bluuebunny/crossref_metadata_2025 huggingface dataset:
Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection
Authors: Lieberman Judy , Shankar Premlata , Manjunath N. , Andersson Jan
Published: July 2002

CD8 T cells play an important role in protection and control of HIV-1 by direct cytolysis of infected cells and by suppression of viral replication by secreted factors. However, although HIV-1–infected individuals have a high frequency of HIV-1–specific CD8 T cells, viral reservoirs persist and progressive immunodeficiency generally ensues in the absence of continuous potent antiviral drugs. Freshly isolated HIV-specific CD8 T cells are often unable to lyse HIV-1–infected cells. Maturation into competent cytotoxic T lymphocytes may be blocked during the initial encounter with antigen because of defects in antigen presentation by interdigitating dendritic cells or HIV-infected macrophages. The molecular basis for impaired function is multifactorial, due to incomplete T-cell signaling and activation (in part related to CD3ζ and CD28 down-modulation), reduced perforin expression, and inefficient trafficking of HIV-specific CD8 T cells to lymphoid sites of infection. CD8 T-cell dysfunction can partially be corrected in vitro with short-term exposure to interleukin 2, suggesting that impaired HIV-specific CD4 T helper function may play a significant causal or exacerbating role. Functional defects are qualitatively different and more severe with advanced disease, when interferon γ production also becomes compromised.
The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials
Authors: Kottaridis Panagiotis D. , Gale Rosemary E. , Frew Marion E. , Harrison Georgina , Langabeer Stephen E. , Belton Andrea A. , Walker Helen , Wheatley Keith , Bowen David T. , Burnett Alan K. , Goldstone Anthony H. , Linch David C.
Published: July 2002

In acute myeloid leukemia (AML), further prognostic determinants are required in addition to cytogenetics to predict patients at increased risk of relapse. Recent studies have indicated that an internal tandem duplication (ITD) in the FLT3 gene may adversely affect clinical outcome. This study evaluated the impact of a FLT3/ITD mutation on outcome in 854 patients, mostly 60 years of age or younger, treated in the United Kingdom Medical Research Council (MRC) AML trials. An FLT3/ITD mutation was present in 27% of the patients and was associated with leukocytosis and a high percentage of bone marrow blast cells (P < .001 for both). It had a borderline association with a lower complete remission rate (P = .05) and a higher induction death rate (P = .04), and was associated with increased relapse risk (RR), adverse disease-free survival (DFS), event-free survival (EFS), and overall survival (OS) (P < .001 for all). In multivariate analysis, presence of a mutation was the most significant prognostic factor predicting RR and DFS (P < .0001) and was still significant for OS (P = .009) and EFS (P = .002). There was no evidence that the relative effect of a FLT3/ITD differed between the cytogenetic risk groups. More than one mutation was detected in 23% of FLT3/ITD+ patients and was associated with worse OS (P = .04) and EFS (P = .07). Biallelic disease or partial/complete loss of wild-type alleles was present in 10% of FLT3/ITD+ patients. The suggestion is made that detection of a FLT3/ITD should be included as a routine test at diagnosis and evaluated for therapeutic management.
Inhibitor of histone deacetylation, depsipeptide (FR901228), in the treatment of peripheral and cutaneous T-cell lymphoma: a case report
Authors: Piekarz Richard L. , Robey Rob , Sandor Victor , Bakke Susan , Wilson Wyndham H. , Dahmoush Laila , Kingma Douglas M. , Turner Maria L. , Altemus Rosemary , Bates Susan E.
Published: July 2002

Depsipeptide, FR901228, has demonstrated potent in vitro and in vivo cytotoxic activity against murine and human tumor cell lines. In the laboratory, it has been shown to be a histone deacetylase (HDAC) inhibitor. In a phase I trial of depsipeptide conducted at the National Cancer Institute, 3 patients with cutaneous T-cell lymphoma had a partial response, and 1 patient with peripheral T-cell lymphoma, unspecified, had a complete response. Sézary cells isolated from patients after treatment had increased histone acetylation. These results suggest that inhibition of HDAC is a novel and potentially effective therapy for patients with T-cell lymphoma.
Treatment of High-Risk Neuroblastoma With Triple-Tandem High-Dose Therapy and Stem-Cell Rescue: Results of the Chicago Pilot II Study
Authors: Kletzel Morris , Katzenstein Howard M. , Haut Paul R. , Yu Alice L. , Morgan Elaine , Reynolds Marleta , Geissler Grant , Marymount Maryanne H. , Liu Dachao , Kalapurakal John A. , Shore Richard M. , Bardo Diana M.E. , Schmoldt Jennifer , Rademaker Alfred W. , Cohn Susan L.
Published: July 2002

PURPOSE: To investigate whether intensive induction therapy followed by triple-tandem cycles of high-dose therapy with peripheral-blood stem-cell rescue and local irradiation will improve event-free survival for patients with high-risk neuroblastoma.

PATIENTS AND METHODS: From August 1995 to January 2000, 25 consecutive newly diagnosed high-risk neuroblastoma patients and one child with recurrent MYCN-amplified disease were enrolled onto the Chicago Pilot II Protocol. After induction therapy and surgery, peripheral-blood stem cells were mobilized with three cycles of high-dose cyclophosphamide and granulocyte colony-stimulating factor. Patients then underwent triple-tandem cycles of high-dose therapy with peripheral-blood stem-cell rescue followed by radiation to the primary site.

