bluuebunny/crossref_metadata_2025 huggingface dataset:
Rate of evolution of a quantitative character.
Authors: Nagylaki T
Published: May 2006

The rate of change of the mean of a quantitative character is evaluated exactly and also under the hypothesis of linear biparental regression. Generations are discrete and nonoverlapping; the monoecious population mates at random. The genotypic and environmental contributions to the character are additive and stochastically independent. The character is influenced by arbitrarily many multiallelic loci and has constant genotypic values; dominance, epistasis, and the linkage map are also arbitrary. The population is initially in linkage equilibrium, and there is no position effect. If the biparental regression is linear, then the regression coefficient is simply Vgam/V, and hence the single-generation change in the mean is deltaZ = (Vgam/V)S, where Vgam and V denote the gametic and total variances in the character and S designates the selection differential. The corresponding exact result is DeltaZ = (C/W) + (B/W2), where C, W and B represent the gametic covariance of the character and fitness, the mean fitness, and a correction term, respectively. If selection is weak, then DeltaZ approximately C/W. Furthermore, deltaZ = C/W if either there is no environmental contribution and the gametic effects are additive or the character is fitness itself. In the latter case, C is the gametic variance in fitness. Thus, even in linkage equilibrium, weakness of selection generally does not suffice to validate the linear-regression result. This conclusion holds even for additive loci.
Regulation of PACE propeptide-processing activity: requirement for a post-endoplasmic reticulum compartment and autoproteolytic activation.
Authors: Rehemtulla A , Dorner A J , Kaufman R J
Published: May 2006

PACE (paired basic amino acid cleaving enzyme) is a subtilisin-like serine protease involved in processing of propeptides in the constitutive secretory pathway. We here demonstrate that the transmembrane and cytoplasmic domains of PACE are required for retention in the secretory pathway but not for propeptide-cleaving activity. Addition of the endoplasmic reticulum retention signal Lys-Asp-Glu-Leu (KDEL) to the carboxyl terminus of the truncated molecule resulted in intracellular retention of the protein and loss of activity, indicating that the endoplasmic reticulum is an inappropriate environment for propeptide processing. In addition, mutation of a consensus PACE cleavage site within the amino-terminal region prevented processing of PACE to a mature form and destroyed activity. These data indicate that PACE is synthesized as a proprotein which requires autoproteolytic removal of an 81-residue pro sequence for optimal activity. A mutant form of PACE that lacked the pro sequence was nonfunctional, and addition of a pro sequence from a homologous subtilisin-like serine protease, PC2, did not restore activity. By analogy to the bacterial subtilisin family, the propeptide of PACE may guide the folding of PACE into an active enzyme.
Cloning and expression of mouse integrin beta p(beta 7): a functional role in Peyer's patch-specific lymphocyte homing.
Authors: Hu M C , Crowe D T , Weissman I L , Holzmann B
Published: May 2006

Lymphocytes express integrin receptors, termed lymphocyte Peyer's patch high endothelial venule (HEV) adhesion molecules (LPAMs), that mediate their organ-specific adhesion to specialized HEVs found in mucosal lymphoid organs (Peyer's patches). LPAM-1 consists of a murine integrin alpha 4 noncovalently associated with integrin beta p. Here, we describe the cloning and expression of a mouse cDNA encoding beta p, which is an 806-amino acid transmembrane glycoprotein. The genomic Southern blot analysis indicates that beta p is the murine homologue of human beta 7. The function of alpha 4 beta 7 as a Peyer's patch-specific adhesion molecule was tested directly by expression of the murine beta 7 cDNA in an alpha 4+ beta 7-B-cell line or coexpression of the alpha 4 and beta 7 cDNAs in an alpha 4-beta 7-T-cell line. The transfected cells exhibited a new Peyer's patch-specific adhesive phenotype that could be specifically blocked by monoclonal antibodies against alpha 4 and beta 7. Moreover, an anti-beta 7 monoclonal antibody specifically blocked binding of normal lymphocytes to Peyer's patch HEV but did not inhibit their binding to peripheral lymph node HEVs, indicating that beta 7 is a unique component of the Peyer's patch-specific homing receptor.
Intrinsic signal optical imaging in the forepaw area of rat somatosensory cortex.
Authors: Gochin P M , Bedenbaugh P , Gelfand J J , Gross C G , Gerstein G L
Published: May 2006

The responses of somatosensory cortex (S-I) to tactile stimulation of the forepaw were assessed by intrinsic signal optical imaging. The tips of digits two or five were alternately touched with mechanical tappers while video photographs were taken of S-I illuminated by an 800-nm light source. The resulting images showed two highlighted areas about 300 microns in diameter and 500 microns apart. Generation of these images required less than 1 hr. Electrode penetrations placed in the areas highlighted during stimulation provided multiunit recordings with receptive fields appropriate for the stimulated digit and not the other digit. Penetrations between the high-lighted areas yielded receptive fields on intervening digits. These results demonstrate that intrinsic signal optical images are obtainable in S-I and confirm the functional somatotopy previously reported using electrical recording. Furthermore, the short time required to produce the images and the obtainable spatial resolution suggest that optical recording could be employed for the study of cortical reorganization in this brain region.
Linkage mapping by simultaneous screening of multiple polymorphic loci using Alu oligonucleotide-directed PCR.
Authors: Zietkiewicz E , Labuda M , Sinnett D , Glorieux F H , Labuda D
Published: May 2006

