bluuebunny/crossref_metadata_2025 huggingface dataset:
Homozygous familial hypercholesterolemia mutant with a defect in internalization of low density lipoprotein.
Authors: Miyake Y , Tajima S , Yamamura T , Yamamoto A
Published: May 2006

During studies on binding of low density lipoprotein (LDL) to fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH), a unique line was derived from subject M.N. This line could bind as much LDL as normal cells, or even more. However, like fibroblasts from other patients with the homozygous form of FH, it failed to show regulation of cholesterol synthesis. Analyses of the LDL receptors showed that this line could not mediate internalization of receptor-bound LDL. Studies on the fibroblasts of the parents of this subject showed that the inability to internalize LDL was hereditary and that the subject was a pure homozygote for this defect. The plausibility of this finding was supported by the fact that her parents were first cousins. The possible existence of a homozygous state of this defect was predicted by Goldstein et al. [Goldstein, J. L., Brown, M. S. & Stone, N. J. (1977) Cell 12, 629-41], but an actual case of the internalization defect in a pure homozygous form had not been found. There were no differences from normal cells in the nature of the LDL binding activity of this line, such as in its specificity, affinity, or Ca2+ requirement.
Nonenzymatic glycosylation of peripheral nerve protein in diabetes mellitus.
Authors: Vlassara H , Brownlee M , Cerami A
Published: May 2006

A new affinity chromatography system that selectively retains glycosylated amino acids has been utilized to determine the amount of nonenzymatic glycosylation present in peripheral nerve from diabetic and control rats and dogs. The mean value for glycosylated amino acids in diabetic rats was 2.8 times greater than the mean value in normal rats (P less than 0.001). In diabetic dogs, mean values were 2.15 times greater than normal values (P less than 0.05). Amino acid analysis of reduced, glycosylated amino acids previously isolated by affinity chromatography showed that glycosylated lysine and its hydrolysis rearrangement products were the major borohydride-reducible adduct present. In addition, another glycosylated product was noted to be present in major proportions. This radioactive product did not chromatograph with any of the available glycosylated amino acid standards. The finding that diabetes results in a nearly 3-fold increase of peripheral nerve glycosylation is consistent with a number of previous investigations in which glycosylation was measured in hemoglobin, serum albumin, and urinary amino acids and peptides from diabetics and normals. The results reported here provide evidence that increased nonenzymatic glycosylation is occurring in a tissue where physiological, morphological, and clinical degeneration characteristically develop as a result of diabetes mellitus.
Covariant chronogeometry and extreme distances: Elementary particles
Authors: Segal I. E. , Jakobsen H. P. , Ørsted B. , Paneitz S. M. , Speh B.
Published: May 2006

We study a variant of elementary particle theory in which Minkowski space,
M
0
, is replaced by a natural alternative, the unique four-dimensional manifold
¯M
with comparable properties of causality and symmetry. Free particles are considered to be associated (
i
) with positive-energy representations in bundles of prescribed spin over
¯M
of the group of causality-preserving transformations on
¯M
(or its mass-conserving subgroup) and (
ii
) with corresponding wave equations. In this study these bundles, representations, and equations are detailed, and some of their basic features are developed in the cases of spins 0 and ½. Preliminaries to a general study are included; issues of covariance, unitarity, and positivity of the energy are treated; appropriate quantum numbers are indicated; and possible physical applications are discussed.
In vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.
Authors: Goldenberg C J , Raskas H J
Published: May 2006

Early region 2 of the adenovirus 2 genome (map position 61-75) specifies two poly(A)+ nuclear RNAs (28S and 23S) that appear to be precursors of the 20S cytoplasmic mRNA [Goldenberg, C. J. & Raskas, H. J. (1979) Cell 16, 131-138]. Splicing of these nuclear RNAs in vitro has been obtained with a whole cell extract prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The splicing reaction was demonstrated by two procedures: (i) hybridization of pulse-labeled RNA fractionated by size and (ii) annealing of RNAs with radioactive DNA probes followed by nuclease digestion. The first procedure provided evidence that sequences from the large 2300-nucleotide intron (74.6-68.8) were excised and 5' transcripts were spliced to the mRNA body. Utilizing both S1 and Exo VII nucleases, the second procedure demonstrated excision of sequences from the smaller 720-nucleotide intron (68.5-66.3), the splicing of sequences from the second leader (68.8) to the mRNA body, and the formation of an mRNA body of 1700 nucleotides, the size found in vivo. These findings provide evidence that an in vitro system that splices viral RNAs to yield products comparable to those found in vivo is now available.
In vitro incorporation of L-canavanine into vitellogenin of the fat body of the migratory locust Locusta migratoria migratorioides.
Authors: Pines M , Rosenthal G A , Applebaum S W
Published: May 2006