RESULTS: Twenty-two of the 26 patients successfully completed induction therapy and were eligible for the triple-tandem consolidation high-dose therapy. Sufficient numbers of peripheral-blood stem cells were collected in all but one patient. Seventeen patients were able to complete all three cycles of high-dose therapy and peripheral-blood stem-cell rescue, two patients completed two cycles, and three patients completed one cycle. There was one toxic death, and one patient died from complications of treatment for graft failure. With a median follow-up of 38 months, the 3-year event-free survival and survival rates are 57% ± 11% and 79% ± 10%, respectively.

CONCLUSION: The results of this pilot study demonstrate that it is feasible to intensify consolidation with triple-tandem high-dose chemotherapy and peripheral-blood stem-cell rescue and local irradiation, and suggest that this treatment strategy may lead to improved survival for patients with high-risk neuroblastoma.
Monoclonal antibodies to choline acetyltransferase of rat brain
Authors: Ichikawa Tomoyuki , Ishida Isao , Deguchi Takeo
Published: July 2002

Monoclonal antibodies to rat brain choline acetyltransferase were produced by the hybridoma technique. Two stable cell lines, Ab‐57 and Ab‐60, secreted immunoglobulin of subclass IgG1. The monoclonal antibodies bound to choline acetyltransferase without blocking catalytic activity. Affinity of Ab‐57 was 100‐times higher than that of Ab‐60. Both antibodies bound to the rat enzyme in a mutually exclusive fashion. The antibodies showed cross‐species reactivity with choline acetyltransferase from several mammalian brains.
Rapid conversion of newly‐synthesized orotate to uridine‐5‐monophosphate by rat liver cytosolic enzymes
Authors: Dileepan Kottarappat N. , Kennedy James
Published: July 2002

It had been noted previously that the activity of mitochondrial dihydroorotate dehydrogenase was lower in crude tissue preparations containing cytosol than in isolated mitochondria. Closer examination reveals that the apparent lower enzyme activity is due to rapid conversion of newly‐synthesized orotate to uridine‐5‐monophosphate by the cytosolic enzymes, orotate phosphoribosyltransferase and orotidylate decarboxylase.
Interactions of antibodies against soluble phosphofructokinase with the soluble and particulate enzymes from yeast
Authors: Huse Klaus , Kopperschläger Gerhard
Published: July 2002

Antibodies obtained from rabbits against soluble yeast phosphofructokinase also react with the particulate yeast phosphofructokinase. Their effects on the activity of the soluble enzyme recognized as inactivation or slight activation depend on the specific immune response of an individual animal yielding antisera with different proportions of inactivating and activating antibodies. The availability of particulate phosphofructokinase to complex inactivating antibodies specifically allows a separation of activating and inactivating antibodies from each other by a simple extraction procedure.
Reserpine labels the catecholamine transporter in synaptic vesicles from bovine caudate nucleus
Authors: Near Joseph A. , Mahler Henry R.
Published: July 2002

Tritiated reserpine binds to synaptic vesicles from bovine caudate with high affinity (K
app
d = 1.25 nM, B
max = 3.3 pmol/mg protein). This interaction is both ATP‐dependent and sensitive to the protonophores CCCP and nigericin, suggesting that a proton electrochemical gradient is required for binding. Dopamine, epinephrine, norepinephrine and serotonin all inhibit reserpine binding at concentrations similar to those required for inhibition of dopamine uptake. Treatment with saponin to release vesicle contents results in complete loss of accumulated dopamine but retention of bound reserpine. these results indicate that reserpine binds to the catecholamine transport system of synaptic vesicles with high affinity and specificity.
Inhibition of glutamate dehydrogenase activity in rabbit renal mitochondria by phosphoenolpyruvate
Authors: Bryła Jadwiga , Marzena Matyaszczyk
Published: July 2002

The effect of phosphoenolpyruvate on glutamate dehydrogenase activity was studied in both intact and Triton X‐100‐treated rabbit renal mitochondria. The intramitochondrial phosphoenolpyruvate content was modulated by application of both 3‐MPA, an inhibitor of phosphoenolpyruvate carboxykinase, and BTCA, which inhibits the tricarboxylate‐transporting system. The data indicate that: (i) phosphoenolpyruvate is a potent inhibitor of glutamate dehydrogenase activity; and (ii) its inhibitory effect on the enzyme may be abolished by leucine and ADP, activators of glutamate dehydrogenase.
Characterisation of an intermediate in neurophysin biosynthesis in the guinea pig
Authors: Gordon-Weeks Ruth , Jones Peter M. , Robinson Iain C.A.F.
Published: July 2002

In addition to oxytocin (OT), vasopressin (AVP) and their respective neurophysins (NPs), another [35S]cysteine incorporating component is present in the guinea pig neurohypophysis. Gel filtration and Con A affinity chromatography revealed that this component was larger than NP and was glycosylated. NP‐immunoreactivity was assessed using antisera which distinguish the OT‐ and AVP‐related NPs. Whilst the anti‐NP antiserum detected only one component (guinea pig NP), the anti‐NP antiserum detected both NP and the glycosylated 35S‐labelled component. These results suggest that a significant amount of NP in guinea pig neural lobes bears a glycopeptide extension and represents a partially processed form of the AVP precursor in this species.
Recognition of signal sequences
Authors: Finkelstein Alexei V. , Bendzko Peter , Rapoport Tom A.
Published: July 2002