We present the use of our recently described multiple-loci polymorphic DNA markers ("alumorphs") for linkage mapping of the human genome. By using the polymerase chain reaction (PCR) with an Alu-specific primer we could reveal, in a single experiment, up to 20 genomic polymorphisms seen as the presence or absence of amplified DNA fragments originating from genomic segments flanked by Alu repeats. Using this approach we examined genomic DNA samples from two families with a history of pseudovitamin D-deficiency rickets (PDDR), an autosomal recessive disorder. An indication of linkage with the PDDR phenotype was found for one of the polymorphic bands, denoted 30A. A significant linkage [logarithm-of-odds (lod) score greater than 3.0] was obtained between this polymorphism and a number of chromosome 12q markers tightly linked to PDDR. The 30A band specifically hybridized to DNA digests from hybrid cell lines carrying a human chromosome 12, thus independently assigning the 30A marker to this chromosome. Since Alu elements are ubiquitous in human DNA, the use of alternative Alu-specific primers, which reveal different sets of Alu-flanked loci, should provide an efficient and rapid approach to human genetic mapping.
Biosynthetic regulation of proteoglycans by aldohexoses and ATP.
Authors: Kanwar Y , Yoshinaga Y , Liu Z , Wallner E , Carone F
Published: May 2006

Biosynthetic regulation of renal glomerular heparan sulfate-proteoglycans by various aldohexoses (mannose, glucose, and galactose) was investigated. Isolated kidneys were perfused for 5 hr with medium containing [35S]sulfate, to label sulfated proteoglycans, or [35S]methionine, to label total glomerular proteins. All the hexoses, above 10 mM concentration, caused a significant decrease in the de novo synthesis of [35S]sulfate-labeled proteoglycans. The relative effectiveness of the hexoses was as follows: mannose much greater than glucose greater than galactose. The proteoglycans were of relatively lower molecular weights and exhibited reduced charge-density characteristics. Autoradiographic studies revealed a 2- to 3-fold decrease of grain density over the glomerular basement membrane and mesangial compartments, and immunoprecipitable heparan sulfate-proteoglycans were similarly decreased 2- to 3-fold. There was no significant decrease in the total [35S]methionine-labeled glomerular proteins or immunoprecipitable type IV collagen and laminin. Cellular ATP levels were dramatically reduced in all groups, and the maximal depletion was caused by mannose. Addition of ATP (0.1-1.0 mM) to the perfusion medium resulted in the normalization of the de novo synthesis and of the biochemical characteristics of heparan sulfate-proteoglycans. The relevance of decreased de novo synthesis of proteoglycans due to the depletion of ATP in hyperglycemic states is discussed in terms of increased glomerular permeability to plasma proteins, as seen in diabetes mellitus.
Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1.
Authors: Thorens B
Published: May 2006

Glucagon-like peptide 1 (GLP-1) is a hormone derived from the preproglucagon molecule and is secreted by intestinal L cells. It is the most potent stimulator of glucose-induced insulin secretion and also suppresses in vivo acid secretion by gastric glands. A cDNA for the GLP-1 receptor was isolated by transient expression of a rat pancreatic islet cDNA library into COS cells; this was followed by binding of radiolabeled GLP-1 and screening by photographic emulsion autoradiography. The receptor transfected into COS cells binds GLP-1 with high affinity and is coupled to activation of adenylate cyclase. The receptor binds specifically GLP-1 and does not bind peptides of related structure and similar function, such as glucagon, gastric inhibitory peptide, vasoactive intestinal peptide, or secretin. The receptor is 463 amino acids long and contains seven transmembrane domains. Sequence homology is found only with the receptors for secretin, calcitonin, and parathyroid hormone, which form a newly characterized family of G-coupled receptors.
Chromosome location of Oryza sativa recombination linkage groups.
Authors: Gustafson J P , Dillé J E
Published: May 2006

In situ hybridization, a powerful tool for the molecular cytogeneticist, can be used to physically map repetitive, low-copy, and unique DNA sequences in plant chromosomes. With the availability of a recombination map in Oryza sativa L. and an improved in situ hybridization technique, this study was designed to establish the relationship between the genetic and physical distances of the rice restriction fragment length polymorphism map. Analysis indicated that considerable variation can exist between genetic and physical maps. A 183-centimorgan linkage map for chromosome 2 covered less than 50% of the chromosome and did not include the centromere, whereas a 91-centimorgan linkage map for chromosome 1 covered approximately 80% of the chromosome. The results indicated that there are potential "hot" and "cold" spots of recombination and polymorphisms in rice, which involve both genes and restriction fragment length polymorphisms.
Occurrence of the alpha subunits of G proteins in cerebral cortex synaptic membrane and postsynaptic density fractions: modulation of ADP-ribosylation by Ca2+/calmodulin.
Authors: Wu K , Nigam S K , LeDoux M , Huang Y Y , Aoki C , Siekevitz P
Published: May 2006