L-Canavanine competes with L-arginine for incorporation into vitellogenin secreted in vitro by the fat body of the female locust Locusta migratoria migratorioides. Incorporation of L-[guanidinooxy-14C]canavanine into vitellogenin has been established unequivocally by combined arginase and urease hydrolyses of the acid hydrolysate of antibody-precipitated canavanyl vitellogenin. Continued exposure of the fat body to canavanine decreases in vitro protein secretion but the proportion of canavanyl vitellogenin to native vitellogenin increases. Canavanine-mediated inhibition of fat body protein secretion is dependent on both the canavanine concentration and the arginine retention by the fat body. Canavanine replaces about 10% of the arginyl residues of canavanyl vitellogenin. The electrophoretic mobility of canavanyl vitellogenin is greater than that of native vitellogenin but the ability of this aberrant protein to react with vitellogenin antibody is unimpaired.
Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intercellular collagen matrix.
Authors: Chazov E I , Alexeev A V , Antonov A S , Koteliansky V E , Leytin V L , Lyubimova E V , Repin V S , Sviridov D D , Torchilin V P , Smirnov V N
Published: May 2006

Human umbilical endothelial cells (ECs) were grown on fibrillar type I collagen in 16.4-mm multiwell tissue culture plates. Human platelets were added to the wells, and platelet adhesion to collagen was examined by scanning electron microscopy and radioisotopic technique in the absence of ECs and in preconfluent and confluent EC cultures. Single adherent platelets of different shapes as well as small aggregates were seen on collagen surface. Human plasma fibronectin added to the system stimulated platelet adhesion and their spreading on collagen. ECs had no effect on the percentage of platelets adherent to collagen-coated gaps in preconfluent culture but decreased the number of spread platelets. It is demonstrated that collagen-coated gaps can bind 14C-labeled liposome--antibody and 14-C-labeled liposome--fibronectin conjugates. ECs grown on fibrillar collagen are suggested as useful models for screening of antiplatelet drugs and for the study of drug targeting to the areas of vascular injury for prevention of thrombosis.
Identification of mouse mammary epithelial cells by immunofluorescence with rabbit and guinea pig antikeratin antisera.
Authors: Asch B B , Burstein N A , Vidrich A , Sun T T
Published: May 2006

Few markers are available to identify the three types of mammary epithelial cells--ductal, alveolar, and myoepithelial--especially in pathological conditions and in cell cultures. We have used antisera to human keratins in immunofluorescence to facilitate the identification of the three mouse mammary epithelial cell types. In frozen tissue sections and primary cell cultures, a rabbit antikeratin antiserum specifically stained cytoplasmic filaments in all three types of epithelial cells. A guinea pig antiserum against the same keratin preparation, however, reacted preferentially with filaments in myoepithelial cells and readily detected this cell type in normal, dysplastic, and malignant mammary tissues and cell cultures. Neither antisera reacted with fibroblasts or any other mesenchymal cells. The combined use of the two antikeratin antisera thereby permits rapid surveys of tissue sections and cultures for the localization of not only all epithelial cells but also the subpopulation of myoepithelial cells. Moreover, when mammary cultures established from late-pregnant or lactating mice were stained simultaneously with guinea pig antikeratin and rabbit anticasein antisera, three populations of epithelial cells were mutually exclusive: those stained by anticasein antiserum, those stained by guinea pig antikeratin antiserum, and those stained by neither, consistent with properties of alveolar, myoepithelial, and ductal cells, respectively. These antisera thus offer a tool for studying different epithelial cell types during mammary development, tumorigenesis, and malignant progression.
Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin.
Authors: Sobue K , Muramoto Y , Fujita M , Kakiuchi S
Published: May 2006

A calmodulin-binding protein called "caldesmon" was purified from chicken gizzard muscle as the major calmodulin-binding protein in this tissue. Its molecular weight estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 150,000, and two of these polypeptides constituted the native molecule. Caldesmon is an actin-binding protein also, binding F-actin reversibly in the presence or absence of Ca2+. The interaction of caldesmon with F-actin was abolished by the binding of calmodulin with the caldesmon. Because the interaction between caldesmon and calmodulin was Ca2+-dependent but the interaction between caldesmon and F-actin was not, Ca2+ acts as a flip-flop switch between the formations of two complexes, caldesmon.calmodulin and caldesmon.F-actin: increasing the formation of the former complex at increased Ca2+ level and the formation of the latter complex at decreased Ca2+ level. The equilibrium of the formations of both complexes was achieved at a Ca2+ concentration near 1 microM.
Crucial role of thyroid hormone in x-ray-induced neoplastic transformation in cell culture.
Authors: Guernsey D L , Borek C , Edelman I S
Published: May 2006

Incubation of mouse embryo fibroblasts (C3H/10T1/2) in media depleted of thyroid hormone for 1 week rendered the cells completely resistant to the transforming action of an x-ray dose, 4 grays, that yields transformation frequencies (no. foci per surviving cells) of approximately 10(-3) in media supplemented with triiodothyronine (T3) (1 nM). Studies on the timing of the additions or removal of the hormone indicate that T3 was maximally effective when added 12 hr before irradiation and that progression from the time of irradiation to the appearance of foci (6 weeks) was independent of the presence or absence of the hormone. The dependence of x-ray-induced transformation on the concentration of T3 in the medium was virtually the same as that for augmentation of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the involvement of protein synthesis in the process is the abolition of T3- and x-ray-dependent transformation by cycloheximide at a concentration (100 ng/ml) that inhibits 50% of protein synthesis. We propose that thyroid hormone induces the synthesis of a host protein that is an obligatory participant in x-ray-mediated transformation.
Targeted killing of cultured cells by receptor-dependent photosensitization.
Authors: Mosley S T , Goldstein J L , Brown M S , Falck J R , Anderson R G
Published: May 2006

