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PubMed1967

Studies on rabbit lymphocytes in vitro. VI. The induction of blast transformation with sheep antisera to rabbit IgA and IgM.

Sheep antisera specific for the three major immunoglobulin groups of the rabbit, i.e. IgG (gamma-chain), IgA (alpha-chain), and IgM (micro-chain), are each able to induce blast transformation of the peripheral lymphocytes of the rabbit when added to in vitro cultures. The per cent of lymphocytes transformed with each antiserum indicate that one-fourth of the peripheral lymphocytes carry or have the capacity to synthesize molecules of all three of the major immunoglobulin groups, and that the remaining three-fourths carry or have the capacity to synthesize only two (IgG and IgM). The data do not permit direct conclusions concerning the ability of a single cell to produce molecules belonging to more than one immunoglobulin group at the same time, or the ability of a given cell to make a transition from the synthesis of molecules of one immunoglobulin group to those of another group.

Animals↗
PubMed2000

Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis.

The adenylate cyclase (CyaA) of Bordetella pertussis delivers the N-terminal catalytic domain into the cytosol of a large number of eukaryotic cells, in particular, professional antigen-presenting cells. This allows the delivery of CD8(+) T-cell epitopes to the major histocompatibility complex class I presentation pathway. We have previously shown that immunization of mice with CyaA carrying a single CD8(+) T-cell epitope leads to antiviral protection as well as to protective and therapeutic antitumor immunity associated with the induction of specific cytotoxic T-lymphocyte (CTL) responses. Here, we evaluated the capacity of CyaA carrying one to four copies of the CD8(+) CD4(+) T-cell epitope from the nucleoprotein of the lymphocytic choriomeningitis virus to induce T-cell responses. Both CTL and Th1-like specific responses were detected in mice immunized with recombinant CyaA with or without adjuvant. Although the insertion of the larger peptides resulted in partial loss of the invasive capacity of recombinant CyaA, insertion of several copies of the same epitope led to a strong enhancement of Th1 responses and, to a lesser degree, CTL responses. These results underscore the potency of CyaA for vaccine design with a new impact on diseases in which the Th1 response has been described to have a beneficial effect.

Adenylate Cyclase Toxin↗
PubMed1976

Effect of "fractionated" insulins on total plasma insulin binding capacity and insulin requirements in severe diabetes.

Serial observations of insulin requirement and total plasma insulin binding capacity have been carried out on six xevere diabetics whose treatment was changed from standard soluble and isophane (NPH) insulins to "fractionated" preparations (Nordisk Insulin Ltd). No correlation was found between initial insulin dose and binding capacity, but changes in these two functions during the study period were closely correlated, both for the group as a whole and for individual patients in whom falls in insulin dose occurred. It is concluded that highly purified insulins could be valuable in the treatment of insulin-resistant cases, that their use is frequently associated with a gradual reduction in insulin dose, and that estimation of total plasma insulin binding capacity may indicate which patients are most likely to benefit from fractionated insulin. No sudden change in insulin requirement was seen on changing to purified insulin preparations.

Adult↗
PubMed2003

In vitro inhibitory effect of EGTA on macrophage adhesion: endodontic implications.

Ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) is an specific calcium ion chelator proposed as endodontic irrigant. This study investigates the effect of EGTA on substrate adherence capacity of rat inflammatory macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that EGTA decreased substrate adherence capacity of inflammatory macrophages in a time- and dose-dependent manner. The EGTA concentration that caused half-maximal inhibition (IC50) was 202 +/- 32 mM (p < 0.01). EDTA was more potent than EGTA in inhibiting macrophage adherence (IC50 = 185 +/- 22 mM). Calcium chloride (10 mM) decreased the EGTA inhibitory effect on adherence index by 60.2% (p < 0.01). We conclude that EGTA significantly decreased substrate adherence capacity of macrophages.

Animals↗
PubMed1997

EDTA inhibits in vitro substrate adherence capacity of macrophages: endodontic implications.