We have examined the isolated postsynaptic density (PSD) fraction for the presence of a G protein. First, we found specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate to the PSD. Second, pertussis toxin-activated ADP-ribosylation of the isolated PSD fraction resulted in the appearance of a G protein with an apparent molecular mass of 41 kDa, and two G proteins with apparent molecular masses of 41 kDa and 39 kDa in synaptic membrane (SM) fraction and total homogenate (H). The amount of the 41-kDa G protein per unit protein was in the order of SM greater than H greater than PSD. Anti-G(i0 antibodies recognized the 41-kDa G protein in both PSD and SM, whereas anti-G(o) antibodies reacted with the 39-kDa G protein in the SM. The absence of G(o) protein in the PSD suggested that there was no contamination with SM. Moreover, unlabeled PSD incubated with an extract of SM that contained the labeled G proteins resulted in no label in the subsequently reisolated PSD, suggesting that the G protein found in the PSD was not due to adsorption of the G protein onto the PSD during its isolation from the SM. PSD pretreated with EGTA gave an 11-fold increase in the ADP-ribosylation reaction of the G(i) protein; similar effects on the G(i) and G(o) proteins of SM were obtained. Restoration of Ca2+/calmodulin to the PSD, but not of either Ca2+ or calmodulin alone, removed the effect of EGTA, indicating a strong complex formation between G(i) and Ca2+/calmodulin that decreased the ADP-ribosylation reaction. Preincubation with the Ca(2+)-channel blocker nifedipine decreased the ADP-ribosylation reaction in the PSD. We conclude that G(i) is present in the PSD, that it may interact with calmodulin and that it is involved in the regulation of voltage-dependent Ca2+ channel. We present a theory of the involvement of the G protein and calmodulin in postsynaptic neurophysiological events.
Defective signal transduction by the CD2 molecule in immature T-cell receptor/CD3- thymocytes.
Authors: Turka L A , Fletcher M C , Craighead N , Thompson C B , June C H
Published: May 2006

The CD2 accessory molecule mediates an activation pathway in mature T cells, transducing signals similar to those observed following stimulation of the T-cell receptor/CD3 (TCR/CD3) complex. CD2 is also one of the earliest cell surface markers to appear during thymic ontogeny and has been proposed to be a stimulatory pathway for immature thymocytes that have not yet expressed TCRs on their surface (TCR/CD3-). To examine this hypothesis highly purified TCR/CD3- human thymocytes were stimulated using mitogenic combinations of anti-CD2 monoclonal antibodies or individual biotinylated anti-CD2 monoclonal antibodies crosslinked with avidin. TCR/CD3+ thymocytes responded readily to either stimulus as determined by anti-phosphotyrosine immunoblotting, and the pattern of tyrosine phosphorylated substrates was similar to that of mature T cells. In contrast, TCR/CD3- thymocytes responded weakly and with a distinct substrate pattern. In addition, the altered signal transduced by CD2 in TCR/CD3- thymocytes did not lead to a rise in intracellular calcium, failed to induce interleukin 2 receptor expression, and did not serve as a comitogen with phorbol ester or interleukin 2, functions that were all intact in TCR/CD3+ thymocytes. Failure of TCR/CD3- thymocytes to respond to CD2 stimulation was not due to an intrinsic defect in these cells as they responded normally to phorbol ester plus calcium ionophore. In TCR/CD3- thymocytes, CD2 stimulation also failed to affect steady-state mRNA levels of the recombination-activating genes RAG1 and RAG2, whereas in TCR/CD3+ cells activation of the CD2 pathway terminated their expression. Together, these data support the concept that CD2 engagement does not deliver a stimulus to TCR/CD3- thymocytes and suggests that this molecule may not directly participate in the earliest stages of thymic development.
Shc proteins are phosphorylated and regulated by the v-Src and v-Fps protein-tyrosine kinases.
Authors: McGlade J , Cheng A , Pelicci G , Pelicci P G , Pawson T
Published: May 2006

The mammalian shc gene encodes two overlapping proteins of 46 and 52 kDa, each with a C-terminal Src homology 2 (SH2) domain and an N-terminal glycine/proline-rich sequence, that induce malignant transformation when overexpressed in mouse fibroblasts. p46shc, p52shc, and an additional 66-kDa shc gene product become highly tyrosine phosphorylated in Rat-2 cells transformed by the v-src or v-fps oncogene. Experiments using temperature-sensitive v-src and v-fps mutants indicate that Shc tyrosine phosphorylation is rapidly induced upon activation of the v-Src or v-Fps tyrosine kinases. These results suggest that Shc proteins may be directly phosphorylated by the v-Src and v-Fps oncoproteins in vivo. In cells transformed by v-src or v-fps, or in normal cells stimulated with epidermal growth factor, Shc proteins complex with a poorly phosphorylated 23-kDa polypeptide (p23). Activated tyrosine kinases therefore regulate the association of Shc proteins with p23 and may thereby control the stimulation of an Shc-mediated signal transduction pathway. The efficient phosphorylation of Shc proteins and the apparent induction of their p23-binding activity in v-src- and v-fps-transformed cells are consistent with the proposition that the SH2-containing Shc polypeptides are biologically relevant substrates of the oncogenic v-Src and v-Fps tyrosine kinases.
Inhibition of the self-renewal capacity of blast progenitors from acute myeloblastic leukemia patients by site-selective 8-chloroadenosine 3',5'-cyclic monophosphate.
Authors: Pinto A , Aldinucci D , Gattei V , Zagonel V , Tortora G , Budillon A , Cho-Chung Y S
Published: May 2006