This paper describes a method, designated "receptor-dependent photosensitization," by which the receptor-mediated endocytosis of low density lipoprotein (LDL) can be used to deliver a photosensitizing agent, pyrene, to cultured human and animal cells. The hydrophobic core of LDL is extracted and replaced with pyrene covalently coupled to cholesteryl oleate. This reconstituted LDL enters cells in significant amounts only when the cells express LDL receptors, resulting in the accumulation of pyrene cholesteryl oleate within lysosomes. Subsequent exposure of the cells to ultraviolet light leads to cell death. Cells killed by this technique include normal and simian virus 40-transformed human fibroblasts, human A-431 epidermal carcinoma cells, Chinese hamster ovary cells, and mouse L cells, all of which express LDL receptors. Mutant fibroblasts from a patient with homozygous familial hypercholesterolemia, which lack LDL receptors, do not take up significant amounts of the pyrene-containing LDL and are not killed by subsequent exposure to light. The current experiments establish the feasibility of receptor-dependent photosensitization as an efficient and selective method for killing cultured human and animal cells.
Genetic and molecular diversity in nondeletion Hb H disease.
Authors: Higgs D R , Pressley L , Aldridge B , Clegg J B , Weatherall D J , Cao A , Hadjiminas M G , Kattamis C , Metaxatou-Mavromati A , Rachmilewitz E A , Sophocleous T
Published: May 2006

Restriction endonuclease mapping of nondeletion alpha-thalassemia determinants from a variety of racial groups showed no detectable abnormalities within a 40-kilobase region of the zeta-alpha globin gene cluster. By using a zeta-specific probe, we defined three different types of interactions that give rise to Hb H disease, each involving a nondeletion alpha-thalassemia haplotype. mRNA analysis showed further diversity within these groups, indicating that there are at least three nondeletion determinations.
Heterogeneity of newly inserted and preexisting murein in the sacculus of Escherichia coli.
Authors: de Pedro M A , Schwarz U
Published: May 2006

In vivo studies of murein biosynthesis show that newly synthesized murein unexpectedly differs in its chemical composition from preexisting murein. New murein is loosely crosslinked with the preexisting sacculus; in a maturation process involving further transpeptidation, the final stage of crosslinkage in murein is achieved. Newly inserted murein initially carries pentapeptide subunits, which are the donor of the secondary transpeptidation reaction. In mutants with defective penicillin-binding protein 4 the secondary transpeptidation step is abolished. Uncoupling of the secondary transpeptidation reaction from crosslink formation during the initial insertion of new murein was also found in a mutant with a defect in lipoprotein biosynthesis. We conclude that the initial transpeptidation of murein and crosslink formation during the maturation of newly inserted murein are catalyzed by two different enzyme systems.
Role of the plasma membrane proton pump in pH regulation in non-animal cells.
Authors: Sanders D , Hansen U P , Slayman C L
Published: May 2006

Possible methods by which eukaryotic cells can regulate intracellular pH (pHi) in response to experimental acid loading were investigated by using as a model cell the fungus Neurospora. Attention was focused on the role of membrane transport in such regulation, starting from the fact that this organism possesses a powerful electrogenic proton extrusion pump. Intracellular acidification was forced by introducing butyric acid into the recording medium, and subsequent changes in pHi and membrane potential were determined with intracellular microelectrodes. In separate experiments, membrane current-voltage curves were obtained and resolved--by an explicit kinetic model--into distinct pump and leak components. Decreased pHi causes increased outward pumping of H+ ions, in a manner quantitatively consistent with their role as a substrate for the proton pump. This increased pumping is often manifest as a transient hyperpolarization at the onset of cytoplasmic acidification. With a considerably slower time course, decreased pHi also produces a large increase in membrane leak conductance, which brings about net membrane depolarization and further stimulates the pump (by virtue of the reduced back electromotive force). Although the identity of the ion responsible for increased leak conductance is not yet known, the evident modulation of conductance seemingly plays an important role in stabilizing the intracellular pH: Stimulation of the pump alone would have little net effect on pHi because it would result simply in enhanced backflux of H+ (to which the membrane is most permeable in normal circumstances). An increased leak to nonprotons, however, would allow the pump to accomplish net H+ ejection.
Nucleosome phasing and micrococcal nuclease cleavage of African green monkey component alpha DNA.
Authors: Musich P R , Brown F L , Maio J J
Published: May 2006

The micrococcal nuclease cleavage of intact nuclear chromatin from African green monkey cells and of the completely deproteinized sequences was studied by using high-resolution analytical and DNA sequencing gels and secondary restriction enzyme analysis. When deproteinized component alpha DNA was used as substrate, not all phosphodiester bonds in the 172-base-pair repeat units were cleaved with equal frequency by the nuclease. A distinct preference for the cleavage of A-T rather than G-C bonds was observed; however, A + T-richness in itself did not confer susceptibility to cleavage by micrococcal nuclease. The results suggested that, in deproteinized DNA, nuclease cleavage at particular dinucleotides may be influenced more by the effect of adjacent sequences than by the composition of the dinucleotide. In contrast to complex cleavage patterns of the deproteinized component alpha DNA which arose because of multiple cleavage sites in the repeat unit, micrococcal nuclease cleaved component alpha nuclear chromatin at one site per nucleosome repeat, near position 126 in the nucleotide sequence. This simple chromatin cleavage pattern is consistent with the discrete nucleosomal structure of component alpha in chromatin and a direct phase relationship between the component alpha DNA sequence repeats and the nucleosome protein structural repeats.
Conserved 5S rRNA complement to tRNA is not required for protein synthesis.
Authors: Pace B , Matthews E A , Johnson K D , Cantor C R , Pace N R
Published: May 2006

