Isolation of adenosine 5'-diphosphate-L-glycero-D-mannoheptose, the assumed substrate of heptose transferase(s), from Salmonella minnesota R595 and Shigella sonnei Re mutants

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.     

An error estimation of Michaelis-Menten (Monod)-type kinetics

Due to research on biochemistry and genetic engineering, mathematical models of microbial growth have become more complicated but Michaelis-Menten or Monod type expressions have still been used for conversion rates, uptake rates, etc. It is worth examining the error that can be caused by these quasi-steady-state-hypotheses. This paper presents a simple but very effective rationale function that describes the error of the quasi-steady-state hypothesis in enzyme kinetics. A simplified fermentation kinetic model was used for comparison of microbial growth but no analytical error function has been found for batch cultivation. In the case of continuous fermentation the error can be given in an analytical form. Many simulations, based on real SCP experiments, show a significant effect of the quasi-steady-state hypothesis. Since the rate constants of intracellular events are not really known, we have to be very careful when taking into account Michaelis-Menten type expressions in the building of complicated models. Correspondence to: L. Szigeti     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

The light microscopic demonstration of hydroperoxidase-positive Phi bodies and rods in leukocytes in acute myeloid leukemia

Summary Unique fusiform or spindle-shaped particles (Phi bodies) and rods with hydroperoxidase (catalase and/or peroxidase) activity are present in human granulocyte precursors only in acute myelogenous leukemia (AML). These newly recognized particles are much more numerous and prominent than Auer rods. They may be rapidly and readily identified using the microscope in marrow or peripheral blood films when the procedures recommended in this paper for fixation, incubation for hydroperoxidase demonstration in 3,3-diaminobenzidine (DAB)/H₂O₂ medium, copper salt treatment and counterstaining (optional) with the Papanicolaou method are employed. Films prepared in the same manner but treated with benzidine/H₂O₂ medium for myeloperoxidase did not reveal these particles.We believe that Phi bodies are pathognomonic of AML since they are almost invariably present in AML patients with active disease. Their presence serves to distinguish AML from acute lymphocytic leukemia and from chronic granulocytic leukemia in blast crisis. Since the particles disappear in disease remission and reappear upon relapse, the recommended procedure is not only useful in diagnosis but in guiding therapy.When a very rapid diagnosis is needed, it is not necessary to counterstain the preparations, but the nuclei, cytoplasm and plasmalemma can readily be observed in the granulocyte precursors when they are counterstained by the Papanicolaou method. This treatment does not diminish the clarity of the Phi bodies and rods which stain by virtue of their peroxidatic activity.This cytochemical diagnostic procedure should be considered for adoption by hematology laboratories.This investigation was supported by NIH research grants DE02668, CA11265, DE04730, and RR05333     

Immune challenge mediates vocal communication in a passerine bird: an experiment

Secondary sexual characters may have evolved in part to signalresistance to parasites. Avian song has been hypothesized tobe involved in this process, but the role of parasites in modulatingacoustic communication systems in birds remains largely unknown,owing to lack of experiments. We studied the relationship betweenparasitism, testosterone, song performance, and mating successin male collared flycatchers (Ficedula albicollis) by experimentallychallenging their immune system with a novel antigen. We predictedthat a challenge of the immune system would reduce song performance,and that this reduction would be conditional on the size ofa visual sexual signal, the forehead patch that was previouslyfound to reflect resistance. An antagonistic linkage betweentestosterone and immune function would predict that a challengeof the immune system should suppress testosterone level. Animmunological treatment by sheep red blood cells (SRBCs) triggereda decrease in body mass, testosterone level, and song rate,but other song traits were not significantly affected by theantigen challenge. Initial testosterone level was associatedwith forehead patch size and all song traits except song rate.SRBC injection caused stronger reduction in song rate amongmales with smaller forehead patches, and the change in songrate was also predictable by song features such as strophe complexityand length. We show that song rate and other song characteristicsmay be important cues in male-male competition and female choice.These results suggest that parasite-mediated sexual selectionhas contributed in shaping a complex acoustic communicationsystem in the collared flycatcher, and that testosterone mayplay an important role in this process. Parasitism may drivea multiple signaling mechanism involving acoustic and visualtraits with different signal function.     

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and α3β1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with α3β1 and αVβ3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-αV and -β1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against αVβ3 and αVβ5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble α3β1, αVβ3, and αVβ5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that αVβ3 and αVβ5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin αVβ5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas α3β1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and αVβ3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of αVβ5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with α3β1 and KSHV. Preincubation of KSHV with soluble heparin and α3β1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (αVβ3, α3β1, and αVβ5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.     

Transient presence and functional interaction of endogenous GABA and GABAA receptors in developing rat optic nerve.

GABA (gamma-aminobutyric acid) is a major inhibitory synaptic neurotransmitter with widespread distribution in the central nervous system (CNS). GABA can also modulate axonal excitability by activation of GABAA receptors in CNS white matter regions where synapses and neuronal cell bodies are not present. Studies on cultured glia cells have revealed the synthesis of GABA in rat optic nerve O-2A progenitor cells that give rise to oligodendrocytes and type 2 astrocytes in vitro. We report here that: (i) GABA is detected by immuno-electron microscopy in intact rat optic nerve and is localized to glia and pre-myelinated axons during the first few weeks of postnatal development, but is markedly reduced or absent in the adult; and (ii) neonatal optic nerve is depolarized by GABAA receptor agonists or by the inhibition of GABA uptake. These results demonstrate the presence of functional GABAA receptors, and GABA uptake and release mechanisms in developing rat optic nerve, and suggest that excitability of developing axons can be modulated by endogenous neurotransmitter at non-synaptic sites.