The physiologic balance between the two regulatory subunit isoforms, RI and RII, of cAMP-dependent protein kinase is disrupted in cancer cells; growth arrest and differentiation of malignant cells can be achieved when the normal ratio of these intracellular signal transducers of cAMP is restored by the use of site-selective cAMP analogs. In this study we evaluated the effects of the site-selective cAMP analog 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) on clonogenic growth of blast progenitors from 15 patients with acute myeloblastic leukemia and 3 patients affected by advanced myelodysplastic syndrome. Leukemic blast progenitors undergo terminal divisions, giving rise to colonies in methylcellulose. The self-renewal capacity of blast progenitors is conversely reflected in a secondary methylcellulose assay after exponential growth of clonogenic cells in suspension cultures. In all the samples tested, 8-Cl-cAMP, at micromolar concentrations (0.1-50 microM), suppressed in a dose-dependent manner both primary colony formation in methylcellulose and the recovery of clonogenic cells from suspension culture. Strikingly, in the samples from the entire group of patients, 8-Cl-cAMP was more effective in inhibiting the self-renewing clonogenic cells than the terminally dividing blast cells (P = 0.005). In addition, in four out of six cases studied, 8-Cl-cAMP was able to induce a morphologic and/or immunophenotypic maturation of leukemic blasts. An evident reduction of RI levels in fresh leukemic cells after exposure to 8-Cl-cAMP was also detected. Our results showing that 8-Cl-cAMP is a powerful inhibitor of clonogenic growth of leukemic blast progenitors by primarily suppressing their self-renewal capacity indicate that this site-selective cAMP analog represents a promising biological agent for acute myeloblastic leukemia therapy in humans.
Identification of a second insulin-like growth factor in a fish species.
Authors: Shamblott M J , Chen T T
Published: May 2006

An internal portion of insulin-like growth factor (IGF) amplified from the total cDNA of rainbow trout (Oncorhynchus mykiss) liver by a PCR was used to screen a rainbow trout liver cDNA library, and recombinant clones encoding two distinct IGFs were isolated. On the basis of a 98.7% nucleotide and 98.3% predicted amino acid identity to coho salmon IGF-I, one cDNA sequence was identified as rainbow trout preproinsulin-like growth factor I (rtIGF-I). The second cDNA sequence shared 46.1% and 43.3% identity with rtIGF-I at the nucleotide and predicted amino acid levels, respectively, and was identified as rainbow trout preproinsulin-like growth factor II (rtIGF-II). Predicted amino acid sequence comparisons of rtIGFs with those of human IGFs indicate that rtIGF-I is more similar to human IGF-I than to human IGF-II, and that rtIGF-II is more similar to human IGF-II than to human IGF-I. Southern blot analysis of rainbow trout genomic DNA probed with rtIGF-I and -II cDNA suggests that these two forms of IGF originate from separate genes. The presence of a teleost IGF-II suggests that the divergence of IGFs occurred early in vertebrate evolution.
Defective guanyl nucleotide-binding protein beta gamma subunits in a forskolin-resistant mutant of the Y1 adrenocortical cell line.
Authors: Mitchell J , Northup J K , Schimmer B P
Published: May 2006

Forskolin-resistant mutants derived from Y1 adrenocortical cells display decreased responsiveness both to receptor and postreceptor stimulators of adenylyl cyclase and decreased amounts of the alpha subunits of the GTP-binding proteins (G proteins) that mediate stimulation (Gs) and inhibition (Gi) of adenylyl cyclase--namely, Gs alpha and Gi alpha-2. This phenotype is suggestive of a mutation that affects the processing or plasma membrane incorporation of G protein alpha subunits. Since the membrane attachment of heterotrimeric G proteins has been ascribed in part to the beta gamma subunits, we examined the quantity and functional activity of beta gamma subunits in wild-type Y1 and forskolin-resistant Forsk-10r-9 and Forsk-10r-3 cells. We now show that two assays previously used to examine the activity of purified beta gamma subunits--namely, to support either rhodopsin-catalyzed guanyl nucleotide exchange on Gt alpha or pertussis toxin-catalyzed ADP-ribosylation of Gt alpha--can be used with detergent extracts of cells. In both assays the beta gamma activity in Forsk-10r-9 and Forsk-10r-3 extracts was decreased by 53-76% compared with wild-type Y1 extracts. When normalized for immunoreactive beta subunit, the beta gamma activity in the Forsk-10r-9 samples was decreased by 55-57% compared with the wild-type Y1 samples. These results suggest that a mutation of one of the G protein beta or gamma subunits may result in the multiple defects of adenylyl cyclase activity and apparent loss of G protein alpha subunits seen in the forskolin-resistant mutant cells. The frequency with which these spontaneous mutations arise in the Y1 cell line suggests that they may contribute more generally to genetic abnormalities in signal transduction.
Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells.
Authors: Raffioni S , Bradshaw R A
Published: May 2006

