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The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.
Anat Rec 1992 Jan
PMID:Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L-[35S]- and L-[methyl-3H]-methionine. 153 54

A prokaryotic expression vector containing the rec A promoter and a translational enhancer element from the gene 10 leader of bacteriophage T7 was used to direct efficient synthesis of rat intestinal fatty acid binding protein (I-FABP) in E. coli. Expression of I-FABP in E. coli has no apparent, deleterious effects on the organism. High levels of expression of I-FABP mRNA in supE+ strains of E. coli, such as JM101, is associated with suppression of termination at its UGA stop codon. This can be eliminated by using a supE-strain as MG1655 and by site-directed mutagenesis of the cDNA to create an in frame UAA stop codon. E. coli-derived rat I-FABP lacks its initiator Met residues. It has been crystallized with and without bound palmitate. High resolution x-ray crystallographic studies of the 131 residue apo- and holo-proteins have revealed the following. I-FABP contains 10 anti-parallel beta-strands organized into two orthogonally situated beta-sheets. The overall conformation of the protein resembles that of a clam--hence the term beta-clam. The bound ligand is located in the interior of the protein. Its carboxylate group forms part of a unique five member hydrogen bonding network consisting of two ordered solvent molecules as well as the side chains of Arg106 and Gln115. The hydrocarbon chain of the bound C16:0 fatty acid has a distinctive bent conformation with a slight left-handed helical twist. This conformation is maintained by interactions with the side chains of a number of hydrophobic and aromatic amino acids. Apo-I-FABP has a similar overall conformation to holo-I-FABP indicating that the beta-clam structure is stable even without bound ligand. The space occupied by bound ligand in the core of the holo-protein is occupied by additional ordered solvent molecules in the apo-protein. Differences in the side chain orientations of several residues located over a potential opening to the cores of the apo- and holo-proteins suggest that solvent may play an important role in the binding mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Expression of rat intestinal fatty acid binding protein in E. coli and its subsequent structural analysis: a model system for studying the molecular details of fatty acid-protein interaction. 226 73

Sheep and goat binucleate cells (BNC) play a central role in placental growth and development. This study reports a simple method for isolating 60-70% pure populations of BNC of high viability. After incubation of the isolated BNC with a brief pulse of 14C-leucine or 3H-fucose or 3H-galactose, electron microscope autoradiography showed that label was eventually incorporated into the characteristic BNC granules via the Golgi body. Fucose and galactose initially showed a much higher Golgi body label than leucine, which was at first predominantly localised in the endoplasmic reticulum. 35S-methionine incorporation by BNC suspensions was extensive enough to allow an immunoprecipitation investigation which demonstrated that the protein hormone ovine placental lactogen and the SBU-3 antigen were synthesised de novo. Previous studies with isolated BNC have shown a remarkable range of substances to be released into the incubation medium but not necessarily synthesised during the incubation. The results demonstrate unequivocally that isolated BNC's are capable of total synthesis in vitro of two of the proteins that these same cells are known to secrete in vivo.
Anat Rec 1990 Jan
PMID:Characterization of the synthetic capacities of isolated placental binucleate cells from sheep and goats. 229 81

The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetyl-glucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both, in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.
Anat Rec 1989 Jun
PMID:Biosynthesis and secretion of enamel proteins in the rat incisor. 277 7

The pattern and timing of the breakdown and loss of matrix proteins were studied in developing rat incisor enamel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fluorography, radioautography, and in vitro incubations of proteins isolated from freshly dissected, crushed pieces of enamel. For biochemical studies, the technique of Robinson et al. (1974, 1977, 1983) was used to transect the enamel organ and enamel into a series of strips at 1 mm intervals along the length of the tooth. The proteins in each strip were extracted and either quantified by Lowry analysis or applied to 12% slab (enamel) or 5-15% continuous gradient (enamel organ) SDS-polyacrylamide gels and separated by electrophoresis. The biochemical studies indicated that the amount of protein contained within an enamel strip increased gradually by volume across the secretory stage, reached a peak early during the maturation stage, and then declined rapidly thereafter. The distribution of enamel proteins on SDS-polyacrylamide gels changed markedly throughout this period. These changes included increases and decreases in the intensity of staining of proteins at certain molecular weights (e.g., 18 kDa) and the appearance and disappearance of some proteins not seen clearly near the start of the secretory stage of amelogenesis (e.g., 32 and 10 kDa). Labeling studies with 35S-methionine suggested that the "stacked" arrangement of proteins typical of forming enamel (secretory stage) actually represented a very dynamic association of proteins, with new ones being added at the top of the stack and then breaking down with time to become those seen at lower molecular weights. Across the secretory stage, new proteins were always added to the top of the stack, but during early maturation this activity slowed dramatically, allowing the breakdown of aging proteins to be visualized more clearly. Radioautographic studies with 3H-methionine indicated that the breakdown of newly secreted proteins also was correlated with a movement of label from the site of secretion into deeper, previously unlabeled, areas of forming enamel. In vitro studies revealed that the rate and degree of breakdown of enamel proteins varied markedly, depending on the stage of amelogenesis from which the proteins were extracted. Secretory stage enamel proteins showed slow in vitro degradation with accumulation of proteins near 18 kDa. Early maturation stage enamel proteins showed more rapid breakdown with little accumulation of proteins near 18 kDa, whereas late maturation stage enamel proteins showed complete degradation by 2 days of incubation in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
Anat Rec 1989 Jun
PMID:Degradation and loss of matrix proteins from developing enamel. 277 8

