Effects of dexamethasone on carnitine metabolism in liver and extrahepatic tissues

The in vivo effects of dexamethasone administration on liver and extrahepatic tissue carnitine concentrations were assessed in 48-h-starved rats. In heart and kidney, but not in liver, dexamethasone significantly increased total carnitine concentration. Acute (2.5 h) treatment with 2-tetradecylglycidate (TDG), a specific inhibitor of carnitine palmitoyl transferase 1, not only increased total hepatic carnitine concentrations, but also permitted an effect of dexamethasone (a further increase in hepatic carnitine concentration). The results are discussed in terms of acute (substrate-mediated) and chronic (hormonal) control of carnitine turnover.     

Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-β protein (Aβ) in vivo is a critical pathological event in Alzheimer's disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimer's disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Aβ(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Aβ peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.     

The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies

Immunization with extracellular sexual stages of the malaria parasites can induce the production of antibodies which block the development of the parasites in the midgut of a mosquito after a blood meal. We have generated a number of monoclonal antibodies against gametes and zygotes of the human malaria Plasmodium falciparum. Two monoclonal antibodies (mAb) reacting with a 230-kDa gamete surface protein (mAb 1B3 and 2B4 both isotype IgG2a) were found to block transmission of P. falciparum to mosquitoes. Blocking was complement dependent and this was verified in vitro by the rapid lysis of newly formed gametes and zygotes in the presence of the mAb and active complement. Both mAb reacted by immunofluorescence with the surface of gametes and zygotes from isolates of P. falciparum from various geographical areas. Each mAb immunoprecipitated a 230-kDa protein from 125I-labeled surface proteins of newly formed gametes and zygotes and immunoblotted a protein doublet of about molecular mass 260 and 230 kDa from gametocytes and gametes of P. falciparum. Only the 230-kDa protein is expressed on the surface of newly formed macrogametes and zygotes. The 230-kDa gamete surface protein forms a molecular complex with two proteins of 48 and 45 kDa. The 48- and 45-kDa gamete surface proteins have previously been shown to be targets of mAb which block infectivity of P. falciparum to mosquitoes. The present study now demonstrates that antibodies against the 230-kDa gamete surface protein block transmission of P. falciparum to mosquitoes. The 230-kDa gamete protein is thus a potential candidate for a gamete vaccine.     

Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides. Correlation of effect with biologic activity of IL-2

Current malaria vaccine strategies focus on subunit vaccines that contain one or a limited number of malaria Ag. However, there is widespread nonresponsiveness to many of these Ag probably resulting from Ir gene control. Using a congenic mouse model, we demonstrated that human rIL-2 (as an adjuvant) can overcome Ir gene controlled low immune responsiveness to peptide malaria Ag vaccine candidates [R32tet32, R32LR, and Th2R-NP (NANP)5NA] as determined by the antibody response, providing it is emulsified with the Ag during immunization. This effect is not caused by IL-2 merely acting as a foreign protein and stimulating noncognate help; it requires biologic activity of the IL-2, as determined by studying the effect of inactive rIL-2, which has minimal biologic activity but which has retained its antigenicity. IL-2 does not appear to be working by an effect on priming of specific Th, and IL-2 cannot overcome an Ir gene controlled low T cell proliferative response. IL-2 may have a role to play in human vaccine development where a high titer antibody response to a subunit vaccine is required.     

Human IFN-gamma production is inhibited by a synthetic peptide homologous to retroviral envelope protein

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the in vitro production of IFN-gamma from human peripheral mononuclear cells. The results showed that CKS-17 coupled to a carrier protein, BSA, inhibited production of IFN-gamma in a dose-dependent manner. Controls, consisting of BSA, which had undergone the coupling procedure or neurotensin coupled to BSA in an identical manner as CKS-17, showed no such inhibition. Reduction in IFN-gamma production could not be attributed to decreased viability of cells, delay of IFN-gamma production or to involvement of suppressor cells. Moreover, inhibition of IFN-gamma production was not related to the inhibition of DNA synthesis. The inhibition appeared to be a direct effect of CKS-17 on IFN-gamma-producing cells. Kinetic studies revealed that this suppression occurred when CKS-17 was introduced to the culture concurrent with or within 48 h after introduction of IFN inducers. Preincubation experiments showed that the presence of CKS-17 in the culture medium was not necessary to exert its inhibitory effect. These results suggest that a portion of retroviral envelope proteins possess important immunomodulatory actions.     

Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

ELISA assays have been developed for (1–3)N-acetylgalactosaminyltransferase (blood group A transferase) and (1–3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc1–2Gal-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuc1–2(GalNAc1–3)Gal-R-BSA or Fuc1–2(Gal1–3)Gal-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.     

Spectroscopic studies on cadmium (II)- and cobalt(II)-substituted metallothionein from the crab Cancer pagurus. Evidence for one additional low-affinity metal-binding site

The binding of diamagnetic Cd(II) and paramagnetic Co(II) ions to the metal-free form of crab, Cancer pagurus, metallothionein (MT) was studied by various spectroscopic techniques. Both reconstituted and native Cd(II)-MT containing 6 mol Cd(II)/mol protein display electronic absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra which were indistinguishable. The stoichiometric replacement of Cd(II) ions in native Cd(II)6-MT by paramagnetic Co(II) ions enabled the geometry of the metal-binding sites to be probed. The electronic absorption and MCD spectra of Co(II)6-MT revealed features characteristic of distorted tetrahedral tetrathiolate Co(II) coordination for all six metal-binding sites. The stepwise incorporation of Cd(II) and Co(II) ions into this protein was monitored by electronic absorption and CD, and by electronic absorption and EPR spectroscopy, respectively. The results indicate that the metal-thiolate cluster structure is generated when more than four metal ions are bound. Below this titration point separate tetrahedral tetrathiolate complexes exist. This suggests that the cluster formation occurs in a two-step process. Furthermore, the spectroscopic features in both Cd(II)- and Co(II)-metal derivatives above the full metal occupancy of six suggest the existence of one additional metal-binding site. The subsequent loss of one Cd(II) ion from crab Cancer Cd(II)7-MT in the gel filtration studies demonstrate the low metal-binding affinity of the latter site. While the spectroscopic properties indicate an exclusively tetrahedral type of metal-thiolate sulfur coordination for the binding of the first six metal ions, they suggest that the seventh metal ion is coordinated in a different fashion.