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGF-stimulated PC12 cells by an anti-phosphotyrosine antibody. All three growth factors increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is 15- to 20-fold, 2- to 3-fold, and 8- to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-gamma to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these growth factors. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite outgrowth but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these growth factors, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.
mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina.
Authors: Kim R Y , Gasser R , Wistow G J
Published: May 2006

mu-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that mu-crystallin is expressed at high abundance in lens, but outside the lens mu-crystallin is preferentially expressed in neural tissues, retina, and brain. An almost full-length cDNA for mu-crystallin was cloned from human retina. In human tissues, mu-crystallin mRNA is present in neural tissue, muscle, and kidney. This pattern of expression and relationship to an enzyme involved in unusual amino acid metabolism suggests the interesting possibility that mammalian mu-crystallins could be enzymes participating in processes such as osmoregulation or the metabolism of excitatory amino acids.
[D-Ala2]deltorphin I binding and pharmacological evidence for a special subtype of delta opioid receptor on human and invertebrate immune cells.
Authors: Stefano G B , Melchiorri P , Negri L , Hughes T K , Scharrer B
Published: May 2006

The effects of the opioid neuropeptide [D-Ala2]deltorphin I, isolated from amphibian skin, on immunoregulatory activities were studied in representatives of vertebrates and invertebrates. The high potency of this compound parallels that of [Met]enkephalin, which was previously demonstrated in vertebrate plasma and invertebrate hemolymph. The addition of [D-Ala2]deltorphin I at 10(-11) M to human granulocytes or immunocytes of the mollusc Mytilus edulis resulted in cellular adherence and conformational changes indicative of cellular activation. This value is in line with the concentrations obtained with [Met]enkephalin, tested in the presence of the specific neutral endopeptidase 24.11 inhibitor phosphoramidon, and this opioid's synthetic analog [D-Ala2, Met5]enkephalin which, like [D-Ala2]deltorphin I, is resistant to proteolytic degradation. Both ligands appear to be acting on the same population of immunocytes. The same relationship was estimated to exist in the insect Leucophaea maderae, in which the high viscosity of the hemolymph makes the quantification of reactive cells more difficult than in Mytilus. In addition, [D-Ala2]deltorphin I is as potent as beta-endorphin in affecting the proliferation of lymphocytes in response to mitogen. Saturation experiments with unlabeled ligands and the radioligands [3H][D-Ala2]deltorphin I and [3H][D-Ala2,Met5]enkephalinamide revealed the presence of two high-affinity binding sites on human granulocytes, one sensitive to the nonequilibrium delta opioid antagonist [D-Ala2,Leu5,Cys6]enkephalinamide and the other relatively insensitive. The results obtained with [D-Ala2]deltorphin I support the view that the special role played by endogenous [Met]enkephalin in immunobiological activities of vertebrates and invertebrates is mediated by a special subtype of delta opioid receptor.
Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel.
Authors: Gellens M E , George A L , Chen L Q , Chahine M , Horn R , Barchi R L , Kallen R G
Published: May 2006

The principal voltage-sensitive sodium channel from human heart has been cloned, sequenced, and functionally expressed. The cDNA, designated hH1, encodes a 2016-amino acid protein that is homologous to other members of the sodium channel multigene family and bears greater than 90% identity to the tetrodotoxin-insensitive sodium channel characteristic of rat heart and of immature and denervated rat skeletal muscle. Northern blot analysis demonstrates an approximately 9.0-kilobase transcript expressed in human atrial and ventricular cardiac muscle but not in adult skeletal muscle, brain, myometrium, liver, or spleen. When expressed in Xenopus oocytes, hH1 exhibits rapid activation and inactivation kinetics similar to native cardiac sodium channels. The single channel conductance of hH1 to sodium ions is about twice that of the homologous rat channel and hH1 is more resistant to block by tetrodotoxin (IC50 = 5.7 microM). hH1 is also resistant to mu-conotoxin but sensitive to block by therapeutic concentrations of lidocaine in a use-dependent manner.
Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).
Authors: Schmid K , Hediger M A , Brossmer R , Collins J H , Haupt H , Marti T , Offner G D , Schaller J , Takagaki K , Walsh M T
Published: May 2006

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.
Linkage of atherogenic lipoprotein phenotype to the low density lipoprotein receptor locus on the short arm of chromosome 19.
Authors: Nishina P M , Johnson J P , Naggert J K , Krauss R M
Published: May 2006