The present peroxidase-antiperoxidase immunohistochemical study demonstrated that approximately 50% of the total chromaffin cells of the rat adrenal medulla exhibited NPY-like immunoreactivity. The immunoreactive material was localized in the core of the chromaffin granules as well as diffusely in the cytoplasm. By combination of immunohistochemistry with noradrenaline-fluorescence microscopy, all NPY-immunoreactive chromaffin cells are nonfluorescent, indicating that all NPY-chromaffin cells co-store adrenaline. A comparison of two consecutive sections, each of which was processed for the immunostaining with anti-NPY and anti-Met-Enk-Arg-Gly-Leu antisera, respectively, indicated that NPY and preproenkephalin A and its derivatives coexist in approximately one-fifth of the total NPY-immunoreactive cells. In addition to the NPY-immunoreactive cells, a plexus of NPY-immunoreactive nerve fibers with varicosities was found in the subcapsular regions of the adrenal gland. The nerve fibers were often associated with small blood vessels and extended into the zona glomerulosa. Single NPY-immunoreactive fibers were sparsely distributed in the deeper regions of the cortex and in the medulla. Ganglion cells in the adrenal gland were not seen exhibiting intensely positive NPY-like immunoreactivity. The NPY-immunoreactive nerve fibers contained abundant small clear vesicles mixed with a few small and large granular vesicles. The immunoreactive material appeared on the granular cores as well as in the axoplasm. The NPY fibers were closely apposed to smooth muscle cells and pericytes of small blood vessels in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)
Anat Rec 1986 Mar
PMID:Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers of rats. 351 14

We have purified the beta subunit of the DNA polymerase III holoenzyme to homogeneity from an overproducing strain (Blanar, M., Sandler, S., Armengod, M., Ream, L., and Clark, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4622-4626). From this procedure we can obtain 100 mg quantities of protein. The beta isolated from the overproducer is indistinguishable from that isolated from wild-type cells in terms of its activity and molecular weight. Partial amino acid sequence analysis has confirmed the DNA sequence of the dnaN gene (Ohmori, H., Kimura, M., Nagata, T., and Sakakibara, Y. (1984) Gene (Amst.) 28, 159-170) and established the sites for initiation and termination of translation. No processing that removes amino acid residues from beta occurs since the active protein begins with the initiating methionine and terminates at the position predicted from the DNA sequence. Our knowledge of the precise amino acid composition has been used to determine the extinction coefficient of beta to be 17,900 and 18,700 cm-1 M-1 at 280 and 277 nm, respectively. The extinction coefficient at 280 nm is reduced to 14,700 cm-1 M-1 under denaturing conditions in guanidine HCl. Conditions have been optimized so that 1 N-ethylmaleimide residue can be incorporated per beta monomer with full preservation of activity.
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PMID:Chemical characterization and purification of the beta subunit of the DNA polymerase III holoenzyme from an overproducing strain. 352 43

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.
Anat Rec 1987 Feb
PMID:Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors. 357 31

Beta-endorphin-related opiate peptides or the opiate antagonist naloxone were chronically infused for periods of 24 to 48 hours to the lateral cerebral ventricle of adult male rats using Alza osmotic minipumps. Previous studies have suggested a "chemotactic"-like effect of opiate peptides for supraependymal macrophages in the region of the third ventricle of the brain. The present study demonstrates a stimulatory effect of beta-endorphin infusion on the appearance of lymphocyte and neutrophil-like cells, in addition to macrophages, in the region of the third ventricle, suggestive of an intracerebral inflammatory response. None of the other molecules, including alpha-endorphin, methionine-enkephalin, naloxone, or sterile saline produced similar cellular responses after infusion, although some of the latter substances may have induced the appearance of supraependymal neuron-like cells in the area. Observations suggest that the chronic presence of beta-endorphin, a biologically active opiate peptide, will interact with cells of the immune system, which have the ability to gain access to the cerebrospinal fluid.
Anat Rec 1984 Sep
PMID:Chronic infusion of opiate peptides to rat cerebrospinal fluid with osmotic minipumps. 609 99

The pelvic flexure portion of the equine large colon is the proposed location of a pacemaker mechanism. This study was conducted to ascertain whether the distribution of certain putative neurotransmitters differs at the pelvic flexure compared to other sampling sites. Tissue samples were collected from the intestinal tracts of six horses. Serial sections from these samples were reacted with primary antisera specific for substance P, vasoactive intestinal polypeptide (VIP), methionine-Enkephalin, and calcitonin gene-related peptide (CGRP). The regional distribution of immunoreactive neuronal elements was uniform for each of the neuropeptides except VIP. Although neurons exhibiting VIP-like immunoreactivity were abundant throughout the colon, they were somewhat more plentiful near the apex of the pelvic flexure and the left dorsal colon. These neurons may participate in the initiation and propagation of the propulsive/retropulsive contraction waves, which emanate from this location and are believed to lend a sphincter-like capacity to the pelvic flexure. The submucosal plexus was replete with neurons with intense substance P and VIP-like reactivity. Reactive fibers left submucosal ganglia to project to the intestinal mucosa, reflecting a possible secretogogic role for these neurons. This role may be especially important for the horse as a hindgut fermenter. There were abundant methionine-Enkephalin and substance P-like reactive varicosities throughout the myenteric plexus, many of which established a pericellular plexus of varicose fibers. The abundance of these varicosities, which may correlate with a high degree of neuronal integration, did not vary regionally. These data may enhance our understanding of both normal colonic peristalsis and motility disorders caused by a depletion of these neuropeptides.
Anat Rec 1993 Jun
PMID:Neuropeptide distributions in the colon, cecum, and jejunum of the horse. 768 32


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