Change of the vibrational dynamics near the glass transition in polyisobutylene: Inelastic neutron scattering on a nonfragile polymer.
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Journal Article↗
Biomedical subjects
D Richter
Publications and source records attributed to D Richter.
Dendritic and axonal mRNA trafficking.
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Animals↗
The rat insulin-degrading enzyme. Molecular cloning and characterization of tissue-specific transcripts.
The primary structure of the rat insulin-degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl-terminal consensus sequence A/S-K-L, for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.
Amino Acid Sequence↗
Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A.
A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.
Animals↗
Improved imputation of non-responses to mailback questionnaires.
Poor response rates, perhaps as low as 10 to 30 per cent, certainly bring into question the validity of any survey suffering such a malady. Repeated mailouts are capable of providing information on those persons who are resistant to responding. If one is willing to assume an exponential drop-off in response rates, then imputation is possible for the entire group of non-responders. Furthermore, a second exponential drop-off in favourable (or non-favourable) responses to particular items within the questionnaire leads to closed form estimates of population parameters and their associated standard errors. Three or more mailouts also provide a test of validity of the model assumed.
Bias↗
[Growth inhibition of intraocular proliferative explants under in vitro conditions by verapamil].
BACKGROUND: For the treatment of non neoplastic proliferations in the fundus, a nontoxic, proliferation-inhibiting long-term therapy would be desirable in addition to vitreoretinal surgery. In theory, the use of calcium channel blockers as a means of prevention appears promising. METHODS: In order to test the action of Verapamil in the above application, 42 preretinal, epiretinal and subretinal membranes were removed during pars plana vitrectomy between May, 1991 and June, 1992. Following crushing, these were cultured under cell culture conditions. RESULTS: Cell division outside the membranes was seen in 27 cases. In 13 cases the minimum cell count was approximately 1000 per primary culture. Subcultures were prepared from each of the latter, the cells having been divided into four portions. Two of the samples were diluted with Verapamil in a concentration of 0.25 microgram/ml. When cell fusion began, the number of intact cells in each sample was determined. Verapamil inhibited growth significantly (significance level of 0.93, P < 0.01). CONCLUSIONS: A larger number of cases would have to be studied to permit more comprehensive conclusions.
Adolescent↗
A novel rat serotonin receptor: primary structure, pharmacology, and expression pattern in distinct brain regions.
Serotonin mediates various central and peripheral functions by its ability to bind to and activate a variety of receptors that belong to four pharmacologically defined classes termed 5-HT1 to 5-HT4. Using a polymerase chain reaction-derived probe, a clone, GPRFO, was isolated from a rat brain cDNA library. This cDNA encodes a putative G protein-coupled receptor that exhibits highest similarity to receptors for biogenic amines. RNA blot analysis indicated that the corresponding gene is expressed in the diencephalon. Consistent with the RNA blot data, GPRFO mRNA has been detected by in situ hybridization in the centrolateral, central medial, and intermediodorsal thalamic nuclei. Highest amounts of GPRFO mRNA, however, have been observed in a small area, i.e., the hippocampal rudiments and the stria longitudinalis. COS-7 cells transiently transfected with the GPRFO cDNA acquire saturable high-affinity binding sites for [3H]serotonin (KD = 41 nM). The pharmacological properties of the receptor differ from those of the known serotonin receptor subtypes, suggesting that the GPRFO cDNA encodes a novel serotonin receptor that is expressed in distinct rat brain regions.
Amino Acid Sequence↗
Changes in plasma and hepatic lipids, small intestinal histology and pancreatic enzyme activity due to aging and dietary fiber in rats.
Rats were fed either a control diet or a control diet supplemented with wheat bran, psyllium husk or oat bran to increase intake of fiber. Groups of rats were killed after 3.5, 10, 15, or 18.5 mo of consuming the diets. Plasma cholesterol and triglyceride concentrations were significantly higher in 18.5-mo-old than younger animals. Fiber supplementation did not prevent the age-related increase in lipids. Cecal weight, including contents, was higher in the psyllium husk and oat bran groups than control, and smooth muscle thickness in the ileum of psyllium husk and oat bran animals was greater than control. The score for torn villi in the small intestine was lower than expected in the wheat bran group. Amylase activity in the pancreas declined significantly with age in all groups. In aging animals fiber supplementation may enhance ileal compensation for decreases in proximal intestinal function but does not prevent age-related changes in the gut or in lipid concentrations.
Aging↗
Expression patterns of rat somatostatin receptor genes in pre- and postnatal brain and pituitary.
