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Biomedical subjects

A Ludwig

Publications and source records attributed to A Ludwig.

At least 91 records · Page 5Linked to original sources
PubMed1998

Submicron oil-in-water emulsion formulations for mefloquine and halofantrine: effect of electric-charge inducers on antimalarial activity in mice.

Stearylamine, oleic acid, phosphatidylserine and dicetylphosphate have been studied to determine their capacity to induce electric charge on non-ionic submicron emulsions containing halofantrine and mefloquine. The in-vivo antimalarial activity of drug-loaded emulsions, evaluated in mice, was affected by the nature of the additives used. In particular, the electric-charge inducers markedly affected the pharmacological activity of mefloquine, but not of halofantrine. After subcutaneous administration ED50 values (the doses affording 50% protection) were 3 and 15 mg kg(-1), respectively, for halofantrine and mefloquine emulsions without charge inducers. The mefloquine-loaded emulsions with charge inducers were active at 10 mg kg(-1) for dicetylphosphate, 17 mg kg(-1) for phosphatidylserine, 23 mg kg(-1) for oleic acid and 27 mg kg(-1) for stearylamine, again after subcutaneous administration. This work has enabled the formulation of stable emulsions, incorporating drugs with high antimalarial activity, which are proposed for parenteral delivery of these fairly soluble drugs.

Animals↗
PubMed1998

The structure of the murine calcium channel gamma-subunit gene and protein.

The gamma-subunit of voltage-gated calcium channels is a membrane protein that is associated with the skeletal muscle type of voltage gated calcium channels. Using a subunit-specific polyclonal antibody, the gamma-protein was detected in mouse skeletal muscle but not in brain, where at least five additional types of voltage-gated calcium channels are expressed. Murine genomic clones containing the full coding sequence of the gamma-subunit were isolated, the exons were mapped and sequenced. The murine gamma-subunit is encoded by a single copy gene containing 4 translated exons which are distributed over approximately 14 kilobases of DNA. The intron placement within the mouse gene correlates with the previously revealed organization of the human gamma-subunit gene, although the primary structures of the gamma-subunits are only moderately conserved between the murine, human, rat and rabbit proteins (75% identity).

Alternative Splicing↗
PubMed1998

[WinDiet. Support decision system for the assessment of nutritional status and diet prescription].

A "Sistema de Apoio à Decisão para Avaliação do Estado Nutricional e Prescrição de Dietas" called "WinDiet" was developed. The system was developed on a computer compatible to IBM-PC standard. For system processing procedures through the computer, that is, interpretation, compilation, table generation en others, it was selected the Delphi version 1.0 software, from Borland International, for its using facilities, data basis accomplishment, communication environment and mainly an ample availability of mathematic, impression and programming resources besides allowing for all the programming operations to be accomplished on Windows environment. Knowing the procedures for nutritional evaluation and diet prescription was possible by consulting to text books, periodicals and the area experts. This system allows for individual nutritional assessment applying the anthropometric methods such as weight, height, skinfold thickness measurings, the lab methods such as albumin, transferrin, height/creatinin index and others, the dietary methods by food consumption recall, and the clinical methods that give emphasis to pathological antecedents and indicative sings of nutritional status. The software also turns possible to prescribe and calculate diets from recommendation besides having an actual data basis which may be changed or modified. Yet it maintains the individual description from all evaluation and menus, emitting its complete reports. It was verified that it reduce significantly the working time and increase the precision of estimate and nutritional diagnosis. Thus, the WinDiet may be used in nutritionist's offices, ambulatories, hospitals as well as on nutritional researches and education.

Diet Therapy↗
PubMed1997

The CXC-chemokine neutrophil-activating peptide-2 induces two distinct optima of neutrophil chemotaxis by differential interaction with interleukin-8 receptors CXCR-1 and CXCR-2.

