bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and characterization of human liver cDNA encoding a protein S precursor.
Authors: Hoskins J , Norman D K , Beckmann R J , Long G L
Published: May 2006

Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approximately 0.01%. Blot hybridization of electrophoretically fractionated poly(A)+ RNA revealed a major mRNA approximately 4 kilobases long and two minor forms of approximately 3.1 and approximately equal to 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid "leader" peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal gamma-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approximately 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.
Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation.
Authors: Mangelsdorf D J , Pike J W , Haussler M R
Published: May 2006

In vitro translation of cellular poly(A)+ RNA coupled with immunoprecipitation was developed as a technique for characterizing 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and assessing receptor mRNA activity. Cell-free translation of poly(A)+ RNA isolated from chicken intestine revealed two immunoprecipitable forms of avian receptor at 60 kDa and 58 kDa. These two species were identical in electrophoretic mobility to those detected directly in intestinal cytosol by immunoblot analysis. Liver, a tissue devoid of 1,25-(OH)2D3 binding activity, contained no apparent translatable receptor mRNA. 1,25-(OH)2D3 receptors were also synthesized in vitro employing poly(A)+ RNA obtained from several cultured mammalian cell lines. Selective immunoprecipitation revealed a single form of receptor at 54 kDa in mouse fibroblasts (3T6) and pig kidney cells (LLC-PK1) and a 52-kDa species in human breast carcinoma (T47D). Each of these in vitro translated mammalian 1,25-(OH)2D3 receptors migrated identically with its cellular counterpart that was synthesized in vivo employing metabolic labeling of cell protein with [35S]methionine. In vitro translation of poly(A)+ RNA derived from mouse 3T6 cells treated with 1,25-(OH)2D3 for 24-48 hr disclosed a 5-fold increase in receptor mRNA activity over untreated control cells. These results are consistent with the conclusions that 1,25-(OH)2D3 receptors are protein species ranging from 52 to 60 kDa and that, though their functional and immunological domains have been evolutionarily conserved, an inverse relationship apparently exists between phylogenetic status and receptor mass. The data also support the hypothesis that the presence of 1,25-(OH)2D3 leads to a significant increase in receptor mRNA activity in 3T6 cells, indicative of receptor autoregulation.
Substrate specificity of the chymotrypsin-like protease in secretory granules isolated from rat mast cells.
Authors: Le Trong H , Neurath H , Woodbury R G
Published: May 2006

The substrate specificity of rat mast cell protease I (RMCP I), a chymotrypsin-like serine protease localized in the secretory granules of mast cells, was compared to that of bovine alpha-chymotrypsin by using several peptide and protein substrates of known amino acid sequences. Although the overall specificities of the two proteases appeared similar, subtle but significant differences were observed. RMCP I was more prone than chymotrypsin to hydrolyze peptide bonds consisting of Leu-Xaa or two hydrophobic residues--e.g., Phe-Phe. Additionally, the hydrolysis of angiotensin I catalyzed by chymotrypsin, but not by RMCP I, resulted in the generation of angiotensin II as an intermediate product. In contrast to the solubilized enzyme, the RMCP I activity within the insoluble granules was completely stable for at least 2 months in suitable buffers at pH 8.0 or pH 7.2, at 4 degrees C. Carboxypeptidase A activity associated with isolated mast cell granules was completely inhibited by 10 mM o-phenanthroline. Polypeptides smaller than apomyoglobin (17,199 Da) were rapidly hydrolyzed by granule-bound RMCP I, whereas apomyoglobin and other larger proteins were not hydrolyzed. In contrast, the free protease readily hydrolyzed the larger proteins. Neither normal rat serum nor alpha 1-antitrypsin, both of which inhibited the activity of free RMCP I, was effective in inhibiting granule-associated RMCP I. The results indicate that granule-bound RMCP I is not released into solution from isolated secretory granules under physiological conditions of ionic strength and pH and that the granule structure limits the size of proteins that can be hydrolyzed by the protease.
Molecular cloning and primary structure of myelin-associated glycoprotein.
Authors: Arquint M , Roder J , Chia L S , Down J , Wilkinson D , Bayley H , Braun P , Dunn R
Published: May 2006