The hypothesis assumes that every continuous, entirely hydrophobic sequence of sufficient length, which is not involved in strong intramolecular contacts with other parts of the nascent protein chain, will function as a signal for translocation across the endoplasmic reticulum membrane or across the inner bacterial membrane. The signal peptide is proposed to be deeply immersed into a hydrophobic cleft of the receptor. Accordingly, only the entirely nonpolar peptides can be absorbed and, despite different primary structures, all of them would assume the same conformation dictated by the structure of the receptor pocket.
Subchloroplastic localization of NAD kinase activity: evidence for a Ca2+, calmodulin‐dependent activity at the envelope and for a Ca2+, calmodulin‐independent activity in the stroma of pea chloroplasts
Authors: Simon Patrice , Bonzon Marc , Greppin Hubert , Marmé Dieter
Published: July 2002

Chloroplasts were prepared from pea seedlings and tested for NAD kinase activity. More than half of a Ca2+, calmodulin‐dependent activity and most of a Ca2+, calmodulin‐independent activity of the homogenate were associated with chloroplasts. The Ca2+, calmodulin‐dependent activity could be detected by adding Ca2+ and calmodulin to the incubation medium containing intact chloroplasts. This activity could not be separated from the chloroplasts by successive washes or by phase partition in aqueous two‐polymer phase systems. After chloroplasts fractionation, the Ca2+, calmodulin‐dependent NAD kinase activity was localized at the envelope, and the Ca2+, calmodulin‐independent activity was recovered from the stroma. In view of these results and of a previous report [Simon (1982) Plant Cell Rep. 1, 119–122] the occurrence and presumed role of calmodulin in the chloroplast are discussed.
Increase in cell‐surface N‐acetylglucosaminide β(1→4)galactosyltransferase activity with retinoic acid‐induced differentiation of F9 embryonal carcinoma cells
Authors: Nakhasi Hira L. , Nagarajan Lalitha , Anderson Wayne B.
Published: July 2002

Exposure of F9 cells to all‐trans‐retinoic acid over a period of 6 days resulted in 4‐fold induction of cell surface N‐acetylglucosaminide β(1→4)galactosyltransferase (GT) activity. The retinoic acid‐induced GT activity was further enhanced by treatment of the cells with 8‐bromo cyclic AMP. The ability of retinoic acid alone, or retinoic acid in combination with 8‐bromo cyclic AMP, to induce GT activity was inhibited by both actinomycin D and cycloheximide. These findings indicate that the induction of galactosyltransferase activity noted with differentiation of F9 cells involves de novo synthesis of new enzyme protein.
Interaction of a tryptophan‐containing peptide with chromatin core particles
Authors: Colot Vincent , Toulme Jean-Jacques , Helene Claude
Published: July 2002

The binding of a tetrapeptide lysyltryptophylglycyllysine to nucleosome core particles has been investigated using UV absorption and fluorescence spectroscopy. Modifications of the absorption spectra and fluorescence quenching of the tryptophyl residue are consistent with stacking between the indole ring and nucleic acid bases. Therefore DNA interactions with histones do not prevent stacking of the tryptophyl residue with nucleic acid bases in the peptide—core particle complexes. The number of peptide binding sites is reduced to half that of naked DNA.
Heat shock causes diverse changes in the phosphorylation of the ribosomal proteins of mammalian cells
Authors: Kennedy Iain M. , Burdon Roy H. , Leader David P.
Published: July 2002

When HeLa cells or BHK cells were subjected to heat shock at 42°C (for 2 h) or 45°C (for 10 min) there was extensive dephosphorylation of ribosomal protein S6. Concomitantly ribosomal protein L14, which is not significantly phosphorylated in normal cells, became phosphorylated, as did a non‐structural protein of M
r = 27 000, associated with the ribosomes. The latter effects were not prevented by cycloheximide or actinomycin D. When cells shocked at 45°C for 10 min were returned to 37°C for 2 h there was rephosphorylation of ribosomal protein S6 and dephosphorylation of the 27 kDa protein, but not of ribosomal protein L14.
Galactolipase activity and free fatty acid levels in chloroplasts
Authors: Kaniuga Zbigniew , Gemel Joanna
Published: July 2002

Both galactolipase activity and free fatty acid content are much higher in chloroplasts of chilling‐sensitive plants (bean, broad bean, tomato and maize) than in those of chilling‐resistant ones (spinach, pea and lettuce). However, monogalactosyldiacylglycerol and digalactosyldiacylglycerol content in chloroplasts of these two groups of plants relates neither to free fatty acid levels nor galactolipase activity. It seems that chloroplast galactolipase activity and free fatty acid level may be useful biochemical parameters discriminating chilling‐sensitive from chilling‐resistant plants.
Ca2+ binding and charge movements in membranes of platelets and sarcoplasmic reticulum
Authors: Levitsky D.O. , Loginov V.A. , Lebedev A.V. , Levchenko T.S. , Leytin V.L.
Published: July 2002