The disodium salt of ethylenediamine tetraacetate (EDTA) is a calcium ion chelator used in endodontics to enlarge root canals. This study investigated the effect of EDTA on substrate adherence capacity of rat inflammatory macrophages to determine if EDTA leakage to periapical tissues during root canal therapy can alter macrophage function. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index (AI) was calculated. Results showed that EDTA decreased substrate adherence capacity of inflammatory macrophages in a time and dose-dependent manner. The lowest EDTA concentration that caused a significant inhibition of AI was 50 mM (p < 0.05), and the EDTA concentration that caused half-maximal inhibition (IC50) was 194 +/- 20 mM (p < 0.01). Calcium chloride (10 mM) increased the adherence index of macrophages by 17.1% (p < 0.05) and decreased the EDTA inhibitory effect on AI by 49.5% (p < 0.05). We conclude that an EDTA concentration lower than that used in endodontics decreased the substrate adherence capacity of macrophages significantly. Adhesion is the first step in the phagocytic process and in antigen presentation, but leakage of EDTA to periapical tissues during root canals preparation may inhibit macrophage function and reduce periapical inflammatory reactions.

Animals↗
PubMed1997

Calcium hydroxide inhibits substrate adherence capacity of macrophages.

The purpose of this study was to investigate the effect of calcium hydroxide on substrate adherence capacity of rat inflammatory macrophages to determine if calcium hydroxide can alter macrophage function. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index (AI) was calculated. Results showed that calcium hydroxide decreased substrate adherence capacity of inflammatory macrophages in a time and dose-dependent manner. The lowest calcium hydroxide concentration that caused a significant inhibition of AI was 1 mM (p < 0.05), and the concentration of calcium hydroxide that caused half-maximal inhibition (IC50) was 1.54 mM (p < 0.01). We conclude that calcium hydroxide decreased substrate adherence capacity of macrophages. When adhesion as the first step in the phagocytic process and in antigen presentation is taken into account, calcium hydroxide could inhibit macrophage function and reduce inflammatory reactions in periapical tissues or in dental pulp when it is used in root-canals therapy or in direct pulp capping and pulpotomy, respectively. Moreover, this effect could explain, at least in part, the mineralized tissue-inducing property of calcium hydroxide.

Animals↗
PubMed1979

[Microbial emission, immission and changes in the germ count in the cooling water during operation of wet cooling towers III. communication: laboratory tests for the determination of the reduction kinetics of Escherichia coli in cooling tower plumes (author's transl)].

The present paper reports on laboratory tests for the determination of the reduction rate of micro-organisms. The investigations were carried out with a pure culture of E. coli, a germ type from the enterobacterial group. By a suitable variation of the thermodynamic parameters it was possible to simulate the conditions through which the cooling tower plume passes inside the chimney of the cooling tower and when it mixes with ambient air. The results allow the following changes in the germ counts in the plume of a wet cooling tower to be expected: 1. As long as the cooling tower plume contains droplets, the capacity of the carried germs to form colonies does not change materially. 2. In the case of unsaturated plumes we must distinguish between two situations: a) The tests in which the germ suspension was sprayed into unsaturated air without any admixture of secondary air, did not demonstrate any dependence of the reduction rate on the relative humidity. b) In contrast to this, the tests in which the germ-carrying droplets were evaporated by admixing a stream of unsaturated secondary air, showed that the capacity to form colonies clearly decreased with diminishing relative air humidity. Case b) is of importance for the dissemination and immission of germs from cooling towers, because here, too, the visible plume initially laden with drops is dried by the admixture of unsaturated ambient air.

Escherichia coli↗
PubMed1987

Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase.

Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).

Amino Acyl-tRNA Synthetases↗
PubMed1994

Retroviral capture of c-erbB proto-oncogene sequences: rapid evolution of distinct viral genomes carrying mutant v-erbB genes with different transforming capacities.