The notion that tRNA and 5S rRNA interact through evolutionarily conserved complementary sequences has been tested by nucleolytic modification of the 5S rRNA, using the modified rRNA to reconstitute the large ribosomal subunit, and assaying for poly(uridylic acid)-directed polyphenylalanine synthesis. The 5S rRNA sequence C-G-A-A (residues 43-46) and several residues surrounding it are not essential for protein synthesis.
Structure of two human alpha-tubulin genes.
Authors: Wilde C D , Chow L T , Wefald F C , Cowan N J
Published: May 2006

The ability of a chicken alpha-tubulin cDNA probe to cross-hybridize with human DNA under stringent conditions has been exploited to screen two independently constructed human genomic libraries. Nine clones were isolated, accounting for 60% of the bands observed in a whole genomic Southern blot of human DNA. Two clones were selected for further analysis by restriction mapping, orientation experiments using 3'- or 5'-specific probes, and electron microscopy of heteroduplexes. One clone, 2 alpha, contains an alpha-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19 alpha, contains an alpha-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19 alpha has only two intervening sequences that correspond to the first two in clone 2 alpha. However, these intervening sequences differ in size between clones 2 alpha and 19 alpha and show no detectable sequence homology. The sum of the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length of the cDNA molecule.
Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane.
Authors: Inouye S , Soberon X , Franceschini T , Nakamura K , Itakura K , Inouye M
Published: May 2006

The positively charged amino-terminal region of the signal peptide has been proposed to have an important role at an initial step of protein secretion across the membrane (loop model). To test this hypothesis, the charge on the amino-terminal region of the signal peptide of the prolipoprotein of the Escherichia coli outer membrane was altered by using synthetic oligonucleotides from +2 to +1, 0, and -1 by guided site specific mutagenesis of a plasmid DNA carrying an inducible lipoprotein gene. The wild-type sequence of this sectio, Met-Lys-Ala-Thr-Lys (+2), was thus changed to Met-Lys-Asp-Thr-Lys (I-1; +1), Met-Ala-Thr-Lys (I-2; +1), Met-Asp-Thr-Lys (I-3; 0), and Met-Glu-Asp-Thr-Lys (I-4; -1). After induction of lipoprotein production, cells were pulse labeled with [35S]methionine for 10 sec. The lipoprotein of I-1, I-2, and I-3 was assembled in the membrane, although the rates of lipoprotein production progressively decreased as the charge on the signal peptide became more negative. Conversely, in the case of I-4, only a small amount of lipoprotein assembled in the membrane while a large amount of glycerol-unmodified prolipoprotein accumulated in the cytoplasm. This soluble prolipoprotein was gradually and posttranslationally secreted across the membrane to be modified and assembled in the membrane. These results indicate that the positively charged amino-terminal region of the signal peptide plays an important role in efficient protein secretion across the membrane.
In vitro stimulation of phosphate uptake in isolated chick renal cells by 1,25-dihydroxycholecalciferol.
Authors: Liang C T , Barnes J , Balakir R , Cheng L , Sacktor B
Published: May 2006

Renal cells isolated from vitamin D-deficient chicks had an increased Na+-dependent phosphate uptake when preincubated with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3]. Phosphate uptake in the absence of Na+ and methyl alpha-glucoside uptake dependent on Na+ were not affected. Phosphate uptake was stimulated 15% by 0.010 pM 1,25-(OH)2D3. Maximal enhancement of 30% was obtained with 100 pM. The uptake when fully stimulated by preincubation in vitro approximated the uptake of cells isolated from chicks that were previously repleted with 1,25-(OH)2D3 in vivo. Cells from repleted chicks were not stimulated additionally when preincubated with 1,25-(OH)2D3 in vitro. The increase in phosphate uptake could be measured after a 1-hr preincubation period; full response required at least 2 hr. Phosphate uptake induced by 1,25-(OH)2D3 was blocked by cycloheximide and actinomycin D. Enhancement of phosphate uptake was relatively specific for the 1,25-(OH)2D3 analog of vitamin D3. The potency order was 1,25-(OH)2D3 greater than 25-(OH)D3 = 1-(OH)D3 greater than 24,25-(OH)2D3 greater than D3. Kinetically, 1,25-(OH)2D3 increased the Vmax of the phosphate uptake system; the affinity for phosphate was unaffected. 3H-Labeled 1,25-(OH)2D3 was taken up by the isolated renal cells. It was estimated that the stimulation of phosphate uptake might be initiated by very few molecules of 1,25-(OH)2D3 per cell. It is proposed that 1,25-(OH)2D3 contributes importantly to the mechanisms by which phosphate transport is regulated in the kidney.
Origin of evolutionary novelty in proteins: how a high-cysteine chorion protein has evolved.
Authors: Rodakis G C , Kafatos F C
Published: May 2006