Myelin-associated glycoprotein (MAG) may play a role in the cellular interactions leading to myelination. Using monoclonal antibodies and conventional antisera against MAG, we have isolated a cDNA clone from an expression library prepared from rat brain mRNA. The identity of the clone was confirmed by the exact match between its nucleotide sequence and two peptide sequences of 13 and 9 amino acids that we obtained by Edman degradation of two CNBr fragments of MAG. The cDNA clone hybridized to two size species of mRNA in rat approximately 3.5 kilobases in length. These mRNAs were present in brain but not liver and were expressed most abundantly at the time of active myelination (day 14). The mRNA for MAG was present at barely detectable levels in hypomyelinating jimpy mice compared to normal littermate controls. Therefore the MAG cDNA clone is both brain and myelin specific. DNA sequence analysis revealed that our MAG cDNA was derived from the same mRNA as clone p1B236, a randomly selected, brain-specific, partial cDNA isolated by Sutcliffe et al. [Sutcliffe, J. G., Milner, R. J., Shinnick, T. M. & Bloom, F. E. (1983) Cell 33, 671-682]. Analysis of the predicted protein sequence suggests that MAG has a long extracellular domain (499 amino acids), followed by a short transmembrane segment (20 amino acids) and an intracellular carboxyl-terminal domain (90 amino acids). The molecule has several glycosylation sites, three internal repeats homologous to a repeat in the neural cell adhesion molecule (N-CAM), and sites for phosphorylation near the carboxyl terminus. The primary structure reported here provides a molecular framework for further investigations into the function of the MAG molecule.
In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum
Authors: Bureau Thomas E. , Brown R. Malcolm
Published: May 2006

The cytoplasmic and outer membranes of
Acetobacter xylinum
(ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme (EC 3.2.1.17) and trypsin (EC 3.4.21.4) were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase (UDP-glucose:1,4-β-D-glucan 4-β-D-glucosyltransferase; EC 2.4.1.12) activity was assayed as the conversion of radioactivity from UDP-[
14
C]glucose into an alkali-insoluble β-1,4-D-[
14
C]glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (
i
) enzymatic hydrolysis followed by TLC, (
ii
) methylation analysis followed by TLC, and (
iii
) GC/MS. Further, the weight-average and number-average degree of polymerization of the
in vitro
product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the
in vitro
product is cellulose II, which is in contrast to the
in vivo
product—namely, cellulose I.
Two 3' sequences direct adult erythroid-specific expression of human beta-globin genes in transgenic mice.
Authors: Behringer R R , Hammer R E , Brinster R L , Palmiter R D , Townes T M
Published: May 2006

Previous experiments have demonstrated that the human beta-globin gene is correctly regulated in transgenic mice. The beta-globin gene is not expressed in yolk sac-derived erythroid cells in early embryonic development but is expressed concomitantly with the adult mouse beta-globin genes in 14- to 16-day fetal liver and adult reticulocytes. In an attempt to localize sequences that direct erythroid-specific expression, fragments of the human beta-globin gene were inserted in the opposite orientation 200 base pairs (bp) upstream of an intact human A gamma marker gene, which is not expressed on its own in mouse fetal liver. In the experiments reported here, two beta-globin 3' sequences activated the marker gene specifically in fetal liver. One sequence is located in a 250-bp Pst I fragment 550-800 bp downstream from the poly(A) site; the other is located near an EcoRI site in the third exon. These two sequences are active individually, and their combined effect is greater than their effects alone. beta-Globin 5' sequences from -815 to -50 were also analyzed for activity in this assay. The 5' sequences did not activate the marker gene when tested alone but did stimulate expression that was already directed to adult erythroid tissue by the two 3' sequences. These results suggest that three separate sequences are involved in human beta-globin gene regulation. The two 3' sequences act as adult erythroid enhancers and the 5' sequence stimulates expression that is already determined to be erythroid specific.
Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).
Authors: Winkles J A , Friesel R , Burgess W H , Howk R , Mehlman T , Weinstein R , Maciag T
Published: May 2006

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.
Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA.
Authors: Gutman A , Kornblihtt A R
Published: May 2006

We describe here a third region of variability in human fibronectin (FN) due to alternative RNA splicing. Two other positions of alternative splicing have been reported previously (ED and IIICS). The third region involves a 273-nucleotide exon encoding exactly one 91-amino acid repeat of type III homology, located between the DNA- and the cell-binding domains of FN, which is either included in or excluded from FN mRNA. The two mRNA variants arising by an exon-skipping mechanism are present in cells known to synthesize the cellular form of FN. However, liver cells, which are the source of plasma FN, produce only messengers without the extra type III sequence. Therefore, the region described here resembles, both structurally and functionally, the previously described ED (for extra domain) region, located toward the C terminus of the molecule, between the cell- and heparin- (hep 2) binding domains. We conclude that both the extra type III repeat (named EDII) and ED represent sequences restricted to cellular FN. Combination of all the possible patterns of splicing in the three regions described to date may generate up to 20 distinct FN polypeptides from a single gene.
Thyroid hormones regulate levels of thyrotropin-releasing-hormone mRNA in the paraventricular nucleus.
Authors: Koller K J , Wolff R S , Warden M K , Zoeller R T
Published: May 2006