The CXC-chemokines interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2), and melanoma growth-stimulatory activity (MGSA) are chemoattractants with high selectivity for neutrophils. Although IL-8 has been shown to act as an extremely potent mediator, reports on NAP-2 and MGSA are still contradictory. Here we show for the first time that NAP-2 and MGSA induce two distinct optima of neutrophil chemotaxis. A first optimum is elicited within a concentration range as low as it is characteristic for IL-8. However, a second optimum appears at more than 200-fold higher stimulus concentrations, at which IL-8 is inactive. Investigating the involvement of the two chemokine receptors CXCR-1 and CXCR-2 in NAP-2-mediated chemotaxis, we observe that the cells become desensitized to the first optimum of the chemokine after selective downregulation of CXCR-2, while both optima disappear upon simultaneous downregulation of both receptors. Blocking monoclonal antibodies (MoAbs) specific for CXCR-2 or CXCR-1 either suppress the first optimum of NAP-2-induced chemotaxis or drastically reduce the second one, respectively. These results provide evidence that both receptors are involved in NAP-2-induced neutrophil chemotaxis, with CXCR-2 rendering the cells responsive to low dosages of the chemokine, and with CXCR-1 extending their responsiveness to NAP-2 dosages higher by several orders of magnitude.

Antibodies, Monoclonal↗
PubMed1997

Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain.

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different alpha1 and four different beta subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The beta subunit-specific antibodies identified the presence of beta1a in skeletal muscle; beta2 in heart; and beta2, beta3, and beta4 in brain. The beta3 protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The beta4 protein was also widely expressed, most prominently in the cerebellum. beta2 protein was expressed at only low levels. In situ hybridization with beta subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the alpha1A, alpha1B, alpha1C, and alpha1D subunits showed a broad distribution of alpha1A and alpha1B transcripts, whereas the expression level of alpha1C and alpha1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that beta4 may associate predominantly, but probably not exclusively, with the alpha1A subunit, and beta3 with the alpha1B subunit. In certain brain areas such as the habenula, the beta3 subunit may associate with other alpha1 subunits too. Furthermore, the beta2 subunit may form complexes with different alpha1 subunits in brain and cardiac muscle. These results demonstrate that a given beta subunit may associate with different alpha1 subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Animals↗
PubMed1997

[Lung edema and erosive gastroduodenitis as a sequela of inappropriate use of an adrenaline dose aerosol after wasp sting].

Epinephrine (adrenaline) is an important drug in the treatment of severe anaphylactic reactions. Along with other drugs such as H1-antihistamines and glucocorticosteroids, it is found in every first aid kit for at-risk individuals, such as those who are allergic to insect stings. Subcutaneous or intramuscular injections if carried out by an untrained individual or the patient himself might give rise to potential problems. Therefore, it is common to prescribe epinephrine pressure aerosol as a safer alternative. If epinephrine aerosol is overused, it can cause serious problems. A patient developed by self-medication following a wasp sting lung edema as well as an erosive gastroduodenitis. She consumed two aerosol vials each of which contained about 73 mg of adrenaline. In order to avoid such incidents it is crucial that every doctor provides his or her patient with sufficient oral and written information regarding the correct use fo epinephrine inhalers.

Administration, Inhalation↗
PubMed1997

Alternatively spliced IS6 segments of the alpha 1C gene determine the tissue-specific dihydropyridine sensitivity of cardiac and vascular smooth muscle L-type Ca2+ channels.

Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca2+ channel at lower concentrations than the cardiac Ca2+ channel, although their alpha 1 subunit, which binds the DHPs, is derived from the same gene. This alpha 1C gene gives rise to several splice variants, among which the alpha 1C-b variant is affected by lower concentrations of nisoldipine than the alpha 1C-a variant. Functional expression of chimeras of alpha 1C-a and alpha 1C-b subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the alpha 1C-a subunit were expressed in heart but not in aorta, whereas the IS6 segment of the alpha 1C-b subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 alpha 1C-a and IS6 alpha 1C-b in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca2+ channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the alpha 1C gene.

