Biomedical subjects

R Kellner

Publications and source records attributed to R Kellner.

At least 55 records · Page 3Linked to original sources

PubMed1996

Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. Cloning, heterologous expression, and localization.

The membrane-bound guanylyl cyclase in vertebrate photoreceptor cells is one of the key enzymes in visual transduction. It is highly sensitive to the free calcium concentration ([Ca2+]). The activation process is cooperative and mediated by a novel calcium-binding protein named GCAP (guanylyl cyclase-activating protein). We isolated GCAP from bovine rod outer segments, determined amino acid sequences of proteolytically obtained peptides, and cloned its gene. The Ca2+-bound form of native GCAP has an apparent molecular mass of 20.5 kDa and the Ca2+-free form of 25 kDa as determined by SDS-polyacrylamide gel electrophoresis. Recombinant GCAP was functionally expressed in Escherichia coli. Activation of guanylyl cyclase in vertebrate photoreceptor cells by native acylated GCAP was half-maximal at 100 nM free [Ca2+] with a Hill coefficient of 2.5. Activation by recombinant nonacylated GCAP showed a lower degree of cooperativity (n = 2.0), and half-maximal activation was shifted to 261 nM free [Ca2+]. Immunocytochemically we localized GCAP only in rod and cone cells of a bovine retina.

Amino Acid Sequence↗

PubMed1996

Determination of alpha-amylase activity using Fourier transform infrared spectroscopy.

A new method for the determination of alpha-amylase activity in aqueous solutions and human serum with FTIR-spectroscopy is proposed. The chemical reaction catalyzed by the enzyme under study can be followed directly when applying FTIR-spectroscopic detection also in the case, where no colored or electrochemical active species are generated or consumed during the course of the reaction of alpha-amylase with simple starch. Therefore the determination of the alpha-amylase activity could successfully be performed by recording two FTIR-spectra, one immediately after mixing the sample and a substrate (starch-) solution and the other after a 20 min reaction time. From these two FTIR-spectra a difference spectrum was calculated hereby eliminating an unspecific absorption of the matrix. The intensities of the resulting difference spectra corresponded to the extent of the reaction which took place during the investigated time interval and hence could be related to the activity of the enzyme in the sample. The developed method is linear from 80 to 1400 U/l (r.s.d.=5% for 700 U/l) in aqueous solutions and was also successfully applied to the determination of alpha-amylase activity in human serum where a linear working range from 100 to 800 U/l (r.s.d.=11% for 150 U/l) was achieved.

Journal Article↗

PubMed1996

Two synpatic vesicle proteins of 25 kDa: a comparison of the molecular properties and tissue distribution of svp25 and o-rab3.

Two synaptic vesicle proteins of the electric ray Torpedo--svp25 and o-rab3--are compared with respect to their biochemical properties and tissue distribution. On SDS-PAGE both proteins migrate to the same position of about 25 kDa. As revealed by application of monospecific antibodies and subcellular fractionation both proteins comigrate and cofractionate with the synaptic vesicle compartment. o-Rab3 and svp25 can be separated by lectin chromatography; svp25 is highly glycosylated and binds to concanavalin A sepharose. Upon deglycosylation using glycopeptidase F and O-glycosidase its apparent molecular mass is reduced to about 14 kDa. Partial amino acid sequences obtained by direct microsequencing of purified and deglycosylated svp25 revealed that svp25 is a novel protein that has not yet been characterized in molecular terms. Whereas svp25 was detected in all brain areas investigated, the expression of o-rab3 was found to be restricted to specific regions. An immunoblot analysis demonstrates an exclusive association of both proteins with neural tissues. Our results suggest that cholinergic synaptic vesicles from electric ray electric organ contain at least two membrane-associated proteins of an apparent molecular mass of 25 kDa, the membrane associated o-rab3 and the membrane integral protein svp25. The two proteins can be separated by lectin chromatography for assessment of their biochemical properties.

Animals↗

PubMed1996

Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.

Aspartic Acid Endopeptidases↗

PubMed1995

Purification and properties of human placental ATP diphosphohydrolase.

ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.

Affinity Labels↗

PubMed1995

A family of neuropeptides that inhibit juvenile hormone biosynthesis in the cricket, Gryllus bimaculatus.