The structure of unusual high-cysteine (Hc) proteins (ca. 30 mol %), which are characteristic of the chorion of the silkmoth Bombyx mori, has been determined by determining the sequence of a corresponding cDNA clone. The Hc protein sequence has evolved from a family of more ordinary chorion genes, in large part through fixation of mutations leading to enhanced cysteine content. Mutations of different types are differentially distributed in different parts of the sequence. In two conservative parts, those encoding the amino-terminal signal peptide and the highly structured central region of the protein, only base substitutions have been accepted. By contrast, in two alternating parts, which encode variable arms flanking the central region, deletions and duplications of tandemly repetitive sequences are prominent. Both base substitutions and expansions or deletions of tandemly repetitive elements are important in the evolution of this type of protein; functional constraints of the various protein domains dictate which class of mutations can be accepted.
Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules.
Authors: Ikeda H , Aoki K , Naito A
Published: May 2006

We have developed a cell-free system from Escherichia coli for studying illegitimate recombination between nonhomologous DNA molecules. The recombination is stimulated by oxolinic acid, an inhibitor of DNA gyrase. The stimulation is abolished by coumermycin A1 and is not found in extracts of nalidixic acid-resistant (gyrA) mutants. We therefore inferred that DNA gyrase directly participates in illegitimate recombination, at least in the presence of oxolinic acid [Ikeda, H., Moriya, K. & Matsumoto, T. (1981) Cold Spring Harbor Symp. Quant. Biol. 45, 399--408]. The structure of recombinant DNA molecules formed in the presence of oxolinic acid from a cross between phage lambda and plasmid pBR322 DNAs was analyzed by heteroduplex mapping. Among nine isolates tested, two recombinants were formed by the insertion of the plasmid into the lambda genome. The seven other recombinants had more complicated genome structures. Insertion of pBR322 was accompanied by a deletion on one of the genomes. In all cases, the end points of deletions coincided with one end of the pBR322 insertion. Recombination sites seemed to be distributed randomly on the lambda and pBR322 genomes. Analysis of nucleotide sequences of the recombination junctions proved that the crossover took place between nonhomologous DNA sequences. A model for DNA gyrase-mediated illegitimate recombination is discussed.
Cell-free biosynthesis of somatostatin precursors: Evidence for multiple forms of preprosomatostatin.
Authors: Warren T G , Shields D
Published: May 2006

It has been demonstrated [Shields, D. (1980) J. Biol. Chem. 255, 11625-11628] that mRNA isolated from the islets of Langerhans codes for two preposomatostatin molecules of apparent molecular weights 18,000 and 19,000, respectively. Here evidence is presented that in vitro translation of pancreatic islet mRNA in two different cell-free protein-synthesizing systems directs the synthesis of up to nine distinct forms of somatostatin-immunoreactive polypeptides. The multiplicity of the preprosomatostatin molecules was the result of initiation of translation from separate species of mRNA as demonstrated by amino-terminal labeling with N-formyl-[35S]Met-tRNAMetf. Translation of islet mRNA isolated from different individual animals showed that all of the preposomatostatin polypeptides were present amongst the cell-free products, which implies that the multiple forms were not due to genetic variation in the wild population. Based on their apparent molecular weights and distinctly different isoelectric points, the different preprosomatostatin molecules could be classified into two major families. These results suggest that the anglerfish preprosomatostatins are encoded by separate mRNA species and are consistent with the existence of a multigene family for somatostatin.
Antibodies to RNA from autoimmune NZB/NZW mice recognize a similar antigenic determinant and show a large idiotypic diversity.
Authors: Eilat D , Hochberg M , Fischel R , Laskov R
Published: May 2006

We have identified two RNA-specific hybridoma autoantibodies in fusions of spleen cells from unimmunized NZB/NZW female mice with BALB/c myeloma cells. The two fusion experiments were carried out 2 years apart with different myeloma partners. Specificity analyses showed that the two monoclonal antibodies and the total RNA-binding IgG in NZB/NZW serum recognize a G,C-rich sequence of ribonucleotides. The isolated heavy and light chains of the two antibodies and their papain Fab fragments could be distinguished by NaDodSO4/polyacrylamide gel electrophoresis. Rabbit anti-idiotypic antibodies prepared against the two monoclonal proteins showed unique specificities for the antigen-binding sites of their cognate auto-antibodies. Moreover, the anti-idiotypic antisera had little effect on the RNA-binding capacity of the total IgG from NZB/NZW serum. These results suggest that a wide range of different idiotypes is involved in the autoimmune response to a similar antigenic determinant.
Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TW-260/240) are related molecules containing a common calmodulin-binding subunit bound to a variant cell type-specific subunit.
Authors: Glenney J R , Glenney P , Weber K
Published: May 2006