The atherogenic lipoprotein phenotype (ALP) is a common heritable trait characterized by a predominance of small, dense low density lipoprotein (LDL) particles (subclass pattern B), increased levels of triglyceride-rich lipoproteins, reductions in high density lipoprotein, and a 3-fold increased risk of myocardial infarction. Significant two-point linkage was found between ALP and the LDL receptor locus on the short arm of chromosome 19 in 51 relatives of nine probands with ALP pattern B. The maximum logarithm of odds (LOD) score of 4.07 was observed at a recombination fraction of 0.04, assuming 100% penetrance of ALP pattern B, and 4.27 at a recombination fraction of zero, assuming 90% penetrance of pattern B. Haplotyping data and multipoint linkage analysis suggest that the gene [named ATHS for atherosclerosis susceptibility (lipoprotein-associated)] responsible for ALP is located distal to D19S76 near or at the LDL receptor locus. This result suggests the possibility that genetic variation at the LDL receptor locus or a closely linked locus on chromosome 19 may be responsible for metabolic alterations in ALP pattern B that account for a substantial proportion of the familial predisposition to coronary artery disease in the general population.
Could CuB be the site of redox linkage in cytochrome c oxidase?
Authors: Larsen R W , Pan L P , Musser S M , Li Z Y , Chan S I
Published: May 2006

This paper explores the proton pumping function of cytochrome c oxidase [ferrocytochrome-c:oxygen oxidoreductase (EC 1.9.3.1)] based upon redox linkage at the "high-potential" CuB center. A model is proposed that is derived from a redox-linked ligand exchange mechanism previously described for the CuA site. Qualitative analysis of this mechanism indicates that such a mechanism is feasible. However, the relatively short distance between CuB and cytochrome a3 implies that the uncoupling electron transfers are quite facile. In addition, the position of the CuB center with respect to the inner mitochondrial membrane argues against redox linkage at the CuB site.
Activation of potassium channels: relationship to the heat shock response.
Authors: Saad A H , Hahn G M
Published: May 2006

We examined the possibility that whole cell currents are involved and possibly trigger the response of mammalian cells to heat shock. Heat-sensitive cells from a radiation-induced fibrosarcoma (RIF-1) and heat-resistant variants (TR-4, TR-5) were heated at 45 degrees C for 3-30 min. We observed induction of voltage-dependent currents after heating in the heat-resistant cells. These currents decayed to nonmeasurable levels over a period of 6 h. In RIF-1 cells, however, voltage-dependent currents were detectable during heating only; these currents then decayed rapidly. Tetraethylammonium (TEA) cations blocked the currents; changing the concentration of extracellular K+ modified the current-voltage (I-V) relationship. These currents, therefore, resulted from the activation of voltage-dependent K+ channels. Addition of TEA during heating sensitized TR-4 cells to heat but had no effect on the heat response of the RIF-1 cells. Continuous exposure of the RIF-1 cells to 2% (vol/vol) dimethyl sulfoxide (DMSO) for 7 days induced the expression of additional functional, voltage-dependent K+ channels; these gave rise to currents that were measurable after heating. In parallel, these cells became heat resistant. Addition of TEA to DMSO-treated cells blocked channels and returned the heat response almost to the pre-DMSO levels. Our data show a correlation between heat resistance and expression of K+ channels. Because resistance to heat very likely relates to the heat shock response, our data suggest that activation of channels may be a very early event in initiation of the heat shock response.
Rainbow trout chimeras produced by injection of blastomeres into recipient blastulae.
Authors: Nilsson E E , Cloud J G
Published: May 2006

In mammals, the ability to successfully introduce isolated cells into a recipient embryo and to document their development has been an important experimental advance in determining the developmental potential of cells. These techniques are also useful in reestablishing the genome of embryonic cells into a germ line. The objective of the present study was to determine whether blastomeres isolated from rainbow trout (Oncorhynchus mykiss) will incorporate and continue to develop when injected into recipient embryos. In the first experiment, donor cells, previously labeled by injecting fluorescein isothiocyanate-dextran into the zygote, were isolated from blastulae; approximately 1000 of these cells were microinjected into each unlabeled recipient embryo of the same developmental stage. Following subsequent development through gastrulation, microscopic examination revealed that 19 of 114 injected embryos (17%) contained fluorescent cells. These labeled cells were present at numerous sites within embryos, and the pattern of distribution of these cells varied among embryos. In experiment two, blastomeres from normal diploid embryos were injected into triploid blastulae. The injected embryos were incubated until hatching and then sacrificed; cells from these embryos were dispersed and treated with 4',6-diamidino-2-phenylindole. The proportion of diploid cells, as determined by flow cytometry, varied from 2.0% to 12%. From these results we conclude that blastomeres isolated from rainbow trout blastulae will incorporate and continue to develop following injection into recipient embryos.
Light regulates expression of brain-derived neurotrophic factor mRNA in rat visual cortex.
Authors: Castrén E , Zafra F , Thoenen H , Lindholm D
Published: May 2006