The properties of the Ca2+‐pump system of platelet microsomes isolated without Ca2+‐precipitating anions are studied. Passive Ca2+ binding to the microsomes takes place in a noncooperative manner with K
d = 0.7 μM. Half‐maximal stimulation of ATP‐dependent transport occurs at 0.4 μM Ca2+. The velocity of Ca2+ uptake, Ca2+ capacity and the level of phosphoprotein in platelet microsomes are significantly lower than in cardiac microsomes. Energization of platelet and muscle microsomes and activation of intact platelets result in opposite charge redistribution in hydrophobic regions of the membranes. It is concluded that these charge movements are caused by Ca2+ binding to and dissociation from nonpolar binding sites in the membranes.
Do monoclonal antibodies Tü15 and Tü67 detect heterogeneity of human transferrin receptor molecules?
Authors: Uchańska-Ziegler Barbara , Wernet Peter , Liangru Shi , Ziegler Andreas
Published: July 2002

The possible molecular heterogeneity of human transferrin receptors was analyzed using two murine monoclonal antibodies. Tü 15 and Tü67. Both reagents precipitated from lysates of 125I‐labeled HL‐60 cells a major component of 88 kDa which could be identified as the transferrin receptor by comparison with the proteins detected by monoclonal antibody OKT9. Although sequential immunoprecipitations appeared to demonstrate molecular heterogeneity of transferrin receptors, since the Tü15‐reactive species were fully included in the Tü67‐positive population, but not vice versa, the possible association of Tü15‐reactive molecules with transferrin receptor is also discussed.
Protein kinase activity of the insulin receptor from muscle
Authors: Häring H.U. , Machicao F. , Kirsch D. , Rinninger F. , Hölzl J. , Eckel J. , Bachmann W.
Published: July 2002

The insulin receptor is associated with a protein kinase activity. This has been shown for the receptor of liver, fat, and some other tissues which are not primary targets of insulin action. Here kinase activity is demonstrated for the insulin receptor of rat skeletal and cardiac muscle with similar characteristics. Insulin (10−7
) stimulates phosphorylation of the 95‐kDa receptor subunit 3‐ to 18‐fold. The effect is detectable at 10−10
insulin; the ED
50 is approx. 3 × 10−9
. The kinase phosphorylates exogenous substrate as well, and it is recovered after immunoprecipitation of the receptor with antireceptor antibody suggesting that kinase activity is intrinsic to the muscle receptor.
Lipoxygenase‐mediated production of superoxide anion in senescing plant tissue
Authors: Lynch Daniel V. , Thompson John E.
Published: July 2002

Lipoxygenase activity and superoxide (O.−
2) production by microsomal membranes and cytosol from bean cotyledons increased in parallel as senescence progressed. Superoxide production was heat denaturable and dependent on the availability of linoleate, the substrate for lipoxygenase. The specific inhibitor of lipoxygenase, U28938, caused a parallel reduction in enzyme activity and the formation of O−
2. These observations demonstrate that lipoxygenase activity mediates the formation of superoxide anion, and support the contention that membrane senescence is attributable to a sequence of reactions in which lipasederived fatty acids are utilized by lipoxygenase to generate O−
2 and hydroperoxides.
Reoxidation and renaturation studies on the main toxin of Naja naja samarensis
Authors: Gacond J.J. , Bargetzi J.P. , Juillerat M.A.
Published: July 2002

The main toxin of Naja naja samarensis is a very small and rigid protein (M, 6850, 8 cysteines). When fully reduced, it regains its native conformation by a mechanism involving a rapid cysteine oxidation and a slower, less temperature‐dependent disulfide exchange. In a native‐like form of the protein we observed a population whose cysteines were incompletely reoxidized. When intermediates with blocked cysteines were incubated with oxidized and reduced glutathione, the percentage of native‐like molecules increased. These findings suggest a multiple folding pathway.
Multiple relationships between rate of oxidative phosphorylation and Δµ̃H in rat liver mitochondria
Authors: Zoratti Mario , Petronilli Valeria
Published: July 2002

The relationship between rate of ATP synthesis and transmembrane electrochemical proton gradient has been determined in rat liver mitochondria oxidizing succinate, using the respiratory inhibitor malonate or the uncoupler FCCP to decrease Δµ̃H progressively. As previously reported [(1982) Eur. J. Biochem. 126, 443‐451] two different relationships are obtained depending on the method used. Evidence is presented that this result is not due to underestimation of the Δµ̃H maintained by fast‐respiring mitochondria, as recently suggested [(1985) FEBS Lett. 181, 323‐327].
Histone H1° is distributed unlike H1 in chromatin aggregation
Authors: Jin Yong-jiu , Cole R.David
Published: July 2002

Non‐uniform distribution of H1 histone in bovine thymus chromatin was demonstrated previously. Two classes of chromatin differ in aggregation properties and histone content. The class aggregatable by physiological saline is enriched in H1, especially H1ab, the variant known to be most powerful in condensing DNA. Now, the distribution of H1 subtypes is reported for brain chromatin, where H1ab and H1c were distributed as in thymus. In contrast, H1° preferred neither the aggregatable chromatin nor the aggregation‐resistant class. It is suggested that H1° is uniformly distributed with regard to euchromatin and heterochromatin, whereas H1 is cencentrated in heterochromatin.
Monoclonal antibodies that distinguish between human aorta smooth muscle and endothelial cells
Authors: Glukhova Marina A. , Kabakov Alexander Y. , Ornatsky Olga I. , Frid Maria G. , Smirnov Vladimir N.
Published: July 2002