Four nonapeptides that inhibit juvenile hormone synthesis have been isolated by four high performance liquid chromatographic steps from extracts of the brain of the field cricket, Gryllus bimaculatus. The primary structures of these peptides were assigned by Edman degradation and mass spectrometry as Gly-Trp-Gln-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B1), Gly-Trp-Arg-Asp-Leu-Asn-Gly-Gly-Trp-NH2 (Grb-AST B2), Ala-Trp-Arg-Asp-Leu-Ser-Gly-Gly-Trp-NH2 (Grb-AST B3), and Ala-Trp-Glu-Arg-Phe-His-Gly-Ser-Trp-NH2 (Grb-AST B4). Each of the peptides shows high sequence similarity to the locustamyoinhibiting peptide (Lom-MIP), but is structurally different from all the allatostatins so far identified. The synthetic allatostatins Grb-AST B1-4 are potent inhibitors (50% inhibition at 10(-8) to 7 x 10(-8) M) of juvenile hormone III biosynthesis by corpora allata from 3-day-old virgin females of G. bimaculatus using an in vitro bioassay. At 10(-7) M, Grb-AST B1 also strongly inhibits juvenile hormone III biosynthesis by corpora allata from 2-day-old adult males and 1-day-old (males and females) and 4-day-old (females) last instar larvae of G. bimaculatus. The inhibitory effect of Grb-AST B1 was also evident on corpora allata from a related species, Acheta domesticus. Inhibition of juvenile hormone synthesis by Grb-AST B1-4 is reversible.

Amino Acid Sequence↗

PubMed1995

Identification of two allatostatins from the cricket, Gryllus bimaculatus de Geer (Ensifera, Gryllidae): additional members of a family of neuropeptides inhibiting juvenile hormone biosynthesis.

Two peptide inhibitors of juvenile hormone biosynthesis, designated G. bimaculatus allatostatins A1 and A2, have been purified from extracts of the brain of the field cricket Gryllus bimaculatus. The primary structures of these peptides were assigned as Ala-Gln-His-Gln-Tyr-Ser-Phe-Gly-Leu-NH2 (Grb-AST A1) and Ala-Gly-Gly-Arg-Gln-Tyr-Gly-Phe-Gly-Leu-NH2 (Grb-AST A2). Each of the peptides shows C-terminal amino acid sequence similarity to cockroach allatostatins and blowfly callatostatins. The two peptides are potent inhibitors of in vitro juvenile hormone production by corpora allata from virgin females of G. bimaculatus.

Amino Acid Sequence↗

PubMed1995

Isolation and primary structure of a novel adipokinetic peptide from the pyrgomorphid grasshopper, Phymateus leprosus.

Using a heterologous bioassay, monitoring lipid increase in the haemolymph of migratory locusts, two peptides have been purified from methanolic extracts of corpora cardiaca of the pyrgomorphid grasshopper, Phymateus leprosus. The structures of both peptides were elucidated by a combination of Edman degradation, after deblocking the N-terminal pyroglutamic acid residue, and mass spectrometric techniques. One peptide is an octapeptide (pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2) which also occurs in desert locusts; the second peptide is a novel decapeptide member of the AKH-family (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Ser-NH2).

Amino Acid Sequence↗

PubMed1995

Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions.

When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.

Amino Acid Sequence↗

PubMed1995

Imagery rehearsal treatment for chronic nightmares.

Fifty-eight chronic nightmare sufferers were randomly assigned to two groups: treatment (n = 39) and wait-list control (n = 19). Treated Ss were taught a cognitive-behavioral technique called imagery rehearsal in which they learned in a waking state to change a nightmare and then to visualize the new set of images. Subjects were assessed pre-treatment and 3 months followup for nightmare frequency, self-rated distress and subjective sleep quality. Compared to controls, the treatment group showed significant and clinically meaningful decreases in nightmares. Treated Ss decreased nightmares as measured in nights/week (mean = -2.0, SD = 1.7, P = 0.0001) and actual number of nightmares (mean = -4.2, SD = 4.5, P = 0.0001). Significant improvement in self-rated sleep quality occurred in those treated compared with controls (P = 0.004); and, reduction in nightmares was a significant predictor of improvement in sleep (r = 0.55, P = 0.0001). These preliminary results lend support to the theory that, for some chronic sufferers, nightmares may be conceptualized as a primary sleep disorder which can be effectively and inexpensively treated with cognitive-behavioral therapy.

Anxiety↗

PubMed1995

Isolation and primary structures of neuropeptides of the AKH/RPCH family from various termite species.

We have isolated neuropeptides of the AKH/RPCH family from extracts of whole heads of four termite species (Mastotermes darwiniensis, Microhodotermes viator, Hodotermes mossambicus, and Trinervitermes trinervoides) using the effect of mobilizing lipids in Locusta migratoria for bioassay. Isolation was essentially achieved by two steps of reversed-phase chromatography (on phenyl-support followed by C-18). The peptides were identified by Edman degradation after deblocking with oxoprolyl peptidase. Each termite species contained only one AKH/RPCH family member. The primary structure in M. darwiniensis and T. trinervoides is pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, a peptide previously found mainly in cockroaches and code named Pea-CAH-I. The peptide from M. viator has the primary sequence pGlu-Ile-Asn-Phe-Thr-Pro-Asn-Trp-NH2; it is a novel member of the family and is code-named Miv-CC (Microhodotermes viator corpus cardiacum peptide). Phylogenetic relations between the known cockroach and mantid AKH/RPCH octapeptides and the termite peptides from this study could be revealed employing the parsimony method. Based on a computer analysis, using PAUP 3.1.1., we concluded that termites are plesiomorphic with regard to cockroaches, and mantids are the sister taxon to the termite/cockroach group.