The evolution of oncogene-transducing retroviruses was followed by studying the genomes of five new, erbB carrying retroviruses. These viruses, isolated from cells of one chicken infected with Rous Associated virus 1 (RAV-1), had captured c-erbB sequences as a consequence of RAV-1 integration into the host genome. Their genome structures were distinct; however, their v-erbB genes had sustained identical 5' and 3' deletions and the v-erbB-env junctions were identical at the nucleotide level. The results therefore strongly suggest that all five viruses originate from the same capture event. Sequence analyses of the v-erbB genes from three of these viruses revealed that one of them had undergone no further mutation and lacked detectable capacity to transform cells, therefore probably representing an 'early' form of transducing virus. The two other v-erbB genes contained distinct mutations and differed in their potential to induce fibroblast- and erythroblast transformation; they therefore probably represent later derivatives of the virus that captured the erbB oncogene. The data suggest that the initial retrovirus rapidly underwent many alterations after capture of c-erbB sequences, already in the RAV-1 infected bird as well as during subsequent in vitro isolation procedures. The changes involve both major rearrangements of the genome as well as point mutations that activated the erbB oncogene.

Amino Acid Sequence↗
PubMed1997

In vitro study of the effect of sodium hypochlorite and glutaraldehyde on substrate adherence capacity of macrophages.

The purpose of this study was to investigate the in vitro effect of two irrigation solutions used in endodontics (5.25% sodium hypochlorite and 1% glutaraldehyde) on substrate adherence capacity of macrophages to determine if these substances can alter macrophage function. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Substrate adherence capacity assays were carried out in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that both sodium hypochlorite and glutaraldehyde significantly decreased the substrate adherence capacity of inflammatory macrophages. To take into account that adhesion is the first step in the phagocytic process of macrophages and in antigen presentation, sodium hypochlorite and glutaraldehyde could inhibit macrophage function and reduce inflammatory reactions in periapical tissues when they are used in root-canal therapy.

Animals↗
PubMed1980

The effect of maximal exertion on the levels of plasma peptides in cyclists with different physical working capacities.

The studies were carried out on thirty-one sportsmen, cyclists aged 21-28 years, after the termination of a training course just before the start of their season. They exercised on a bicycle ergometer according to the following schedule: 30 N at 60 rev/min for 6 min, 40 N at 70 rev/min for 5 min, 50 N at 80 rev/min for 5 min and 50 N at 90 rev/min until exhaustion. According to the level of training the subjects were divided into three groups. In the three groups of cyclists were determined the concentrations of total plasma peptides and their individual fractions obtained by gel-filtration on Sephadex G-10. It was found that the total plasma peptide concentration varied with the level of fitness. The lowest plasma peptide concentration was observed in cyclists with the lowest level of fitness and it differed from those observed in cyclists with intermediary and high levels of fitness. After exercise the biggest increase in plasma peptide concentration was found in the cyclists with the low level of fitness, and the lowest one in those with the high level of fitness. Individual plasma peptide fractions also revealed differences before and after exercise depending on the level of fitness.

Adult↗
PubMed1991

Characterization of immune inducer and suppressor macrophages from the normal human lung.

Monoclonal antibodies (MoAbs) that are able to discriminate between dendritic cells (MoAb RFD1+) and mature macrophages (MoAb RFD7+) in normal tissues were used in combination with density separation techniques to isolate relatively homogeneous subpopulations of macrophages from human bronchoalveolar lavage (BAL). A characterization of surface antigen expression, and functional capacity was then carried out on each isolated alveolar macrophage (AM) subset. One population with the phenotype RFD1+RFD7- obtained from the non-adherent cell pool showed the characteristics of antigen-presenting cells having absent or poor expression of Fc and C3b receptors, a low content of lysozomal hydrolase and poor phagocytic capacity. This population strongly stimulated T lymphocytes in allogeneic mixed lymphocyte reactions (MLR). A second AM population, isolated by adherence and density centrifugation expressed the phenotype RFD1+RFD7+. These cells showed the same phenotypic characteristics of mature macrophages with strong expression of C3b and Fc receptors, and marked phagocytic capacity. Such AM were very poor stimulators of allogeneic MLR. Under certain circumstances the RFD1+RFD7+ cells were shown to actively repress the stimulatory capacity of the RFD1+RFD7- subpopulation. These results suggest that variations within the functional capacity of AM subsets may be capable of influencing the strength of acquired T cell immune responses of the lung.