Cellular levels of messenger RNA encoding thyrotropin-releasing hormone (TRH) were measured in the paraventricular nucleus of the hypothalamus and the reticular nucleus of the thalamus in male rats after chemical thyroidectomy and thyroid hormone replacement. TRH mRNA levels were measured by quantitative in situ hybridization histochemistry using a 35S-labeled synthetic 48-base oligodeoxynucleotide probe and quantitative autoradiography. Chemical thyroidectomy, produced by the administration of 6-(n-propyl)-2-thiouracil (PrSur), reduced plasma thyroxine below detection limits and significantly increased TRH mRNA in the paraventricular nucleus. Treatment with exogenous L-triiodothyronine (T3) reduced TRH mRNA to the same level in both hypothyroid and euthyroid animals. Neither PrSur treatment nor T3 replacement influenced TRH mRNA levels in the reticular nucleus of the thalamus. Blot hybridization analysis of electrophoretically fractionated total RNA from pituitaries of these animals indicated that thyrotropin-beta mRNA levels were elevated after thyroidectomy and reduced by T3 treatment, showing that the pituitary-thyroid axis was indeed stimulated by PrSur treatment. These results suggest that thyroid hormones are involved, either directly or indirectly, in regulating the biosynthesis of TRH in the thyrotropic center of the hypothalamus.
Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo, and in vitro studies.
Authors: Fisone G , Wu C F , Consolo S , Nordström O , Brynne N , Bartfai T , Melander T , Hökfelt T
Published: May 2006

A high density of galanin binding sites was found by using 125I-labeled galanin, iodinated by chloramine-T, followed by autoradiography in the ventral, but not in the dorsal, hippocampus of the rat. Lesions of the fimbria and of the septum caused disappearance of a major population of these binding sites, suggesting that a large proportion of them is localized on cholinergic nerve terminals of septal afferents. As a functional correlate to these putative galanin receptor sites, it was shown, both in vivo and in vitro, that galanin, in a concentration-dependent manner, inhibited the evoked release of acetylcholine in the ventral, but not in the dorsal, hippocampus. Intracerebroventricularly applied galanin (10 micrograms/15 microliters) fully inhibited the scopolamine (0.5 mg/kg, s.c.)-stimulated release of acetylcholine in the ventral, but not in the dorsal, hippocampus, as measured by microdialysis technique. In vitro, galanin inhibited the 25 mM K+-evoked release of [3H]acetylcholine from slices of the ventral hippocampus, with an IC50 value of approximately 50 nM. These results are discussed with respect to the colocalization of galanin- and choline acetyltransferase-like immunoreactivity in septal somata projecting to the hippocampus.
Isolation and characterization of lymphokine cDNA clones encoding mouse and human IgA-enhancing factor and eosinophil colony-stimulating factor activities: relationship to interleukin 5.
Authors: Yokota T , Coffman R L , Hagiwara H , Rennick D M , Takebe Y , Yokota K , Gemmell L , Shrader B , Yang G , Meyerson P
Published: May 2006

Conditioned medium from the Con A-treated mouse helper T-cell clone Ly1+2-/9 contains activities that enhance the production of IgA by mouse B cells and induce human cord blood cells to form eosinophil colonies. We have isolated a cDNA sequence that expresses IgA-enhancing factor and eosinophil colony-stimulating factor activities from a cDNA library prepared from activated Ly1+2-/9 cells. Based on homology with the mouse cDNA sequence, a human cDNA sequence coding for an interleukin with IgA-enhancing factor and eosinophil colony-stimulating factor activities was isolated from a cDNA library prepared from a human T-cell clone stimulated with anti-T3 antibody and phorbol 12-myristate 13-acetate. DNA sequence analyses revealed that mouse and human cDNA clones encode proteins of 133 and 134 amino acids, respectively, that are identical to cDNA clones encoding the T-cell replacing factor I and B-cell growth factor II activities. These results establish that a single cDNA clone encodes a protein that acts as a growth and differentiation factor for both B cells and eosinophils.
Sequence of a New World primate insulin having low biological potency and immunoreactivity.
Authors: Seino S , Steiner D F , Bell G I
Published: May 2006