A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS‐polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor immunopredpitation reveal it on the endothelial cell surface or in the Triton X‐100 extract.
On the phosphorylation of low molecular mass HMG (high mobility group) proteins in Ehrlich ascites cells
Authors: Lund T. , Holtlund J. , Laland S.G.
Published: July 2002

This paper shows that the low molecular mass HMG proteins 14 and 17 do not seem to be phosphorylated in Ehrlich ascites cells whereas two other small HMG proteins designated HMG I and Y are. Amino acid analysis and peptide mapping of all four proteins demonstrated that HMG I and Y were not phosphorylated modifications of HMG 14 or 17.
Modulation of glycerophosphorylcholine and glycerophosphorylethanolamine in rice shoots by the environment oxygen level
Authors: Menegus Faustino , Fronza Giovanni
Published: July 2002

Glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine, recently hypothesized to play a central role in phospholipid metabolism, are shown for the first time to be present in higher plants, together with two other unknown phosphodiesters. The GPC concentration is fairly high in young rice (Oryza saliva L.) shoots (0.21 mmol·(kg fresh wt tissue)−). It is progressively reduced by growth in air but considerably increased under anoxia, (0.72 mmol·(kg fresh wt tissue)−), concomitantly with the anoxia‐stimulated elongation of the rice coleoptile. The involvment of GPC concentration in membrane functionality is discussed in the light of a recent hypothesis.
Calcium transport in the cellular slime mould Dictyostelium discoideum
Authors: Europe-Finner G.N. , Newell P.C.
Published: July 2002

Transport of Ca2+ into amoebae of Dictyostelium discoideum was studied using 45Ca and a lanthanum stopping technique. Ca2+ uptake was found to be rapid and showed saturation kinetics. No difference was found in Ca2+ uptake between vegetative and aggregation competent cells, the V
max for unstimulated amoebae being approx. 10 nmol/107 cells per min. Ca2+ uptake had the characteristics of passive facilitated diffusion using a saturatable carrier and NaN3 and ouabain were not inhibitory. The chemoattractants cAMP and folate, previously reported to stimulate the uptake of Ca2+ into amoebae, did not stimulate the rate of Ca2+ uptake by this carrier but increased the extent of Ca2+ taken up over the period 10–30 s after chemotactic stimulation. The significance of these findings for the function of Ca2+in chemotactic signalling is discussed.
Insulin receptor kinase in human skeletal muscle
Authors: Obermaier B. , Ermel B. , Biemer E. , Kirsch D. , Machicao F. , Häring H.U.
Published: July 2002

Receptor‐associated protein kinase activity has been shown in all primary target tissues of insulin action in the rat and a function of insulin receptor phosphorylation in signal transmission was proposed. Insulin receptor phosphorylation so far has not been demonstrated in human target tissues of insulin. We describe here insulin receptor kinase activity in human skeletal muscle. Insulin (10−8
) stimulates the phosphorylation of a 95‐kDa protein from skeletal muscle 2‐fold. The phosphoprotein is quantitatively immunoprecipitated with insulin receptor antibody identifying it as the β‐subunit of the insulin receptor. The insulin stimulation of phosphorylation is detectable also at physiological insulin concentrations (10−9
) showing that receptor phosphorylation could be involved in insulin action in human skeletal muscle as well.
The rate‐limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase
Authors: Brzezinski Peter , Thörnström Per-Eric , G. Malmström Bo
Published: July 2002

The level of reduction of cytochrome a and cua during the oxidation of ferrocytochrome c has been determined in stopped‐flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, cua is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and cua to cytochrome a
3‐Cub is a two‐electron process and, in addition, that the binding of oxidized cytochrome c to the electron‐transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate‐limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.
The prohormone processing activity is enriched in a low‐density subpopulation of chromaffin granules
Authors: Maret Gérard E. , Fauchère Jean-Luc
Published: July 2002

Bovine adrenomedullary chromaffin granules can be separated into two subpopulations by differential centrifugation. The subpopulation which sediments at the interface of two sucrose layers, 1.6 and 1.8 M respectively, is found to be enriched about 10‐times in prohormone processing activity, as measured by in vitro degradation of synthetic peptide substrates. The enhanced proteolytic activity is not due to lysosomal contaminations which are very low and only slightly increased in the more active fraction. The low density of the enriched subpopulation suggests that we are dealing with immature granules. The physiological implications of this finding are discussed. Furthermore, the enriched fraction can be used as the starting material for the isolation ofproenkephalin processing enzymes.
Reversible thermal transition of brain myelin proteolipid
Authors: Mateo P.L. , Lopez-Lacomba J.L. , Moreno M.C. , de Cozar M. , Cortijo M. , Monreal J.
Published: July 2002

Brain myelin proteolipid has been investigated using high‐sensitivity differential scanning calorimetry (DSC) under various conditions. Crude proteolipid with a 40% () content of protein gave rise to a reversible transition, centered at about 60°C. The specific enthalpy of the transition was 50 + 5 J·g−1 with a calorimetric to van't Hoff enthalpy ratio of 5.7 + 0.5. To our knowledge this is the first intrinsic membrane protein in which a reversible thermal transition has been detected and investigated by DSC. Similar experiments were carried out using the recombinants of delipidated proteolipid and the pool of natural membrane lipids; in this case the transition was less enthalpic and showed lower cooperativity. The recombinants with lecithins, however, did not show any transition at 60°C.
Phylogenetic comparison of lens crystallins from the vertebrate and invertebrate ‐ convergent or divergent evolution?
Authors: Chiou Shyh-Horng
Published: July 2002