Spectrin, fodrin, and TW-260/240 form a group of structurally and functionally similar but not identical high molecular weight actin-binding proteins from chicken erythrocytes, brain tissue, or intestinal epithelial brush borders. Immunological data and one-dimensional peptide maps of the separated subunits suggest that a common (Mr 240,000) and a variant (Mr 220,000, 235,000, or 260,000) subunit account for the three different heterodimers. These results are in line with the related but distinct morphology of the three proteins observed in micrographs of rotary-shadowed molecules and the finding that the common (Mr 240,000) subunit seems to account for the calcium-dependent calmodulin-binding activity displayed by the three proteins. The possible functions of spectrin-like molecules in nonerythroid cells are discussed.
Retinoids increase transglutaminase activity and inhibit ornithine decarboxylase activity in Chinese hamster ovary cells and in melanoma cells stimulated to differentiate.
Authors: Scott K F , Meyskens F L , Russell D H
Published: May 2006

Transglutaminase (TGase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) and ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase, EC 4.1.1.17) activities were measured after the addition of retinoid analogs to Chinese hamster ovary (CHO) cells released from quiescence and Cloudman S91 (CCL 53.1) mouse melanoma cells stimulated to differentiate with alpha-melanocyte-stimulating hormone (MSH, melanotropin). In both cell culture lines, we detected a biphasic increase in TGase activity and a single peak of ODCase activity within 7 hr after release or stimulation. Retinoid analogs altered the expression of the initial TGase peak in both CHO and melanoma cells. Retinol increased the activity of TGase 1 hr after release in CHO cells, and the activity remained elevated until hr 4. A broad peak of TGase activity also occurred after the addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODCase, and after addition of alpha-difluoromethylornithine plus retinol. In mouse melanoma cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. Retinoic acid alone also increased TGase activity biphasically in these cells without the addition of MSH. These studies suggest that retinoid effects that increase TGase activity may alter the ODCase expression in proliferation and differentiation.
Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate.
Authors: Machy P , Barbet J , Leserman L D
Published: May 2006

Antibody-bearing fluorescent liposomes containing methotrexate became bound to cells expressing determinants recognized by the antibody. The number of bound liposomes could be evaluated by fluorometry, and the internalization of liposomes was evaluated by the methotrexate-mediated inhibition of radio-labeled deoxyuridine incorporation. The effect of methotrexate transferred from the liposomes into the cells was a function not of the number of liposomes bound but of the nature of the cells and of the target molecules. Liposomes bearing antibodies with specificity for the H-2K or Mr 94,000 and 180,000 molecules were much more effective at drug delivery into T than B cells, even though these determinants were expressed by both cell types. B cells were more sensitive to the effect of methotrexate in anti-H-2 I-A and I-E liposomes than in anti-H-2K liposomes. Inhibition of the methotrexate effect by NH4Cl suggested that methotrexate entered the cell by endocytosis of the liposomes. The results are consistent with differential internalization of H-2K, I-A, I-E, and Mr 94,000 and 180,000 cell surface molecules by mitogen-stimulated T and B cells.
beta-Endorphin enhances lymphocyte proliferative responses.
Authors: Gilman S C , Schwartz J M , Milner R J , Bloom F E , Feldman J D
Published: May 2006

The opioid peptides alpha- and beta-endorphin and [D-Ala2, Met5]enkephalin were investigated for their effect on the proliferation of resting and activated rat splenic lymphocytes in vitro. beta-Endorphin enhanced the proliferative response of spleen cells to the T cell mitogens concanavalin A and phytohemagglutinin. The effect of beta-endorphin was dose dependent and occurred at peptide concentrations similar to those found in rat plasma. alpha-Endorphin and [D-Ala2, Met5]enkephalin did not affect the proliferative responses to any mitogen tested. Furthermore, the potentiating effect of beta-endorphin was not reversed by treatment with 10 microM naloxone. None of the peptides had any effect on resting, unstimulated spleen cells or on the response to a mixture of lipopolysaccharide and dextran sulfate, which is specifically mitogenic for B lymphocytes. The pharmacological properties of the beta-endorphin potentiation indicate that the effect may be mediated by a nonopiate but beta-endorphin-specific mechanism. These results suggest a possible role for peripheral beta-endorphin and may provide a link between stress and disease susceptibility.
Radioimmunoassays of hidden viral antigens.
Authors: Neurath A R , Strick N , Baker L , Krugman S
Published: May 2006

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bond adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.
Insulin resistance and decreased insulin response to glucose in lean type 2 diabetics
Authors: Wajngot Alexandre , Roovete Arved , Vranić Mladen , Luft Rolf , Efendić Suad
Published: May 2006