The relative abundances of mRNAs encoding four different somatostatin receptors were examined using PCR techniques during postnatal development of the rat brain and hypophysis. In most tissues, somatostatin receptor 1 and 4 mRNAs are more abundant than those encoding somatostatin receptor 2 and 3. Transcript levels of somatostatin receptor subtype 4 are relatively high in the cortex, hippocampus, and striatum, those of subtype 1 in the cortex and brainstem, and those of subtype 3 in the cerebellum. In situ hybridization revealed the presence of significant amounts of somatostatin receptor 1 mRNA, as early as prenatal day 14, in the trigeminal ganglion and in the neuroepithelial layers surrounding the lateral, third, and fourth ventricles. In the developing cortex a morphological change in the sites of somatostatin receptor 1 gene expression occurs; mRNA is present superficially in the cortex at prenatal stages, appears in all layers shortly after birth, and in adult rats is restricted to the deep cortical layers. In the cerebellum, somatostatin receptor 1 mRNA levels are highest around birth, declining thereafter. In contrast, cerebellar somatostatin receptor 3 transcripts are absent at birth, become detectable around postnatal day 7, and reach a maximal level during maturation.
Amino Acid Sequence↗
Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni.
In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, we examined the effect of (i) single versus multiple vaccinations with irradiated cercariae, (ii) the dose of irradiation (15 or 50 kilorads) administered to the cercariae, and (iii) the genetic background of mouse strains, high-responder (C57BL/6J) versus moderate-responder (CBA/J) mice. We found that the number of vaccinations did not alter antibody specificity but modified the relative antibody titers against particular antigens. The dose of irradiation used to attenuate the immunizing cercariae had a similar effect on antibody titers but in addition influenced antibody specificity. Only mice that had been vaccinated with moderately irradiated cercariae recognized cathepsin B (Sm31) and Sm32. Interestingly, when vaccinated mice of the two strains, C57BL/6J and CBA/J, were compared, differences in antibody responses to particular antigens were observed. Both strains recognized the integral membrane protein Sm23, glutathione S-transferase, and cathepsin B, whereas Sm32 and paramyosin were recognized only by CBA/J mice, and heat shock protein 70 was recognized exclusively by C57BL/6J mice. In this study, we conclusively identified six distinct antigens that are specifically recognized by the humoral immune response of vaccinated mice.
Animals↗
Isotype responses to candidate vaccine antigens in protective sera obtained from mice vaccinated with irradiated cercariae of Schistosoma mansoni.
In experimental schistosomiasis, sera of mice multiply vaccinated with radiation-attenuated cercariae of Schistosoma mansoni passively transfer resistance against cercarial challenge to naive mice. To further characterize these sera, we tested their protective capacities in two mouse strains (C57BL/6J and CBA/J) and compared the antigen-specific isotype compositions of the different protective sera by means of the enzyme-linked immunosorbent assay. By using an array of purified schistosomal antigens, the patterns of antibody titers and isotypes differed for each experimental group and antigen. In the most-protective C57BL/6J sera, high levels of immunoglobulin G1 (IgG1), IgG2a, and IgG2b bound to heat shock protein 70 and the integral membrane protein Sm23, whereas recognition of these antigens by less-protective CBA/J sera was lower. Glutathione S-transferase (GST) was recognized predominantly by IgM antibodies of all vaccinated groups, and a significant portion of this response was directed against carbohydrate epitopes. Antibodies specific for triosephosphate isomerase, paramyosin, and Sm32 (hemoglobinase) were present in less-protective sera and thus seem less relevant for passive transfer of resistance. The results of this study suggest a contribution of IgG antibodies specific for heat shock protein 70 and Sm23, and possibly a contribution of GST-specific IgM antibodies, to the protective effect of sera from C57BL/6J mice vaccinated with irradiated cercariae.
Animals↗
Mitochondrial cardiomyopathy and scapuloperoneal spinal muscular atrophy in a child.
A 14-year old boy was admitted for signs of heart failure and scapuloperoneal muscle weakness. He fulfilled the clinical, functional and diagnostic criteria for dilated cardiomyopathy. There was also a moderate increase in pulmonary vascular resistance. The immunohistochemical examination of the heart muscle revealed a slightly positive phytohemagglutinin reaction and minimal IgM deposits without complement. The electron microscopy examination disclosed increased numbers of abnormal mitochondria disrupting the usual cell structure; the mitochondria were of various sizes with irregular and abnormal structure of the cristae. The scapuloperoneal spinal muscular atrophy was mild and diagnosed according to clinical and electromyographic findings. Light microscope examination of the skeletal muscle revealed hypotrophic fibers. This patient is presumed to have postinflammatory mitochondriopathy and is currently being managed on low-dose digitalis, diuretics and captopril.
Adolescent↗
Rat insulin-degrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP, BNP, and CNP revealed by HPLC and mass spectrometry.