Alternative Splicing↗
PubMed1996

The effects of calcium and other polyvalent cations on channel formation by Escherichia coli alpha-hemolysin in red blood cells and lipid bilayer membranes.

Channel formation by Escherichia coli alpha-hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations. Hemolysin showed full channel-forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E. coli cells were grown in calcium-depleted media (< 1 microM Ca2+). The addition of divalent cations decreased the single-channel conductance by about 50% with half-saturation constants of 5 mM and less, while the mean lifetime of the HlyA channel was not affected. The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half-saturation constant was 1 microM or below. These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition. When cells were grown in calcium-depleted media, the supernatants showed absolutely no hemolytic activity. Addition of small amounts of Ca2+ to the supernatant led to toxin-mediated hemolysis. Its half-saturation constant was 120 microM. Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a K(m) of 1.5 mM. Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect. These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.

Bacterial Proteins↗
PubMed1996

Molecular cloning and expression of the Modulatory subunit of the cyclic nucleotide-gated cation channel.

The cDNA of three variants of a cyclic nucleotide-gated (CNG) channel modulatory subunit (CNG4c-CNG4e) has been cloned. CNG4c, CNG4d, and CNG4e differ slightly from each other within an amino-terminal sequence that was originally reported as part of the bovine retinal glutamic acid-rich protein (GARP). The core region of CNG4 is homologous to the second subunit of the human rod photoreceptor channel (hRCNC2b), suggesting that both proteins are alternatively spliced products of the bovine and human homologue of the same gene. CNG4 transcripts are present in retina, testis, kidney, heart, and brain. Expression of CNG4 in HEK293 cells did not lead to detectable currents. Coexpression of CNG4 with the principal subunit of the bovine testis CNG channel (CNG3) resulted in currents which differed in several aspects from that induced by CNG3 alone. The heterooligomeric CNG3/CNG4 and the homooligomeric CNG3 channels were modified by Ca2+-calmodulin and some calmodulin antagonists. The results suggest that CNG4 forms functional heterooligomeric channels with CNG3 in vitro and probably also in intact tissues.

Amino Acid Sequence↗
PubMed1996

TNF-alpha renders human neutrophils responsive to platelet factor 4. Comparison of PF-4 and IL-8 reveals different activity profiles of the two chemokines.

Platelet factor 4 (PF-4), like IL-8, is a member of the chemokine superfamily of proinflammatory cytokines. However, although the capacity of IL-8 to stimulate functions in neutrophils is well established, reports on PF-4 are still contradictory. In the present study, we have prepared highly purified PF-4 and examined its ability to induce chemotaxis, degranulation, adhesion to gelatin and plasma proteins, and changes in intracellular calcium levels. Even over a broad range of concentrations, PF-4 alone was unable to induce functional changes in PMN. However, neutrophils pre- or co-incubated with physiologically relevant concentrations of TNF-alpha responded to PF-4 by the selective mobilization of the secondary granule marker lactoferrin but not of the primary granule marker elastase. Contrary to IL-8, PMN did not require pretreatment with cytochalasin B for PF-4-induced exocytosis of lactoferrin. The synergistic effect of PF-4 with TNF-alpha was not a priming phenomenon because the cooperative response remained unchanged even when TNF-alpha was added 5 min after the chemokine. In contrast, TNF-alpha-treated PMN did not respond to PF-4 by chemotaxis or by an increase of intracellular calcium levels, and no competition of PF-4 for IL-8 receptors was observed. Our results suggest a mechanism as well as a biologic role of PF-4 in the regulation of neutrophil function, which is different from that of IL-8 and other alpha-chemokines.

Biomarkers↗
PubMed1996

The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine.