Acid Phosphatase↗
PubMed1991

Essential residues in the polar loop region of subunit c of Escherichia coli F1F0 ATP synthase defined by random oligonucleotide-primed mutagenesis.

The conserved, polar loop region of subunit c of the Escherichia coli F1F0 ATP synthase is postulated to function in the coupling of proton translocation through F0 to ATP synthesis in F1. We have used a random mutagenesis procedure to define the essential residues in the region. Oligonucleotide-directed mutagenesis was carried out with a random mixture of mutant oligonucleotides, the oligonucleotide mixture being generated by chemical synthesis by using phosphoramidite nucleotide stocks that were contaminated with the other three nucleotides. Thirty mutant genes coding single-amino-acid substitutions in the region between Glu-37 and Leu-45 of subunit c were tested for function by analyzing the capacity of plasmids carrying the mutant genes to complement a Leu-4----amber subunit c mutant. All substitutions at the conserved Arg-41 residue resulted in loss of oxidative phosphorylation, i.e., transformants could not grow on a succinate carbon source. The other conserved residues were more tolerant to substitution, although most substitutions did result in impaired growth on succinate. We conclude that Arg-41 is essential in the function of the polar loop and that the ensemble of other conserved residues collectively maintain an optimal environment required for that function.

Amino Acid Sequence↗
PubMed2005

Quantum simulation of the heat capacity of water.

Path integral molecular dynamics simulations have been carried out to evaluate heat capacities of water in gas, liquid, and solid phases. For convergence, a 100-ps simulation run with a large path integral variable (P approximately 128) was required even by using the double-centroid virial heat capacity estimator. For all states, the quantum corrections to the heat capacities are significantly large. The calculated heat capacities for vapor and ice I(h) agreed excellently with experimental data, while that for liquid was less than the experimental value by approximately 20% due to the limit of the SPC/F potential model.

Journal Article↗
PubMed2006

Identification of the LEDGF/p75 binding site in HIV-1 integrase.

Lens epithelium-derived growth factor (LEDGF)/p75 is an important cellular co-factor for human immunodeficiency virus (HIV) replication. We originally identified LEDGF/p75 as a binding partner of integrase (IN) in human cells. The interaction has been mapped to the integrase-binding domain (IBD) of LEDGF/p75 located in the C-terminal part. We have subsequently shown that IN carrying the Q168A mutation remains enzymatically active but is impaired for interaction with LEDGF/p75. To map the integrase/LEDGF interface in more detail, we have now identified and characterized two regions within the enzyme involved in the interaction with LEDGF/p75. The first region centers around residues W131 and W132 while the second extends from I161 up to E170. For the different IN mutants the interaction with LEDGF/p75 and the enzymatic activities were determined. IN(W131A), IN(I161A), IN(R166A), IN(Q168A) and IN(E170A) are impaired for interaction with LEDGF/p75, but retain 3' processing and strand transfer activities. Due to impaired integration, an HIV-1 strain containing the W131A mutation in IN displays reduced replication capacity, whereas virus carrying IN(Q168A) is replication defective. Comparison of the wild-type IN-LEDGF/p75 co-crystal structure with that of the modelled structure of the IN(Q168A) and IN(W131A) mutant integrases corroborated our experimental data.

Amino Acid Sequence↗
PubMed2005

Modeling of copper(II) and lead(II) adsorption on kaolinite-based clay minerals individually and in the presence of humic acid.