The organization of the insulin gene of the owl or night monkey (Aotus trivirgatus), a New World primate, is similar to that of the human gene. The sequences of these two genes and flanking regions possess 84.3% homology. An unusual feature of the owl monkey gene is the partial duplication and insertion of a portion of the A-chain coding sequence into the 3' untranslated region. The insulin gene of this primate also lacks a region of tandem repeats that is present in the 5' flanking region of the human and chimpanzee genes. Owl monkey preproinsulin has 85.5% identity with the human insulin precursor and is the most divergent of the primate insulins/preproinsulins yet described. The differences between owl monkey and human preproinsulin include three substitutions in the signal peptide, two in the B chain, seven in the C peptide, and three in the A chain. One of these replacements is the conservative substitution of valine for isoleucine at position A2, an invariant site in all other vertebrate insulins and insulin-like growth factors. The substitutions in owl monkey insulin at B9, B27, A2, A4, and A17 alter its structure so that it has only 20% of the receptor-binding activity and 1% of the affinity with guinea pig anti-porcine insulin antibodies as compared to human insulin.
Increased expression of major histocompatibility complex antigens on lymphocytes from aged mice.
Authors: Sidman C L , Luther E A , Marshall J D , Nguyen K A , Roopenian D C , Worthen S M
Published: May 2006

Many studies have reported age-related changes in immune responses that could be due to alterations in lymphoid cell numbers or functions. Here we report the results of studies using immunofluorescent staining and in vitro assays of cellular function to compare the expression of cell surface antigens on lymphocytes from mice up to 2 years of age. No significant changes were observed in the frequencies of spleen cells bearing class I or class II major histocompatibility complex (MHC) antigens, surface immunoglobulin, or Thy-1, Ly-1, Ly-2, or L3T4 antigens. However, the densities (per cell) of both class I and class II MHC antigens were increased significantly on cells from aged as compared to young mice, whereas the densities of the other cell surface antigens studied were unchanged or slightly decreased. The increased levels of MHC antigen expression in old relative to young mice were shown to be functionally significant regarding immunological stimulation. These data suggest that T-cell clones silent in young individuals may be activated in comparable situations in older animals, leading to immunological alterations perhaps including increased autoreactivity.
Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.
Authors: Bayliss D A , Millhorn D E , Gallman E A , Cidlowski J A
Published: May 2006

We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration.
Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence.
Authors: Lee S Y , Butler D , Kleckner N
Published: May 2006

Transposon Tn10 inserts preferentially at particular insertion "hot spots" that share a symmetrical 6-base-pair consensus sequence: 5' GCTNAGC 3'. The protein that recognizes this sequence is not known but is likely to be the Tn10-encoded transposase protein. We present evidence that the 5-methyl groups of the two thymines in this sequence are essential for efficient transposon insertion; in their absence the sequence is still recognized, but at lower efficiency. We have reached this conclusion by examination of a specific hot spot whose sequence is 5' GCCAGGC 3'. The innermost cytosines of this sequence happen to be substrates for methylation at their 5 positions by the bacterial dcm-encoded methylase. We find that Tn10 transposes into this site 15 times more frequently in a Dcm+ host than in a Dcm- host; in the Dcm- host, insertions still occur, but at a low frequency. Thus, at this site, the absence of pyrimidine 5-methyl groups at the third positions of the consensus sequence is sufficient to convert a strong insertion hot spot into a weaker but still recognizable hot spot. This observation supports the general proposition, suggested previously by comparisons among consensus sequences, that the presence or absence of these 5-methyl groups is one major feature that can make the difference between a strong and a weak Tn10 insertion hot spot.
Molecular and stochastic dynamics of proteins.
Authors: Nadler W , Brünger A T , Schulten K , Karplus M
Published: May 2006

The rapid fluctuations of protein atoms derived from molecular dynamics simulations can be extrapolated to longer-time motions by effective single-particle stochastic models. This is demonstrated by an analysis of velocity autocorrelation functions for the atoms of lysine side chains in the active site of RNase A. The atomic motions are described by a bounded stochastic model with the friction and noise parameters determined from a molecular dynamics simulation. The low-frequency relaxation behavior is shown to result from collisional damping rather than dephasing. Extrapolation of these results to the quasistochastic motion of the heme group in myoglobin provides an explanation of 57Fe Mössbauer spectroscopic data.
The partial molar volume of water in biological membranes.
Authors: Scherer J R
Published: May 2006

A new algorithm is presented for interpreting the hydration dependence of x-ray diffraction measurements. The method assumes that the volume of the hydrocarbon phase of the lipid bilayer is not affected by hydration and that the volume expansion between bilayers at maximum hydration is caused by incorporation of water molecules whose partial molar volume is that of pure bulk water. These simple assumptions lead to a determination of the area expansion (and hence change in hydrocarbon-phase thickness) as a function of hydration. An analysis is made of x-ray data of the L alpha and L beta' phases of dimyristoyl phosphatidylcholine and the L alpha phase of egg phosphatidylcholine. The partial molar volume of water depends critically on the degree of lipid hydration and the presence of voids between the head groups of adjacent lipids. The calculated head-group spacings at minimum hydration are consistent with those obtained from neutron diffraction and indicate that the methyl groups of the choline are almost in contact with corresponding groups in the opposing bilayer. This calls into question the origin of the repulsive forces observed in dehydration experiments.
Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.
Authors: Ricordi C , Lacy P E , Sterbenz K , Davie J M
Published: May 2006