Specific sensory input has profound transient and long-lasting effects on the function of corresponding sensory cortical areas both during development and in adulthood. To study whether neurotrophic factors might play a role in such processes, we investigated the effects of light on the nerve growth factor and brain-derived neurotrophic factor (BDNF) mRNA levels in rat visual cortex. Keeping adult rats in the dark or preventing normal activity of retinal ganglion cells by intraocular injection of tetrodotoxin significantly decreased the levels of BDNF mRNA in the visual cortex but not in other cortical areas. Exposure to light after a period in darkness rapidly restored the mRNA to control levels. These alterations in visual input had no effect on nerve growth factor mRNA. The mRNA of trkB, the putative signal-transducing receptor unit for BDNF, was also decreased in darkness, although less than BDNF mRNA. BDNF mRNA levels increased in the visual cortex of newborn rats after eye-opening. This increase is retarded, although not completely abolished, by rearing the pups in darkness. Thus, the levels of BDNF mRNA are rapidly regulated by sensory input during development and in adulthood. BDNF may therefore play an important role in formation and in activity-dependent modulation of specific connections in the visual cortex.
Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor.
Authors: Zhu G , Decker S J , Saltiel A R
Published: May 2006

A number of proteins involved in intracellular signaling contain regions of homology to the product of the src oncogene that are termed Src-homology (SH) 2 domains. SH2 domains are believed to mediate the association of these proteins with various tyrosine-phosphorylated receptors in a growth factor-dependent manner. We have examined the kinetic characteristics of one of these interactions, the binding of the SH2 domains of phospholipase C gamma 1 with the receptor for epidermal growth factor (EGF). Bacterial fusion proteins were prepared containing the two SH2 domains of PLC gamma 1 and labeled metabolically with [35S]methionine/cysteine. A fusion protein containing both SH2 domains bound to the purified EGF receptor from EGF-treated cells, whereas no binding to receptors from control cells was detected. Binding was rapid, reaching apparent equilibrium by 10 min. Dissociation of the complex occurred only in the presence of excess unlabeled SH2 protein and exhibited two kinetic components. Similarly, analysis of apparent equilibrium binding revealed a nonlinear Scatchard plot, further indicating complex binding kinetics that may reflect cooperative behavior. The binding of the fusion protein containing both SH2 domains was inhibited by a fusion protein containing only the amino-terminal SH2 domain, although at concentrations an order of magnitude higher than that observed with the complete fusion protein. Fusion proteins containing SH2 domains from the GTPase-activating protein, the p85 regulatory subunit of phosphatidylinositol 3'-kinase, or the Abl oncoprotein competed less effectively. Binding of the PLC gamma 1 SH2 fusion protein to a mutant EGF receptor lacking the two carboxyl-terminal tyrosine phosphorylation sites exhibited a significantly lower affinity than that observed with the wild type, suggesting that this region of the receptor may play an important role. This binding assay represents a means with which to evaluate the pleiotropic nature of growth factor action.
Symmetry in the mechanism of bacteriophage lambda integrative recombination.
Authors: Burgin A B , Nash H A
Published: May 2006

During the strand-exchange events of bacteriophage lambda integration, pairs of phosphodiester bonds are broken and then rejoined to form novel DNA linkages. The reaction proceeds in vitro in the absence of an external energy source; the bond energy needed to rejoin broken strands of DNA must therefore be conserved during cleavage. Although some of this conservation involves a covalent intermediate between DNA and the recombinase Int, it is possible that such an intermediate is formed with only one of the two phosphodiesters. In such an asymmetric mechanism, the second phosphodiester would be attacked by a nucleophile that is exposed by cleavage of the first DNA strand. In contrast, a symmetric mechanism hypothesizes nucleophilic attack by Int on both phosphodiesters. We have distinguished these two mechanisms by removing potential nucleophiles from the integrative recombination reaction. Our data are inconsistent with an asymmetric mechanism. We conclude that during strand exchange both phosphodiesters proceed through a covalent protein-DNA intermediate.
Evidence for the role of dendritic spines in the temporal filtering properties of neurons: the decoding problem and beyond.
Authors: Rose G J , Call S J
Published: May 2006

The question of relations between structure and function acutely applies to the search for the functional role of dendritic spines. While dendritic spines are a prominent and widespread structural feature of neurons in the central nervous system, their function is poorly understood. Because the conducting core of a spine stem can be of extremely small dimensions, a large axial resistance to current flow and "low-pass" filtering of inputs have been hypothesized. Here we show that neurons in the dorsal torus semicircularis of the electric fish Eigenmannia show real-time fluctuations in their transmembrane potential that reflect modulations in the amplitude of a high-frequency sinusoidal carrier signal. In 18 neurons recorded intracellularly and labeled with Lucifer yellow, the decrease in the magnitude of these potentials with increasing rate of amplitude modulation (i.e., low-pass temporal filtering) was positively correlated (r = 0.79, P < 0.001, over a range of one to two octaves in modulation rate) with the mean dendritic spine density (range, 0-0.38 spine per micron of dendritic length) of the cell. The acquisition of synaptic input through dendritic spines may be a general mechanism for achieving the temporal filtering that underlies real-time signal processing in the central nervous system.
Isolation of DNA sequences deleted in lung cancer by genomic difference cloning.
Authors: Wieland I , Böhm M , Bogatz S
Published: May 2006