The aim of this study is to explain how clay minerals adsorb heavy metals individually and in the presence of humic acid, and to model heavy metal adsorption specifically based on surface-metal binary and surface-metal-ligand ternary complexation. The adsorption of Cu(II) and Pb(II) on kaolinite-based clay minerals has been modeled by the aid of the FITEQL3.2 computer program using single- and double-site binding models of the Langmuir approach. Potentiometric titrations and adsorption capacity experiments were carried out in solutions containing different concentrations of the inert electrolyte NaClO4; however, the modeling of binary and ternary surface complexation was deliberately done at high ionic strength (0.1 M electrolyte) for eliminating adsorption onto the permanent negatively charged sites of kaolinite. A "two-site, two pKa" model was adapted, and as for the two surface sites responsible for adsorption, it may be arbitrarily assigned that [triple bond]S1OH sites represent silanol and organic functional groups such as carboxyl having pKa values close to that of silanol, and [triple bond]S2OH sites represent aluminol and organic functional groups such as phenolics whose pKa values are close to that of aluminol, as all the studied clays contained organic carbon. Copper(II) showed a higher adsorption capacity and higher binding constants, while lead(II), being a softer cation (in respect to HSAB theory) preferred the softer basic sites with aluminol-phenol functional groups. Heavy metal cations are assumed to bind to the clay surface as the sole (unhydrolyzed) M(II) ion and form monodentate surface complexes. Cu(II) and Pb(II) adsorption in the presence of humic acid was modeled using a double-site binding model by the aid of FITEQL3.2, and then the whole system including binary surface-metal and surface-ligand and ternary surface-metal-ligand complexes was resolved with respect to species distributions and relevant stability constants. Electrostatic effects were accounted for using a diffuse layer model (DLM) requiring minimum number of adjustable parameters. Metal adsorption onto clay at low pH increased in the presence of humic acid, and the metal adsorption vs pH curves of metal-kaolinite-humic acid suspensions were much steeper (and distinctly S shaped) compared to the wider pH-gradient curves observed in binary clay-metal systems. The clay mineral in the presence of humic acid probably behaved more like a chelating ion-exchanger sorbent for heavy metals rather than being a simple inorganic ion exchanger.

Journal Article↗
PubMed2007

Beta-cell function in individuals carrying the mitochondrial tRNA leu (UUR) mutation.

OBJECTIVES: To assess the beta-cell function in individuals with mitochondrial DNA A3243G mutation with normal glucose tolerance (NGT) or diabetes mellitus (DM). Furthermore, in diabetic individuals, we evaluated the effect of coenzyme Q10 supplementation on insulin secretory response. METHODS: Eight mutation-positive individuals with NGT (n = 4) or DM (n = 4) were studied. beta-Cell function was evaluated by C-peptide levels before and after a mixed liquid meal (Sustacal) challenge and by first-phase insulin response. RESULTS: Fasting and Sustacal-stimulated C-peptide levels were significantly lower in diabetic patients than that in controls (area under the curve: 104.1 +/- 75.7 vs 520.8 +/- 173.8, P = 0.001), whereas in individuals with NGT, this response was preserved (area under the curve: 537.8 +/- 74.3 vs 520.8 +/- 179.8, P = 0.87). The duration of diabetes was negatively correlated with fasting C-peptide levels (r = -0.961, P = 0.038). Among the 3 patients with residual insulin secretion, the short-term treatment with coenzyme Q10 (3 months) improved C-peptide levels in 2 of them. The first-phase insulin response was diminished in 2 individuals with NGT, the oldest ones. CONCLUSIONS: We showed an impaired insulin secretory capacity in individuals carrying the A3243G mutation, this possibly being the primary defect contributing to the development of DM. In addition, our data suggest that this could be a functional defect.

Adult↗
PubMed2000

Evidence of a role for the Q151L mutation and the viral background in development of multiple dideoxynucleoside-resistant human immunodeficiency virus type 1.

The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1(HXB2)). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1(HXB2). In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1(HXB2) partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1(151M), HIV-1(151L) RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1(151L) was found to be less fit than HIV-1(151M), which may explain the preferential selection of HIV-1(151M) observed in vivo. The demonstrated ability of HIV-1(151L/68G) to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1(151L) on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.

Amino Acid Sequence↗