A systematic biochemical comparison has been made of the crystallins isolated from the lenses of five different species belonging to the five major classes of vertebrates. Gel‐permeation chromatography of the lens homogenates on Fractogel TSK HW‐55(S) revealed well‐defined elution patterns with a characteristic distribution of different classes of crystallins from each species. SDS gel electrophoresis and statistical comparison of the amino acid contents indicated that all crystallin groups from different classes share some common subunits and similarity in their amino acid compositions. The results coupled with the relatedness shown in the amino acid compositions of fish γ‐crystallin with those of mammalian γ‐crystallin and the squid crystallin from the invertebrate pointed to the possibility of the existence of a common ancestral protein for all crystallins. This is in favor of the divergent rather than convergent evolution of lens crystallins as commonly assumed in the literature.
A single RNA species injected in Xenopus oocyte directs the synthesis of active tyrosine hydroxylase
Authors: Horellou Philippe , Guibert Bernard , Leviel Vincent , Mallet Jacques
Published: July 2002

Tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of catecholamine, is a tetramer composed of four subunits of the same molecular mass. A full length cDNA clone encoding tyrosine hydroxylase has been inserted into the SP6 expression system. Translation of the corresponding RNA in Xenopus oocyte results in enzymatic activity, demonstrating that a single gene contains all the necessary genetic information to code for a functional enzyme. The potential of this system in the analysis of posttranslational tyrosine hydroxylase modifications is discussed.
Desensitization by covalent modification of the chemoreceptor of Escherichia coli
Authors: Yonekawa Hiroyuki , Hayashi Hiroshi
Published: July 2002

Chemoreceptors in Escherichia coli were studied in situ in chemotactic mutants, deficient in the ability to modify the receptors, by using membrane vesicles prepared from the mutants. The affinity of the receptors for the ligands is related to the level of modification of the receptors. Unmodified serine receptor had a dissociation constant of 0.8 μM, while modified receptor had a dissociation constant that was at least 100‐times higher. The results are discussed in relation to the two‐state model of the chemoreceptor.
Inactivation of the catecholamine transporter during the preparation of chromaffin granule membrane ‘ghosts’
Authors: Gasnier Bruno , Scherman Daniel , Henry Jean-Pierre
Published: July 2002

The activity of the catecholamine transporter of chromaffin granules and the binding to these vesicles of reserpine, a transporter inhibitor, decrease during ghost preparation. In contrast, the number of binding sites of dihydrotetrabenazine, another transporter ligand, is constant. Dihydrotetrabenazine thus binds to an inactive transporter whereas reserpine binds only to active vesicles. Inactivation occurs during lysis of the granules, possibly because of an incomplete resealing. The turnover number of the transporter, determined by dividing the uptake activity by the density of dihydrotetrabenazine binding sites, has a maximal value (140 molecules/min) in intact granules. The reserpine to dihydrotetrabenazine binding ratio (10–25%) is an estimate of the proportion of correctly resealed vesicles.
Identification of three additional members of rat protein kinase C family: gd‐, ϵ‐ and ξ‐subspecies
Authors: Ono Yoshitaka , Fuji Tomoko , Ogita Kouji , Kikkawa Ushio , Igarashi Koichi , Nishizuka Yasutomi
Published: July 2002

Three types of cDNA clone of the protein kinase C family termed δ, ϵ and ξ were newly identified by molecular cloning and sequence analysis. These members have a common structure that is closely related to, but clearly different from the other four known members of the family which have α‐, βI‐, βII‐ and γ‐sequences, although the ξ‐cDNA available at present does not contain a complete reading frame for a protein kinase C molecule. The diverse heterogeneity of the enzyme seems to be an important factor in determining the mode of response of many tissues and cell types to a variety of external stimuli.
The mechanism of H+ transfer by bacteriorhodopsin The properties and the function of intermediate P
Authors: Drachev L.A. , Kaulen A.D. , Skulachev V.P. , Zorina V.V.
Published: July 2002

Regeneration of bacteriorhodopsin (bR) from the M intermediate via a new intermediate P has been studied. In the purple sheets treated with 0.015% Triton X‐100 (the P → bR transition is suppressed), the α‐maximum of absorption of P is located at 560 ± 5 nm, the extinction coefficient being equal to 0.7 ± 0.1 of that of bR. Besides, there is a small but measurable absorbance increase at 330–350 nm, which seems to be due to a β‐maximum of 13‐cis‐retinal residue in P. The similarity of the α‐maximum of P and bR suggests the Schiff base nitrogen to be protonated at the M → P transition. The kinetics of P → bR transition measured at the 335 nm absorbance decrease coincides with that of proton uptake accompanying bR regeneration after flash. A scheme is proposed assuming that H+ absorption from the water phase is coupled to the 13‐cis → all‐trans isomerization of retinal residue in which the Schiff base is already protonated by a proton‐donor group of the protein.
Regulation of calcium uptake in synaptosomes from rat brain by DL‐2‐amino‐5‐phosphonovaleric acid
Authors: Pastuszko Anna , Yee Daniel K. , Wilson David F.
Published: July 2002

