from rough endoplasmic reticulum (ER) leads to Golgi complex leads to immature secretory granules leads to mature secretory granules was as established in previous studies. However, by use of the high resolution emulsion and method of analysis, the precision with which label could be localized within individual source compartments was much greater and the time resolution was much sharper than achieved previously using Ilford L4 emulsion and simple grain density analysis. The main new findings were as follows: (a) the ER was essentially drained of radioactivity by 30 min, the Golgi complex by 1 h, and the immature secretory granules by 2h postpulse. This indicates that the secretory product (prolactin) is rapidly and efficiently transported out of these compartments. (b) approximately 30% of the total radioactivity remains located in the ground cytoplasm over the entire postpulse period examined (up to 4 h), and by 30 min postpulse the grain density in the ground cytoplasm exceeded that of the ER. This indicates the ability to resolve ER-associated label (presumably associated mainly with secretory products) from the cytoplasmic label (presumably associated with nonsecretory proteins). (c) the specific activity of immature secretory granules was much greater than previously appreciated; at 1 h postpulse it was greater than 200 times that of the adjacent Golgi complex cisternae. This large dynamic range in observed grain density demonstrates the ability to effectively correct for radiation spread and thus to detect with great accuracy high concentration of label even from very small structures (20-100 nm) which constitute a small percentage (less than 1%) of the total cell area.

Animals↗