The degradation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) by insulin-degrading enzyme (IDE) has been investigated. As revealed by high-performance liquid chromatography, all three peptides are sequentially cleaved at a limited number of sites, the latter of which were identified by mass spectrometric analyses. The studies revealed that ANP is preferred as substrate over BNP and CNP. ANP degradation is rapidly initiated by hydrolysis at the Ser25-Phe26 bond. Three additional cleavage sites were identified in ANP after prolonged incubation with IDE; in contrast, three and two bonds were hydrolyzed in BNP and CNP, respectively. Analysis of the nine cleavage sites shows a preference for basic or hydrophobic amino acid residues on the carboxyl side of a cleaved peptide bond. In contrast to most of the peptide fragments generated by IDE activity, the initial ANP cleavage product, F-R-Y, is rapidly degraded further by cleavage of the R-Y bond. Cross-linking studies with 125I-ANP in the presence of sulfhydryl-modifying agent indicate that IDE activity is inhibited at the level of initial substrate binding whereas metal-ion chelating agents only prevent hydrolysis. On the basis of its structural and enzymatic properties, IDE exhibits striking similarity to a number of recently-described endopeptidases.
Amino Acid Sequence↗
Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain.
The tetradecapeptide somatotropin-release inhibiting factor somatostatin-14 regulates the release of peptide hormones and also functions as neurotransmitter. The octacosapeptide somatostatin-28, the N-terminally extended form of somatostatin-14, shows similar biological activities yet with different potencies. Both peptides most likely function through distinct receptors. Here we report on the molecular and functional characterization of a somatostatin-28 receptor (SSR-28) cloned from a rat brain cDNA library. The nucleotide sequence contains an open reading frame for a protein of 428 amino acid residues with a predicted molecular mass of 47 kDa. Binding assays using radiolabeled somatostatin-14 and membranes from COS cells transfected with the cloned cDNA show that this receptor, SSR-28, has a higher binding affinity for somatostatin-28 (IC50 = 0.24 nM) than for somatostatin-14 (IC50 = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase mRNA in rat cerebellum and at significantly lower abundance in other brain regions. In situ hybridization indicates that SSR-28 mRNA is present in the granular and Purkinje cell layers of the cerebellum and in the large cells of the hypoglossal nucleus of the brain stem. Signals for SSR-28 mRNA do not overlap with those of a previously cloned rat receptor that preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA is found in the medial cerebellar nucleus, horizontal limb of the diagonal band, various hypothalamic nuclei, and in layers IV and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 mRNAs are developmentally regulated; the levels of the former are highest around birth and levels of the latter are highest at the adult stage.
Amino Acid Sequence↗
Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti.
Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.
Amino Acid Sequence↗
Molecular characterization of a novel rat protein structurally related to poly(A) binding proteins and the 70K protein of the U1 small nuclear ribonucleoprotein particle (snRNP)
A cDNA has been isolated from a rat testis library which encodes a novel protein of 100 kDa that contains domains found in two different proteins involved in the processing of pre-mRNAs. Computer-assisted comparison reveals that one sequence motif of 30 amino-acid residues is very similar to a region conserved in the C-terminal part of eukaryotic poly(A) binding proteins (PABP). A second region of the rat 100 kDa protein, containing alternating basic, mostly arginine, and acidic amino-acid residues, is structurally related to sequence motifs found in the 70K protein of the U1 small nuclear ribonucleoprotein particle (snRNP), which is involved in RNA splicing. Northern blot analysis shows that a corresponding 9.5 kb transcript is highly expressed in rat testis; lower mRNA levels are found in other tissues such as liver, kidney, lung and brain. Ontogenic studies reveal that the expression of the 100 kDa protein-encoding gene and sexual maturation are correlated, being barely detectable during early post-natal life but reaching maximal levels around the first month after birth.
Amino Acid Sequence↗
A protective monoclonal antibody specifically recognizes and alters the catalytic activity of schistosome triose-phosphate isomerase.
mAb M.1 was previously shown to recognize a 28-kDa Ag in all stages of the human helminth parasite, Schistosoma mansoni, and to bind to the surface membranes of newly transformed schistosomula in a transient manner. Here we demonstrate that M.1 passively transfers partial resistance (41-49%) to cercarial challenge in naive mice. Thus, the 28-kDa Ag recognized by M.1 is a putative vaccine candidate. After immunoaffinity purification, tryptic digests of the 28-kDa Ag were prepared and individual peptides were sequenced. Amino terminus sequences of tryptic peptides of the 28-kDa Ag had high (79-87%) sequence homology with the mammalian glycolytic/gluconeogenic enzyme triosephosphate isomerase (TPI). Purified, native 28-kDa Ag from adult parasites was shown to function enzymatically in an analogous manner to yeast and mammalian TPI in the reverse reaction. Addition of M.1 antibody to the enzyme reaction altered the catalytic activity of schistosome TPI. To determine the immunologic cross-reactivity of this vaccine candidate with mammalian TPI, Western blot analysis was performed and demonstrated that M.1 was immunologically specific for the schistosome enzyme.
Amino Acid Sequence↗