In previous studies we have shown that rejection of islet xenografts transplanted between closely related species (rat to mouse) can be prevented by destruction or alteration of antigen-presenting cells in the donor islets and temporary immunosuppression of the recipients. Relatively few studies have been reported on the survival of islet xenografts transplanted between widely discordant species. In the present study, isolated human islets were transplanted beneath the renal capsule of B6 mice made diabetic by the injection of streptozotocin. The effect of culturing the human islets at 24 degrees C for 7 days with or without the administration of anti-L3T4 for 7 days after transplantation was determined. A marked prolongation of the mean survival time was obtained with low-temperature culture alone (greater than 40.2 +/- 9.9 days), with anti-L3T4 alone (greater than 45.2 +/- 6.3 days), and with the combination of these regimens (greater than 51.9 +/- 5.1 days) as compared to controls (7.5 +/- 1.1 days). The surprising finding was the marked effect of low-temperature culture alone on prolonging human islet xenograft survival because this treatment of the donor islets had no effect on the survival of rat islet xenografts. Intact human islets were present in approximately equal to 80% of the recipients after returning to a diabetic state, whereas xenografts of rat islets were completely destroyed. The findings indicate that complete rejection of islets across this widely discordant species barrier is slower than across a closely related barrier and may be occurring by a different rejection process.
Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the anterior pituitary gland.
Authors: Guillemette G , Balla T , Baukal A J , Catt K J
Published: May 2006

The properties of inositol 1,4,5-trisphosphate (InsP3) receptor sites in the anterior pituitary were evaluated by binding studies with InsP3 labeled with 32P to high specific radioactivity. Specific binding of Ins[32P]P3 was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP3 binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0 degree C. Addition of 1 microM unlabeled InsP3 was followed by rapid dissociation of the bound ligand, according to a single exponential function with half-time of about 1 min. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with Kd of 1.1 +/- 0.4 nM and maximal binding capacity of 28 +/- 15 fmol/mg of protein (n = 6). The specificity of Ins[32P]P3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP3 binding sites, the Ca2+-releasing activity of InsP3 was measured in pituitary membrane preparations. In the presence of oligomycin (2.5 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 1 mM ATP caused rapid uptake of Ca2+ by a vesicular component of the membrane fraction. Addition of InsP3 (50-2000 nM) caused a dose-dependent release of Ca2+ with a half-maximal effect at 240 nM. In the presence of 3% polyethylene glycol, GTP also stimulated calcium release. However, a nonhydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate, did not release calcium and completely blocked the effect of GTP. Under physiological conditions within the cytosol, the high-affinity InsP3 binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP3 triggers Ca2+ mobilization in the anterior pituitary gland.
Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase.
Authors: Price P A , Fraser J D , Metz-Virca G
Published: May 2006

Matrix Gla protein (MGP), a low molecular weight protein found in bone, dentin, and cartilage, contains 5 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid (Gla). We have used antibodies raised against MGP and oligonucleotide probes to screen a lambda gt11 cDNA library constructed from the rat osteosarcoma cells (line ROS 17/2) that had been pretreated with 1 alpha,25-dihydroxyvitamin D3. By sequencing several cloned cDNAs, we established a 523-base-pair sequence that predicts an 84-residue mature MGP and a 19-residue hydrophobic signal peptide. The 84-residue mature rat MGP predicted from the cDNA sequence has an additional 5 residues at its C terminus (-Arg-Arg-Gly-Ala-Lys) not seen in the sequence of MGP isolated from bovine bone. The structure of rat MGP provides insight into the mechanisms by which the vitamin K-dependent gamma-carboxylase recognizes substrate. The present studies show that MGP, unlike other vitamin K-dependent proteins, lacks a propeptide. The absence of an MGP propeptide demonstrates that gamma-carboxylation and secretion of vitamin K-dependent proteins need not be linked to the presence of a propeptide or to its proteolytic removal. The propeptides of other vitamin K-dependent proteins are structurally homologous, and there is evidence that this homologous propeptide domain is important to substrate recognition by the gamma-carboxylase. Mature MGP has a sequence segment (residues 15-30) that is homologous to the propeptide of other vitamin K-dependent proteins and probably serves the same role in gamma-carboxylase recognition. Rat MGP also has a second sequence that has recently been identified in all known vitamin K-dependent vertebrate proteins, the invariant unit Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys (EXXXEXC). Since the glutamic residues in this unit are sites of gamma-carboxylation, it has been suggested that the EXXXEXC unit could allow the gamma-carboxylase to discriminate between substrate and product. The demonstration that two structures common to vitamin K-dependent proteins, the homologous propeptides domain and the invariant EXXXEXC unit, are in mature MGP indicates that des-gamma-carboxy-MGP should be an excellent in vitro gamma-carboxylase substrate for analysis of mechanisms involved in substrate recognition and product dissociation.
cDNA sequence, protein structure, and chromosomal location of the human gene for poly(ADP-ribose) polymerase.
Authors: Cherney B W , McBride O W , Chen D F , Alkhatib H , Bhatia K , Hensley P , Smulson M E
Published: May 2006