Amino Acid Sequence↗

PubMed1994

A membrane-bound protein phosphatase type 2C from Paramecium tetraurelia. Purification, characterization, and cloning.

We isolated the first membrane-bound type 2C serine/threonine protein phosphatase from the ciliated protozoan Paramecium tetraurelia (PtPP2C). Three isozymes of 33, 32, and 31 kDa with a specific activity of 1 mumol.min-1.mg1 were purified from the ciliary membrane. All enzymatic properties including (a) insensitivity toward inhibitors of other protein phosphatase families such as okadaic acid and microcystin, (b) absolute requirement for divalent cations, and (c) substrate specificity tested with synthetic phosphopeptides were identical to mammalian PP2C enzymes and identified the PtPP2C as a canonical PP2C in spite of it being about 25% smaller. The NH2-terminal was blocked. Microsequencing of six tryptic peptides established a relationship to other PP2C enzymes. The PtPP2C gene was obtained using degenerate oligonucleotide primers and the polymerase chain reaction. The gene coded for a 33-kDa protein with 300 amino acids and had an (A+T) content of 62%, typical for this protozoan. Nine of 15 Gln residues are encoded by TAA, a universal stop codon which codes for Gln in Paramecium. A large truncation at the COOH-terminal is responsible for the smaller size of the PtPP2C. Only a single transcript of 1 kilobase was detected with a Northern blot indicating that the 32- and 31-kDa proteins were proteolytic products of the 33-kDa enzyme. Sequence comparisons with PP2C enzymes from rat, rabbit, yeast, Arabidopsis, and Leishmania defined a highly diverged enzyme family which shares three conserved domains, I, II, and III, accounting for about 25% of the primary structure. We demonstrated further that the distances between domains I/II and II/III are very similar in all PP2C enzymes (9-13 and 74-80 amino acids, respectively). However, the amino acid sequences of the spacer regions are unrelated. In addition, the COOH-terminal ends of 100-200 amino acids which comprise 30-50% of the enzyme, display no identity. A dendrogramm shows that PtPP2C surprisingly is most closely related to the mammalian PP2C, and enzymes from Leishmania, Arabidopsis, and yeast are more distant relatives.

Amino Acid Sequence↗

PubMed1994

A novel adipokinetic octapeptide found in the damselflies Pseudagrion inconspicuum and Ischnura senegalensis.

A member of the adipokinetic hormone family of peptide was identified in the damselflies Pseudagrion inconspicuum and Ischnura senegalensis using a heterologous (in migratory locusts and American cockroaches) and a homologous (in P. inconspicuum) bioassay. After isolation of the peptide by reversed-phase h.p.i.c. of corpora cardiaca, its structure was determined by automated Edman degradation and matrix-assisted laser-desorption ionization m.s. The sequence of a blocked uncharged octapeptide was established: pGlu-Val-Asn-Phe-Thr-Pro-Gly-TrpNH2. One corpus cardiacum of P. inconspicuum contains about 2.4 pmol of this novel peptide. The synthetic peptide was chromatographically indistinguishable from the natural compound, and on injection in low quantities into the damselfly P. inconspicuum, haemolymph levels of lipids were increased. It is concluded that the novel peptide may be involved in controlling lipid mobilization during flight. Hence the peptide is designated Psi-AKH, Pseudagrion inconspicuum adipokinetic hormone.

Amino Acid Sequence↗

PubMed1994

A possible regulation of negative factor (Nef) activity of human immunodeficiency virus type 1 by the viral protease.

Negative factor (Nef) protein from human immunodeficiency virus type 1 (HIV-1) is cleaved into two well-defined domains by the HIV-1-encoded protease. The cleavage site is located between Trp57 and Leu58 and is well conserved. The two domains are stable in the presence of protease for more than 48 h. The C-terminal core domain contains a well-conserved well-folded region. The cleavage releases the core domain from the myristoylated membrane anchor domain. As is the case for other HIV proteins, cleavage of Nef could be crucial for correct biological function.

Amino Acid Sequence↗

PubMed1994

Bovine retinal rod guanyl cyclase represents a new N-glycosylated subtype of membrane-bound guanyl cyclases.