To identify DNA sequences that are deleted in human lung cancer, genomic subtraction hybridization was used to construct plasmid libraries that are enriched for DNA sequences deleted in the small cell lung carcinoma cell line SK-LC-17. The clones of the libraries contained predominantly single copy sequences, allowing direct screening of normal and tumor DNA by genomic Southern blotting. Of 150 clones tested, three independent clones (del-27, del-118, and del-109) were identified that specifically hybridized with normal human DNA but not with tumor DNA from the cell line SK-LC-17. The corresponding DNA sequences are localized on human chromosomes 5, 8, and X/Y. The DNA regions identified by del-109 and del-118 were also found to be deleted in several other lung carcinoma cell lines. Moreover, del-118 was deleted in a freshly isolated lymph node metastasis of a human lung adenocarcinoma. It is therefore reasonable to speculate that the identified clones are derived from independent genetic loci encoding potential tumor suppressor genes.
Trisomy 7 and trisomy 10 characterize subpopulations of tumor-infiltrating lymphocytes in kidney tumors and in the surrounding kidney tissue.
Authors: Dal Cin P , Aly M S , Delabie J , Ceuppens J L , Van Gool S , Van Damme B , Baert L , Van Poppel H , Van den Berghe H
Published: May 2006

We performed conventional cytogenetic analysis and fluorescence in situ hybridization in short-term cultures of normal and neoplastic kidney tissues. Cell populations carrying an extra chromosome 7 or an extra chromosome 10 as the only chromosome change could be identified in kidney tumors, mostly renal cell carcinomas, and in the surrounding kidney tissue, but not in nonneoplastic kidneys. To identify the type of cells displaying these aneuploidies, we performed in situ hybridization (ISH) with probes specific for the centromeric region of chromosomes 7 and 10 on frozen kidney tissue sections. Trisomy 7 and trisomy 10 were restricted to infiltrating inflammatory cells in the tumor as well as in the surrounding tissue. Trisomy 7 and trisomy 10 were also found in subpopulations of peripheral blood T cells of cancer patients and of normal individuals, as well as in the thymus of five normal fetuses (21-29 weeks), but not in noninvaded reactive lymph node sections of patients without malignancy. When lymphocytes were enriched from kidney tumors and surrounding tissue by either Ficoll/Hypaque density gradient or immunomagnetic selection with anti-CD3, anti-CD4, or anti-CD8 monoclonal antibodies, it was confirmed that they contained a high percentage of trisomy 7 and trisomy 10 cells. Further proof for T-lymphocyte origin of the trisomy 7 and trisomy 10 cells was obtained by simultaneous staining of lymphocytes isolated from tumor tissue with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies and ISH. We conclude that trisomy 7 and trisomy 10, found in renal carcinomas and surrounding kidney tissue, characterize subpopulations of tumor-infiltrating lymphocytes. The biologic significance of this phenomenon is unknown and requires further investigation.
Glutamic acid decarboxylase autoantibodies in preclinical insulin-dependent diabetes.
Authors: De Aizpurua H J , Wilson Y M , Harrison L C
Published: May 2006

Insulin-dependent diabetes mellitus (IDDM) is associated with serum antibodies that precipitate a 64-kDa pancreatic islet cell protein reported to be glutamic acid decarboxylase (GAD; glutamate decarboxylase, EC 4.1.1.15). Previously, antibodies to GAD were found in the rare neurological disorder stiff man syndrome. To demonstrate directly antibodies to GAD, enzymatically active GAD was first purified from fresh human cerebellum. Brain GAD activity was precipitated by noninhibitory antibodies in the sera of 16/26 (62%) subjects defined as having preclinical IDDM (islet cell antibody-positive first-degree relatives of a person with IDDM), 3/13 (23%) with recent-onset IDDM, and 3/3 with the stiff man syndrome. In addition, sera of 5/26 (19%) preclinical and 2/13 (15%) recent-onset IDDM subjects contained antibodies that precipitated GAD but inhibited its activity. Thus, overall, 21/26 (81%) preclinical and 5/13 (38%) recent-onset IDDM subjects had antibodies that precipitated GAD activity. Antibodies to GAD were not detected in sera from subjects with other autoimmune diseases (n = 29) or healthy controls (n = 14). GAD affinity-purified to homogeneity (specific activity, 58 units/mg) was specifically immunoprecipitated as a single 60-kDa species by the IDDM sera. In an ELISA incorporating whole mouse brain GAD captured by the GAD-6 monoclonal antibody the frequencies of GAD antibodies for all subject groups were indistinguishable from those found by precipitation of human brain enzymatic activity. We conclude that (i) GAD is an (auto)antigen in a majority of subjects operationally defined as having preclinical IDDM, (ii) pancreatic islet and brain GAD are likely to be cross-reactive, and (iii) the majority of GAD antibodies are directed away from the catalytic site of the brain enzyme. The lower frequency of GAD antibodies in recent-onset IDDM subjects indicates either that immunoreactivity is lost with near-total beta-cell destruction or that GAD antibodies denote a low risk of progression to clinical disease.