In an attempt to determine the mechanism of decreased glucose tolerance in lean type 2 diabetics, glucose turnover in such subjects and controls was studied under basal conditions and during hyperglycemia induced by intravenous administration of glucose. The diabetics had decreased intravenous glucose tolerance and a fasting plasma glucose of 6-8 mM (108-144 mg/dl). Glucose was infused for 2 hr at 2 mg/kg per min in the controls (
n
= 16) and diabetics (
n
= 9). Furthermore, 11 healthy subjects were infused also with glucose at 4 mg/kg per min to match the glycemia of the diabetics. Glucose production, utilization, and metabolic clearance were assessed by the primed constant tracer infusion technique. In the basal state, diabetics showed normal plasma insulin, C peptide, and glucagon concentrations. Their increased basal plasma glucose levels were associated with normal rates of glucose production and utilization, but the metabolic glucose clearance was 21% lower than in the controls (
P
< 0.001), indicating decreased sensitivity to insulin. During infusion of glucose at 2 mg/kg per min, the hyperglycemia attained in the diabetics (170 mg/dl) was higher than that in controls (115 mg/dl) but comparable to that of the controls exposed to the higher glucose load. With the lower glucose load, metabolic clearance rate decreased more markedly in diabetics, again suggesting insulin resistance. This was further substantiated by the fact that, at the same insulin levels, glucose utilization did not increase more in the diabetics than in the controls, although the glycemia reached was considerably higher in the diabetics. With the lower glucose load, glucose production was suppressed to the same degree in the controls and diabetics, although the attained glycemia was much more marked in the latter. Because both insulin and hyperglycemia can suppress glucose production, some defect in the regulation of glucose production of the diabetics is also indicated. The insulin and C peptide levels were much higher in the controls than in the diabetics at the same levels of glycemia, demonstrating the inadequacy of insulin response to glycemia of the diabetics. Glucagon concentration was equally suppressed in all groups. In conclusion, impaired glucose tolerance of mild type 2 diabetics resulted both from inadequate insulin response and from decreased sensitivity to insulin. The insulin resistance could mainly be ascribed to inadequate glucose uptake, but a defect in glucose-induced suppression of glucose production may also have contributed.
Molecular defect in combined beta-galactosidase and neuraminidase deficiency in man.
Authors: D'Azzo A , Hoogeveen A , Reuser A J , Robinson D , Galjaard H
Published: May 2006

In normal human fibroblasts, an enzymically active 85,000-dalton precursor form of beta-galactosidase is processed, via a number of intermediates, into a mature 64,000-dalton form. In addition there is an enzymically inactive 32,000-dalton component and its 54,000-dalton precursor. In fibroblasts from patients with a combined deficiency of beta-galactosidase and neuraminidase these last two components are absent and hardly any mature beta-galactosidase can be demonstrated. Nevertheless, in the mutant fibroblasts, precursor beta-galactosidase is synthesized and processed normally. The excessive intralysosomal degradation that is responsible for the deficiency of mature beta-galactosidase can be partially corrected by addition of the protease inhibitor leupeptin, which results in the accumulation of 85,000-dalton precursor beta-galactosidase and of a partially processed 66,000-dalton form. When mutant cells were grown in the presence of a "corrective factor" purified from the medium of NH4Cl-stimulated cell cultures, both beta-galactosidase and neuraminidase activities were restored to low control levels. The immunoprecipitation pattern was completely normal after addition of the corrective factor, and mature 64,000-dalton beta-galactosidase accumulated in the mutant fibroblasts. We propose that the combined beta-galactosidase/neuraminidase deficiency is caused by a defective 32,000-dalton glycoprotein which is normally required to protect beta-galactosidase and neuraminidase against excessive intralysosomal degradation and to give these enzymes their full hydrolytic activity.
Labeling and selective recovery of newly synthesized viral DNA from simian virus 40-infected cells incubated with inorganic thiophosphate.
Authors: Sun I Y , Allfrey V G
Published: May 2006

African green monkey CV-1 cells infected with simian virus 40 (SV40) were incubated in the presence of sodium [35S]thiophosphate. The viral DNA was prepared and the newly synthesized SV40 DNA molecules containing phosphorothioate groups were selectively recovered by affinity chromatography on organomercurial-Sepharose columns. The thio-derivatized viral DNA occurred in both superhelical (form I) and relaxed circular (form II) configurations, and it gave the expected cleavage products upon digestion with the restriction endonucleases Kpn I and HindIII. Enzymatic digestion of 35S-labeled SV40 DNA to its component nucleotides and chromatographic separation of the resultant 5'-labeled phosphorothioate esters established the presence of the label in all four major nucleotides, but mainly in thymidylic acid. The ability to label and recovery selectively the newly synthesized polynucleotides by incubating cells with inorganic thiophosphate complements earlier findings [Sun, I. Y.-C. & Allfrey, V. G. (1982) J. Biol. Chem, 257, 1347--1353] that thiophosphate enters intracellular nucleotide pools and serves as an effective precursor for the phosphorylation of nuclear proteins in vivo.
In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase.
Authors: Peacock S , Cenatiempo Y , Robakis N , Brot N , Weissbach H
Published: May 2006

Plasmids pNF1337 and pNF1341, which contain part of the L10 operon including the RNA polymerase beta-subunit gene, have been used as templates in vitro to investigate expression of the beta-subunit gene. For these studies, the synthesis of the first dipeptide of the beta subunit, fMet-Val, was measured instead of that of the entire protein. By using this dipeptide system, we studied the effects of RNA polymerase holoenzyme and L factor (nus A gene product) on fMET-Val synthesis and compared the relative effects of the primary and secondary promoters in the L10 operon on expression of the beta-subunit gene. The results show that the inhibitory effect of RNA polymerase on beta-subunit synthesis and the stimulatory effect of L factor occur before formation of the first dipeptide bond. In this in vitro system, the secondary promoters account for about 50% of the total fMet-Val synthesized. Although the primary promoter is sensitive to guanosine 5'-diphosphate 3'-diphosphate in vitro, the secondary promoters are not affected by this nucleotide.
Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase i).
Authors: Levin Y , Skidgel R A , Erdös E G
Published: May 2006