Recently we described a full-length cDNA for the human nuclear enzyme poly(ADP-ribose) polymerase. Here, we report the chromosomal localization and partial map of the human gene for this enzyme as well as the complete coding sequence for this protein. The nucleotide sequence reveals a single 3042-base open reading frame encoding a protein with a predicted Mr of 113,135. A comparison of this deduced amino acid sequence with the amino acid sequence of three peptides derived from human poly(ADP-ribose) polymerase revealed a match of 27 amino acid residues. A computer-derived structural analysis of the enzyme and a search for similarities with other proteins confirmed that the polymerase belongs to a subfamily of DNA/NAD-binding proteins and DNA-repair proteins. Possible Zn2+-binding "fingers," a nucleotide-binding fold, and a nuclear transport signal were noted. Additionally, chromosomal mapping has identified polymerase-hybridizing sequences on human chromosomes 1 (the active gene), 13, and 14 (processed pseudogenes). Using the polymerase cDNA as a probe, we also have detected several DNA restriction fragment length polymorphisms in normal humans.
Centrosome detection in sea urchin eggs with a monoclonal antibody against Drosophila intermediate filament proteins: characterization of stages of the division cycle of centrosomes.
Authors: Schatten H , Walter M , Mazia D , Biessmann H , Paweletz N , Coffe G , Schatten G
Published: May 2006

A mouse monoclonal antibody generated against Drosophila intermediate filament proteins (designated Ah6/5/9 and referred to herein as Ah6) is found to cross-react specifically with centrosomes in sea urchin eggs and with a 68-kDa antigen in eggs and isolated mitotic apparatus. When preparations stained with Ah6 are counterstained with a human autoimmune serum whose anti-centrosome activity has been established, the immunofluorescence images superimpose exactly. A more severe test of the specificity of the antibody demands that it display all of the stages of the centrosome cycle in the cell cycle: the flattening and spreading of the compact centrosomes followed by their division and the establishment of two compact poles. The test was made by an experimental design that uses a period of exposure of the eggs to 2-mercaptoethanol. This treatment allows observation of the stages of the centrosome cycle--separation, division, and bipolarization--while the chromosomes are arrested in metaphase. Mitosis is arrested in the presence of 0.1 M 2-mercaptoethanol. Chromosomes remain in a metaphase configuration while the centrosomes divide, producing four poles perpendicular to the original spindle axis. Microtubules are still present in the mitotic apparatus, as indicated by immunofluorescence and transmission electron microscopy. When 2-mercaptoethanol is removed, the chromosomes reorient to the poles of a tetrapolar (sometimes tripolar) mitotic apparatus. During the following cycle, the blastomeres form a monopolar mitotic apparatus. The observations of the centrosome cycle with the Ah6 antibody display very clearly all the stages that have been seen or deduced from work with other probes. The 68-kDa antigen that reacts with the Ah6 monoclonal antibody to Drosophila intermediate filament proteins must be a constant component of sea urchin centrosomes because it is present at all stages of the centrosome cycle.
Developmental regulation of the estrogen receptor and the estrogen responsiveness of five yolk protein genes in the avian liver.
Authors: Evans M I , O'Malley P J , Krust A , Burch J B
Published: May 2006

The magnitude of the expression of five yolk protein genes in the avian liver in response to exogenous estradiol is shown to be developmentally regulated. Though each of these yolk protein genes gains the capacity to respond to estradiol during embryonic development, we demonstrate that maximal responses for the different genes are achieved at distinct ages between 1 and 6 weeks after hatching. This observation prompted us to look for possible correlations between yolk protein gene expression and changes in the expression of estrogen receptors that might also occur after hatching. We discovered that indeed the maximal level of nuclear estrogen receptors (assayed following the administration of estradiol) increases progressively over this same period of development from approximately 1000 receptors per cell at 1 week after hatching to approximately 3500 receptors per cell at 6 weeks after hatching. The latter number represents the fully mature state, as comparable levels of receptors are present in the livers of egg-laying hens. Thus, though increases in the expression of estrogen receptors during embryonic liver development have previously been reported, our results indicate that the changes that occur after hatching are quantitatively far more significant to the developmental program for this transcription factor.
Cellular expression of liver and neural cell adhesion molecules after transfection with their cDNAs results in specific cell-cell binding.
Authors: Edelman G M , Murray B A , Mege R M , Cunningham B A , Gallin W J
Published: May 2006