DL‐2‐Amino‐5‐phosphonovaleric acid stimulated calcium influx into synaptosomes isolated from rat brain. The increase in Ca2+ permeability of the synaptosomal membranes induced by this amino acid is not markedly dependent on the membrane potential or Na+ concentration. It is postulated that 2‐amino‐5‐phosphonovaleric acid is an agonist for a receptor(s) which regulates intraneuronal free calcium concentrations by modulating a selective calcium channel. The observed stimulation of calcium uptake may provide an assay system for purification of the endogenous ligand for this receptor and for characterization of its physiological role in neuronal function.
Binding of laminin to brain gangliosides and inhibition of laminin‐neuron interaction by the gangliosides
Authors: Laitinen Jari , Löppönen Riitta , Merenmies Jussi , Rauvala Heikki
Published: July 2002

Binding of laminin to glycolipids of neuronal membranes was studied with a thin‐layer chromatography overlay assay. The major brain ganglioside GD1A was the main binding component, when chromatograms containing the same molar amount of the different brain gangliosides and the brain sulfatide were incubated with laminin at physiological ionic strength. The possible role of laminin binding to brain gangliosides in laminin‐neuron interactions was studied with adhesion assays. It was found that binding of rat brain neurons to laminin is blocked by 10–40,μM brain gangliosides but not by sulfatide. The inhibition by the gangliosides is suggested to be due to competition with the cell surface interaction sites of laminin and not to binding of the gangliosides to the cells. Our findings support the idea that the adhesive and neurite‐promoting effect of laminin is dependent on its interaction with gangliosides at the neuronal cell surfaces.
Domain II of calmodulin is involved in activation of calcineurin
Authors: Hurwitz Mary Y. , Putkey John A. , Klee Claude B. , Means Anthony R.
Published: July 2002

A family of mutant proteins related to calmodulin (CaM) has been produced using cDNA constructs in bacterial expression vectors. The new proteins contain amino acid substitutions in Ca2+‐binding domains I, II, both I and II, or both II and IV. The calmodulin‐like proteins have been characterized with respect to mobility on SDS‐polyacrylamide gels, Ca2+‐dependent enhancement of tyrosine fluorescence, and abilities to activate the CaM‐dependent phosphatase calcineurin. These studies suggest that an intact Ca2+‐binding domain II is minimally required for full activation of calcineurin.
Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol‐rich liposomes
Authors: Moghimi S.Moein , Patel Harish M.
Published: July 2002

In accordance with the finding of our in vivo experiments reported earlier [(1983) Biochim. Biophys. Acta 761, 142–157 (1986) Biochim. Biophys. Acta 888, 184–190], the results of in vitro experiments show that Kupffer cells avidly take up cholesterol‐poor but not cholesterol‐rich liposomes, whereas splenic phagocytic cells take up preferentially cholesterol‐rich rather than cholesterol‐poor liposomes in the presence of serum. Evidence presented here suggests that serum contains opsonins specific for hepatic and splenic phagocytic cells and these opsonins have different affinities for cholesterol‐rich and cholesterol‐poor liposomes.
The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei
Authors: Cronín Ciaran N. , Nolan Derek P. , Paul Voorheis H.
Published: July 2002

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49), 6‐phosphogluconolactonase (EC 3.1.1.31), 6‐phosphogluconate dehydrogenase (EC 1.1.1.44), ribose‐5‐phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose‐5‐phosphate 3′‐epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce d‐ribose‐5‐phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non‐oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce d‐glyceraldehyde 3‐phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose‐bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3‐phosphate produced in the pentose phosphate pathway to glucose 6‐phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde‐3‐phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner‐Doudoroff pathway, 6‐phosphogluconate dehydratase (EC 4.2.1.12) and 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.
Comparison of inhibitor binding in HIV‐1 protease and in non‐viral aspartic proteases: the role of the flap
Authors: Gustchina Alla , Weber Irene T.
Published: July 2002

The crystal structure of HIV‐1 protease with an inhibitor has been compared with the structures of non‐viral aspartic proteases complexed with inhibitors. In the dimeric HIV‐1 protease, two 4‐stranded β‐sheets are formed by half of the inhibitor, residues 27–29, and the flap from each monomer. In the monomeric non‐viral enzyme the single flap does not form a β‐sheet with an inhibitor. The HIV‐1 protease shows more interactions with a longer peptide inhibitor than are observed in non‐viral aspartic protease‐inhibitor complexes. This, and the large movement of the flaps, restricts the conformation of the protease cleavage sites in the retroviral polyprotein precursor.
Interleukin‐4 stimulates collagen gene expression in human fibroblast monolayer cultures Potential role in fibrosis
Authors: Gillery P. , Fertin C. , Nicolas J.F. , Chastang F. , Kalis B. , Banchereau J. , Maquart F.X.
Published: July 2002