Carboxypeptidase N (kininase I, arginine carboxypeptidase; EC 3.4.17.3) cleaves COOH-terminal basic amino acids of kinins, anaphylatoxins, and other peptides. The tetrameric enzyme of Mr 280,000 was purified from human plasma by ion-exchange and arginine-Sepharose affinity chromatography. Treatment with 3 M guanidine dissociated the enzyme into subunits of 83,000 and 48,000 molecular weight, which were separated and purified by gel filtration or affinity chromatography. When tested with hippurylarginine, hippurylargininic acid, benzoylalanyllysine, or bradykinin, the Mr 48,000 subunit was as active as the intact enzyme and was easily distinguished from human pancreatic carboxypeptidase B (EC 3.4.17.2). However, the Mr 48,000 subunit was less stable at acid pH or at 37 degrees C than the intact enzyme was. The carbohydrate-containing Mr 83,000 subunit was enzymatically inactive but stabilized the Mr 48,000 subunit at 37 degrees C. Trypsin, plasmin, and plasma or urinary kallikrein cleaved carboxypeptidase N into lower molecular weight active fragments, which were unstable at 37 degrees C. Cleavage of the Mr 48,000 subunit with the same enzymes increased activity and yielded fragments of Mr 29,000 or less. Antibodies to the Mr 83,000 of Mr 48,000 subunits crossreacted with the intact enzyme, and antibody to carboxypeptidase N also recognized both subunits. However, antibody to the Mr 83,000 subunit did not recognize Mr 48,000 subunit and antibody to the Mr 48,000 subunit did not crossreact with the Mr 83,000 subunit. Thus, this study indicates that carboxypeptidase N is composed of two immunologically distinct subunits, a Mr 48,000 subunit that is responsible for the enzymatic activity and a Mr 83,000 subunit that may stabilize the enzyme in blood.
Anisotropic magnetic interactions in the primary radical ion-pair of photosynthetic reaction centers
Authors: Boxer Steven G. , Chidsey Christopher E. D. , Roelofs Mark G.
Published: May 2006

The quantum yield of triplets formed by ion-pair recombination in quinone-depleted photosynthetic reaction centers is found to depend on their orientation in a magnetic field. This new effect is expected to be a general property of radical pair reactions in the solid state. For 0 < H < 1,000 G, the quantum yield anisotropy is caused by anisotropic electron dipole-electron dipole or nuclear hyperfine interactions, or both. For high fields it is dominated by the anisotropy of the difference
g
-tensor in the radical ion-pair. The magnitude and sign of the contribution of each interaction depend not only on the values of the principal components of each anisotropic tensor but also on the geometric relationship of the principal axes of each tensor to the transition dipole moment used to detect the yield. A detailed formalism is presented relating these quantities to the observed yield anisotropy. The expected magnitude of each anisotropic parameter is discussed. It is demonstrated that the field dependence of the yield anisotropy is consistent with these values for certain reaction center geometries.
Evolution of mouse immunoglobulin lambda genes.
Authors: Selsing E , Miller J , Wilson R , Storb U
Published: May 2006

The mouse has four C lambda and two V lambda genes. We have isolated Charon 4A clones that contain all six lambda genes from a BALB/c germ-line library. We present here the DNA sequences of the C lambda 2, C lambda 3, and C lambda 4 genes and also correct what are apparently errors in previous reports of C lambda 1 protein and DNA sequences. In addition, we have analyzed cloned DNAs by restriction mapping and electron microscopy to determine the relationships among the various lambda genes. By heteroduplex analysis, two gene clusters containing JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 show homology extending from the J regions 5' of C lambda 3/C lambda 2 to just 3' of C lambda 1/C lambda 4. Other than the region between the genes, very little homology exists in the C lambda flanking regions. In contrast, V lambda 1 and V lambda 2 genes show considerable homology extending into the 5' flanking regions. Large inverted repeats are found in the 5' flanking regions of V lambda 1 and C lambda 3, as well as in the 3' flanking regions of both C lambda gene clusters. DNA sequence divergences between the C lambda genes indicate that an ancestral JC lambda x--JC lambda g gene cluster arose at about the time of the first mammals by duplication of a primordial JC lambda gene. The data further suggest that the JC lambda x--JC lambda gene cluster duplicated after the speciation of mouse and man and subsequently diverged into the present day JC lambda 3--JC lambda 1 and JC lambda 2--JC lambda 4 gene clusters. C lambda 4, a pseudogene, became inactive at about the time of duplication of the ancestral JC lambda x--JC lambda y cluster. Comparison of DNA sequence divergence between the V lambda 1 and V lambda 2 genes demonstrates an anomaly. The percentage of amino acid replacement changes is approximately the same for V lambda 1/V lambda 2 as for C lambda 3/C lambda 2, implying that the ancestral V lambda gene was duplicated at the same time, and possibly together with, the JC lambda x--JC lambda y cluster. However, there are fewer silent changes than amino acid replacement changes between the V lambda 1/V lambda 2 genes, suggesting either that a selective pressure acted on the silent sites or that V lambda genes have only recently been duplicated. We also consider the possibility of a gene conversion event subsequent ot a more ancient duplication.