Mouse L cells, which do not express the known primary cell adhesion molecules (CAMs), were permanently transfected with vectors containing the simian virus 40 early promoter and cDNA sequences encoding chicken liver CAM (L-CAM) or each of the three major polypeptide forms of chicken neural CAM (N-CAM). Transfected cells in culture expressing the Ca2+-dependent L-CAM showed uniform surface expression of the molecule. Unlike untransfected L cells, these cells aggregated readily; the aggregation was inhibited by Fab' fragments of antibodies to L-CAM but not by fragments of anti-N-CAM. These cells spread more efficiently in culture than did their untransfected counterparts, forming small colonies of flattened cells that gradually assumed morphologies resembling closely packed L cells. Transfected L cells expressing either the small or large intercellular domain polypeptide (sd or ld) chains of N-CAM aggregated specifically with each other or bound membrane vesicles from chick brain. Both types of binding were specifically inhibited by Fab' fragments of anti-N-CAM antibodies. These cells, in contrast to those transfected with vectors for L-CAM, showed rounded morphologies and spread inefficiently in culture. L cells transfected with vectors specifying the small surface domain polypeptide (ssd) chain of N-CAM showed no phenotypic changes and no evidence for linkage of ssd chains to the cell membrane by phosphatidylinositol intermediates. Instead, these cells synthesized the molecule and released it into the medium. These findings complete the demonstration that different CAMs have specific roles in ligating the cells that synthesize them, and they provide further evidence that L-CAM and N-CAM bind by homophilic mechanisms. The different phenotypic changes observed for each specific CAM are consistent with the hypothesis that CAM synthesis or differing associations of CAM carboxyl-terminal domains with the cell surface and cortex may lead directly or indirectly to specific alterations in the cells bound together by that CAM.
Determinants of membrane protein topology.
Authors: Boyd D , Manoil C , Beckwith J
Published: May 2006

The topology of the integral membrane protein MalF, which is required for maltose transport in Escherichia coli, has been analyzed using fusions of alkaline phosphatase (EC 3.1.3.1). The properties of such fusion strains support a MalF structure previously proposed on theoretical grounds. Several transmembrane segments within MalF can act as signal sequences in exporting alkaline phosphatase. Other transmembrane sequences, in conjunction with cytoplasmic domains, can stably anchor alkaline phosphatase in the cytoplasm. Our results suggest that features of the amino acid sequence (possibly the positively charged amino acids) of the cytoplasmic domains of membrane proteins are important in anchoring these domains in the cytoplasm. These studies in conjunction with our earlier results show that alkaline phosphatase fusions to membrane proteins can be an important aid in analyzing membrane topology and its determinants.
Modulation of endogenous hormone action by recombinant human tumor necrosis factor.
Authors: Warren R S , Donner D B , Starnes H F , Brennan M F
Published: May 2006

Tumor necrosis factor (TNF) has been implicated in the toxic manifestations of overwhelming bacterial infection and in the tissue wasting that often accompanies prolonged infections and malignancy. We have examined a possible role of TNF in the early metabolic alterations following acute tissue injury or sepsis. Recombinant human TNF stimulated rat liver amino acid uptake up to 5-fold in vivo and there was a concomitant increase in plasma glucagon. In vitro TNF had no direct effect on hepatocyte amino acid uptake, but it markedly enhanced the stimulation of amino acid transport by glucagon, without an alteration in binding of glucagon to hepatocytes. This permissive effect of TNF on glucagon action represents an interrelationship between the immune and endocrine systems, and it may help to explain the mechanism of hormonal regulation of both the anabolic and catabolic responses to acute injury.
The neuropeptide cerebellin is a marker for two similar neuronal circuits in rat brain.
Authors: Mugnaini E , Morgan J I
Published: May 2006