A role for the cytokines produced by tissue‐infiltrated inflammatory cells (mainly T‐lymphocytes and must cells) in the pathophysiology of fibrosis has been suggested by several groups. Among the products of these cells, interleukin‐4 (IL‐4) might be one of the factors involved in the initiation of the fibrotic process. We studied the effects of recombinant human IL‐4 on human fibroblast monolayer cultures. IL‐4 (10 and 100 U/ml) induced a dose‐dependent increase of collagen production. Non‐collagen protein synthesis was not significantly altered. A concomitant increase of pro‐α1(I) collagen mRNAs was observed, showing that IL‐4 acts at a pre‐translational level.
IL‐4 and IL‐13 exhibit comparable abilities to reduce pyrogen‐induced expression of procoagulant activity in endothelial cells and monocytes
Authors: Herbert J.M. , Savi P. , Laplace M.-Cl. , Lalé A. , Dol F. , Dumas A. , Labit C. , Minty A.
Published: July 2002

Endotoxin (LPS), interleukin‐1β (IL‐1) and tumor necrosis factor‐α (TNF) increased the expression of tissue factor, a membrane‐anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface, further contributing to the procoagulant activity of the endothelium or monocytes. On endothelial cells and monocytes, interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13), a newly described lymphokine, both strongly inhibited LPS‐induced tissue factor expression, a similar activity also being obtained with regard to the pyrogenic effects of IL‐1 or TNF. When measured in parallel, IL‐4 and IL‐13 counteracted thrombomodulin down‐regulation induced by LPS, IL‐1 or TNF in endothelial cells. These results therefore show that both IL‐4 and IL‐13 protect the endothelial and the monocyte surface against inflammatory mediator‐induced procoagulant changes.
Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells
Authors: Urbé S. , Huber L.A. , Zerial M. , Tooze S.A. , Parton R.G.
Published: July 2002

A specific polyclonal antibody was used to investigate the subcellular distribution of the small GTPase, rab11p, in the neuroendocrine cell line, PC12. We took advantage of a previously described pulse‐chase protocol based on sulfation [1] to examine the distribution of rab11 along the secretory pathway. Using the rab11 antiserum, but not serum depleted of rab11 antibodies, we were able to specifically immunoisolate markers of the constitutive and the regulated secretory pathway in the trans‐Golgi network (TGN) as well as after their exit from this compartment (constitutive secretory vesicles, immature, and mature secretory granules). We therefore conclude that rab11p is associated with the TGN and with TGN‐derived vesicles of both the constitutive and the regulated secretory pathway in PC12 cells.
Calcium, calmodulin and cell cycle regulation
Authors: Means Anthony R.
Published: July 2002

Calcium and its ubiquitous intracellular receptor calmodulin are required for cell proliferation. Studies in a variety of model systems are beginning to identify components of the calcium/calmodulin cascade required for movement of quiescent cells into the cell cycle as well as for proliferating cells to move from G1 to S, G2 to M and through mitosis. Two calcium/calmodulin‐dependent enzymes, the multifunctional calcium/calmodulin‐dependent protein kinase and the protein phosphatase 2B (calcineurin) as well as a spindle pole body protein that binds calmodulin in the absence of calcium have been shown to be essential at specific phases of the cell cycle. In addition, the status of the intracellular calcium pools is critical for normal traverse of the cell cycle.
Concanamycin A blocks influenza virus entry into cells under acidic conditions
Authors: Guinea Rosario , Carrasco Luis
Published: July 2002

The selective inhibitor of the vacuolar proton‐ATPase, concanamycin A, powerfully blocks influenza virus entry into cells, if present during the initial times of virus infection. Attachment of virus particles to cells is not prevented by concanamycin A, rather the exit of influenza virus from endosomes is the step blocked by this macrolide antibiotic. Inhibition of influenza virus entry into cells by concanamycin A or by nigericin takes place under acidic conditions. Moreover, if the pH gradient is abolished by pre‐incubation of cells in acidic pH, influenza virus entry does not occur even in the absence of any inhibitors. These results indicate that acidic conditions per se are not sufficient to promote virus entry into cells; rather this step of virus infection requires a pH gradient.
The role of the CCA sequence of tRNA in the peptidyl transfer reaction
Authors: Tamura Koji
Published: July 2002

Peptidyl transfer is a key step in the process of protein biosynthesis. To examine the role of the universal CCA terminal sequence of tRNA in the process of peptidyl transfer, various mutant transcripts of Escherichia coli valine tRNA were constructed. Peptidyl transferase activity, monitored by the ‘fragment reaction’ with a slight modification, was decreased by mutation at any one base of CCA. The effect of mutation was moderate in the UCA, CUA and CCG mutants. Replacement of A76 by a pyrimidine nucleotide, or replacement of either C74 or C75 by a purine nucleotide caused a marked decrease in the activity. These findings suggested that the universal CCA terminus of tRNA makes a functional interaction with ribosomal RNA by base‐pairing.
Expression of cytochrome P‐450 3A in HT29‐MTX cells and Caco‐2 clone TC7
Authors: Carrière Véronique , Lesuffleur Thécla , Barbat Alain , Rousset Monique , Dussaulx Elisabeth , Costet Philippe , de Waziers Isabelle , Beaune Philippe , Zweibaum Alain
Published: July 2002

HT‐29 sublines and Caco‐2 clones were analyzed for the expression of cytochrome P‐450 3A. The enzyme was found to be expressed in differentiated HT‐29 cells selected by resistance to methotrexate and in one of seven Caco‐2 clones, TC7. Its expression parallels the differentiation process, with highest levels being observed at late confluency. P‐450 3A mRNA and protein patterns, as well as subcellular distribution, are intermediate between those observed in human adult intestine and fetal liver.