Recent studies have shown that Salmonella typhimurium invades the M cells of Peyer's patches (PP) of the murine ileum. The slyA gene of S. typhimurium has also recently been reported to affect virulence of this pathogen in mice and survival in macrophages. We therefore compared the effect on PP tissue of four strains of S. typhimurium: a wild-type strain, two slyA insertion mutants, and a recombinant S. typhimurium derivative carrying multiple copies of slyA. Invasion assays performed 2 and 7 days after orogastric infection revealed significantly lower numbers of bacteria of the slyA mutants and of the SlyA-overproducing strain in PP than of the wild type. However, similar numbers of bacteria of all strains were still present in the lumen of the small intestine after these times. Invasion assays of PP tissue after 90-min ileal loop infection yielded comparable numbers of bacteria of all strains in PP. Transmission and scanning electron microscopy of PP tissue after ileal loop infection demonstrated that the two slyA mutants and the SlyA-overproducing strain were able to attach to, induce membrane ruffling of, and invade M cells in a way morphologically and quantitatively similar to that of the wild type. In contrast to the wild type, both slyA mutants and, to a lesser extent, the SlyA-overproducing strain were significantly impaired in their ability to destroy M cells and adjacent enterocytes. Taken together, these data suggest that slyA is involved in intracellular survival and M-cell cytotoxicity but not in the invasion process and that the amount of SlyA needs to be precisely balanced for virulence.

Animals↗
PubMed1996

Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli.

Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.

Acyl Carrier Protein↗
PubMed1996

In vitro modulation of induced neutrophil activation by different surfactant preparations.

Endotracheal surfactant administration has gained an important role in the treatment of respiratory failure. Polymorphonuclear neutrophil granulocyte (PMN) activation mediated by chemoattractants, such as interleukin-8 (IL-8), neutrophil-activating peptide-2 (NAP-2) and formylated bacterial oligopeptides, has been found to be involved in the pathophysiology of acute respiratory failure. We investigated potential modulating effects of commercial surfactant preparations (Exosurf, Alveofact, Curosurf and Survanta) on spontaneous and chemoattractant-induced PMN function. Isolated cytochalasin B (CytB)-treated PMNs from healthy adults were incubated with increasing concentrations of surfactant. The response of the cells was measured in terms of elastase release from the lysosomes within 30 min. The PMNs showed no direct activation by any of the surfactants tested. However, when cells were stimulated with suboptimal dosages of chemokines, such as IL-8 (2 nM) or NAP-2 (100 nM), or formyl-methionyl-leucyl-phenylalanine (fMLP) (50 nM), and co-incubated with increasing concentrations of surfactant (0.05-8 mg.mL-1) the release of elastase was markedly modulated depending on the surfactant preparation used. Whilst Exosurf and Alveofact showed only modest effects on the elastase release induced by all three mediators, Curosurf and Survanta markedly inhibited the cellular response in a dose-dependent manner. At concentrations above 1 mg.mL-1, Curosurf and Survanta decreased the IL-8-, NAP-2- and fMLP-induced elastase release by 83, 67 and 90%, and by 82, 75 and 80%, respectively. In conclusion, exogenous surfactant may modulate the inflammatory response of the airways by affecting the chemoattractant-induced polymorphonuclear neutrophil activation. Surfactant preparations with inhibiting properties on neutrophil activation may participate in the prevention of neutrophil-induced lung damage.

Adult↗
PubMed1996

Cytolytic toxins from gram-negative bacteria.

Many Gram-negative bacterial pathogens synthesize cytolytic toxins as virulence factors. Most of these toxins generate pores in eukaryotic cell membranes, but there are apparently several different mechanisms of pore formation. Cytolysins of Gram-negative bacteria are usually synthesized as precursor proteins which are converted to the active toxins by modification or proteolytic processing. Such a requirement for activation is not common for cytolysins produced by Gram-positive bacteria. The extracellular secretion of cytolytic toxins from Gram-negative bacteria depends on specific transport systems.

Amino Acid Sequence↗
PubMed1995

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli.

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

Amino Acid Sequence↗