We report here that the neuropeptide cerebellin, a known marker of cerebellar Purkinje cells, has only one substantial extracerebellar location, the dorsal cochlear nucleus (DCoN). By reverse-phase high-performance liquid chromatography and radioimmunoassay, cerebellum and DCoN in rat were found to contain similar concentrations of this hexadecapeptide. Immunocytochemistry with our rabbit antiserum C1, raised against synthetic cerebellin, revealed that cerebellin-like immunoreactivity in the cerebellum is localized exclusively to Purkinje cells, while in the DCoN, it is found primarily in cartwheel cells and in the basal dendrites of pyramidal neurons. Some displaced Purkinje cells were also stained. Although cerebellum and DCoN receive their inputs from different brain regions, their organizations show several similarities, with many neuronal cell types present in one having their presumed homologue in the other. Cerebellin is present in Purkinje and cartwheel cells throughout the cerebellum and the DCoN without site predilection. This should be viewed together with recent data indicating that these two classes of neurons share a similar fate in several murine mutations ("nervous," "lurcher," "Purkinje cell degeneration," and "staggerer"). This situation leads us to conclude that cerebellin represents a marker for a small number of spatially related neuronal cell classes, and that it may play a role in an as yet unknown aspect of a circuit function worthy of further exploration.
Activation of mouse epidermal tumor ornithine decarboxylase by GTP: evidence for different catalytic forms of the enzyme.
Authors: O'Brien T G , Hietala O , O'Donnell K , Holmes M
Published: May 2006

In crude extracts of epidermal papillomas induced by an initiation-promotion protocol, ornithine decarboxylase (OrnDCase) activity was increased by the addition of GTP to the enzyme assay. No effect of GTP on the phorbol ester-induced enzyme isolated from normal epidermis was observed. Kinetic analyses indicated that the major effect of the nucleotide on the tumor-derived enzyme was to lower the apparent Km for L-ornithine. When papilloma OrnDCase was partially purified by gel-filtration chromatography, two forms of the enzyme were resolved, only one of which was found in an epidermal extract from phorbol 12-myristate 13-acetate-treated mice. The enzymatic properties of the two forms of papilloma enzyme were compared. The higher molecular weight form (peak I) was activated by GTP, while the lower molecular weight form (peak II) was not. As expected from the kinetic analyses of the crude papilloma extracts, the apparent Km of peak I enzyme for L-ornithine was very high (1.25 mM) but was much lower in the presence of GTP (0.02 mM). The two forms of papilloma OrnDCase differed in their sensitivities to heat inactivation and the ability of GTP to protect against heat inactivation. The K1/2 for activation of peak I OrnDCase by GTP was 0.1 microM. The activation process was irreversible and did not require Mg2+. When several nucleotides were tested for their ability to activate peak I OrnDCase, only GTP, dGTP, and the nonhydrolyzable derivative GTP[gamma-S] were effective, while GDP, GMP, ATP, and CTP were relatively ineffective. Our results demonstrated the existence of two forms of OrnDCase in epidermal tumor extracts, of which one can be activated by GTP and one cannot. The significance of these findings for the regulation of this enzyme in normal and tumor cells is discussed.
Functional pools of oxidative and glycolytic fibers in human muscle observed by 31P magnetic resonance spectroscopy during exercise.
Authors: Park J H , Brown R L , Park C R , McCully K , Cohn M , Haselgrove J , Chance B
Published: May 2006

Quantitative probing of heterogeneous regions in muscle is feasible with phosphorus-31 magnetic resonance spectroscopy because of the differentiation of metabolic patterns of glycolytic and oxidative fibers. A differential recruitment of oxidative and glycolytic fibers during exercise was demonstrated in 4 of 10 untrained young men by following changes in phosphate metabolites. Concentrations of inorganic phosphate (Pi), phosphocreatine, and ATP were estimated in the wrist flexor muscles of the forearm at rest, during two cycles of three grades of exercise, and in recovery. At high work levels (40% of maximum strength), two distinct Pi peaks were observed and identified with Pi pools at pH 6.9 and pH 5.9-6.4, respectively. These could be accounted for as follows. At the lowest level of work (using 20% of maximum strength), early recruitment primarily of oxidative (type I) and possibly some intermediate (type IIA) muscle fibers occurs with relatively little net lactate production and consequently little decrease in pH. At higher work loads, however, primarily glycolytic (type IIB) muscle fibers are recruited, which have relatively high net lactate production and therefore generate a second pool of Pi at low pH. ATP depletion (35-54%) and Pi losses accompanied the reduction in ability to perform during the first exercise cycle. When the cycle of graded exercise was repeated immediately, the total Pi remained high but gave rise to only one peak at pH 6.8-7.0. These observations indicated exhaustion of glycolytic type IIB fibers, removal of lactate by high local blood flow, and sustained contractions largely by oxidative type I and IIA fibers. A functional differentiation of fiber types could also be demonstrated during recovery if exercise was stopped while two pools of Pi were still apparent. In the first 3 min of recovery, the Pi peak at pH 6.8-6.9 disappeared almost entirely, whereas the Pi peak at pH 6.0 remained unaltered, reflecting the faster recovery of oxidative type I fibers. The potential of magnetic resonance spectroscopy to characterize oxidative and glycolytic fibers, predict capacity for aerobic performance, and signal the presence of muscle pathology is discussed.