The molecular properties of retinal rod guanyl cyclase were investigated. Peptides were derived from a 112-kDa protein previously identified as the particulate bovine retinal rod guanyl cyclase. The peptides showed 100% identity to the deduced amino acid sequence of the cloned human retina-specific membrane guanyl cyclase, whereas identity to the members of the natriuretic peptide receptor guanyl cyclases was 14-59%. The 112-kDa protein was further purified by a new approach using wheat-germ agglutinin chromatography. This indicated N-linked glycosylation in retinal rod guanyl cyclase. N-glycosylation was unexpected from the sequence of the human retina-specific membrane guanyl cyclase, although it is a common property of natriuretic peptide receptors. Therefore, we further analyzed the carbohydrate composition of bovine retinal rod guanyl cyclase by lectin binding using the lectins Galanthus nivalis agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin, Ricinus communis agglutinin, Datura stramonium agglutinin, peanut agglutinin and by chromatography of the purified enzyme using concanavalin-A-Sepharose. The oligosaccharide side chains were of the high-mannose type or hybrid type, probably with mannose, N-acetylglucosamine and sialic acid as terminal sugars. Enzymic deglycosylation by N-glycosidase F was achieved after proteolytic digestion with endoproteinase Glu-C. Lectins neither influenced the basal nor the stimulated guanyl-cyclase activity at low calcium concentrations. Our results indicate that the particulate rod guanyl cyclase represents an unusual new subtype of membrane-bound guanyl cyclases.

Amino Acid Sequence↗

PubMed1994

Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1.

Proteolytic experiments in conjunction with 1H-NMR spectroscopy show that the Nef (negative factor) protein from human immunodeficiency virus type 1 probably consists of two main domains, the N-terminal anchor domain at amino acid positions 2-65 and the C-terminal core domain at positions 66-206. The N-terminal domain is likely to be located at the surface of the protein, while the C-terminal domain has a compactly folded core and is stable in the absence of the anchor domain. It is conceivable that the core domain represents a functional domain of the Nef protein, activated after the removal of the membrane anchor by the human-immunodeficiency-virus protease or cellular proteases. Nef is stable at pH 5-12 and denatures at 317-322 K. The Nef protein remains in its native conformation in dimethyl-sulfoxide/water mixtures up to 35% (by vol.), and in acetonitrile/water up to 14% (by vol.). Nef refolds spontaneously after denaturation with urea or guanidinium hydrochloride. The 1H-NMR parameters and pKa values of five of the nine histidine residues and one of the seven tyrosine residues were determined and were found in four cases to be typical for residues which are not located in the interior of the protein.

Amino Acid Sequence↗

PubMed1994

VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells.

In simple epithelial cells, apical and basolateral proteins and lipids in transit to the cell surface are sorted in the trans-Golgi network. We have recently isolated detergent-insoluble complexes from Madin-Darby canine kidney cells that are enriched in glycosphingolipids, apical cargo and a subset of the proteins of the exocytic carrier vesicles. The vesicular proteins are thought to be involved in protein sorting and include VIP21-caveolin. The vesicular protein VIP36 (36 kDa vesicular integral membrane protein) has been purified from a CHAPS-insoluble residue and a cDNA encoding VIP36 has been isolated. The N-terminal 31 kDa luminal/exoplasmic domain of the encoded protein shows homology to leguminous plant lectins. The transiently expressed protein is localized to the Golgi apparatus, endosomal and vesicular structures and the plasma membrane, as predicted for a protein involved in transport between the Golgi and the cell surface. It is diffusely localized on the plasma membrane but can be redistributed by antibody modulation into caveolae and clathrin-coated pits. We speculate that VIP36 binds to sugar residues of glycosphingolipids and/or glycosylphosphatidyl-inositol anchors and might provide a link between the extracellular/luminal face of glycolipid rafts and the cytoplasmic protein segregation machinery.

Amino Acid Sequence↗

PubMed1994

Purification and properties of the squalene-hopene cyclase from Rhodopseudomonas palustris, a purple non-sulfur bacterium producing hopanoids and tetrahymanol.

The squalene-hopene cyclase of the hopanoid- and tetrahymanol-producing Rhodopseudomonas palustris was released from the isolated membranes by CHAPS and purified to homogeneity by successive chromatography on DEAE Sephacel, Octyl Sepharose, and Blue Sepharose. The enzyme has a molecular weight of 70 kDa as determined by SDS-PAGE and an isoelectric point at about pH 5.0. The enzyme activity has a maximum at 30 degrees C and at pH 6.5. No production of tetrahymanol could be demonstrated by using either crude or purified cyclase preparations.

Amino Acid Sequence↗