Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold

Summary The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV-cell interactions and as a lead in the development of antiviral drugs.     

Oil refinery sludge and green waste simulated windrow composting

Oil refinery sludge (ORS) was mixed with shredded green wastes (GW) at ratios of 1:1 v/v (RI) and 1:3 v/v (RII). The mixtures, of approximately volumes of 1,020 l and 990 l respectively, were introduced in metal cubic containers of 1.0 m³ volume, opened at the top and with small holes punctured in the bottom and the side. The containers were additionally insulated with a layer of rockwool (20 mm). The boxes were emptied, the mixtures were turned and water was added occasionally, in one to two weeks intervals, simulating a windrow composting system. Temperature, physiochemical characteristics, mineral oil and grease (MOG) concentration, polycyclic aromatic hydrocarbons (PAHs) concentration, carbon dioxide emission, methane emission and microorganisms presence were recorded either daily or every time the mixtures were turned, for a period of 120 days. RII recorded temperatures as high as 62°C, reaching 56°C in Day 6 and retained temperatures above 50°C for more than 40 days. RI recorded its highest temperature of 53°C in Day 77. The reason why the two mixtures behaved so differently can be explained by: (i) extended co-digestion phenomena by the microorganisms decomposing the GW in RII, (ii) toxic effect of ORS in RI due to the far larger amounts used (840 kg in RI in comparison with the 460 kg in RII). After Day 36 temperature increased gradually in RI and MOG and PAHs reduction was first noted. At the end of the experiment MOG concentration in RI was 57.2 mg/kg dry weight (dw) (52.1% reduction) where in RII was 34.3 mg/kg dw (62.1% reduction). Emissions of methane and carbon dioxide support the concept of the toxic effect and the delay ignition of the decomposing process in RI. In total, CO₂ and CH₄ emissions from RI were recorded to be 30.8 kg and 18.5 g, respectively, where from RII 59.6 kg of CO₂ and 6.4 g of CH₄ were emitted. An effort was made to determine the effect of temperature alone (as an abiotic treating parameter) in both mixtures. It can be supported that about least 15% of the MOG and PAHs removal.     

Synthesis and antibody recognition of mucin 1 (MUC1)-alpha-conotoxin chimera.

We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The Pro-Asp-Thr-Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a beta-turn conformation. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, which is stabilized by two disulphide bridges in positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, Arg9-His-Tyr-Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4-(2',4'-dimethoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3-Cys13 or Cys2-Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD spectra of the bicyclic MUC1-alpha-[Tyr1]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization.     

Familial hypothyroidism caused by a nonsense mutation in the thyroid-stimulating hormone beta-subunit gene.

Hereditary hypothyroidism caused by thyroid-stimulating hormone (TSH) deficiency is a rare autosomal recessive disease. Affected individuals show symptoms of severe mental and growth retardation that can be prevented by early administration of exogenous thyroid hormone. In this paper, we describe two related Greek families with three children affected by congenital TSH-deficient hypothyroidism. Sequence analysis of the TSH beta-subunit gene (TSHB) showed that the mutation responsible for the hypothyroidism in these families is a nonsense mutation in exon 2. This mutation is a G-to-T transversion at nucleotide 94 that destroys the only TaqI site in the TSHB-coding region and gives rise to a novel 8.5-kb TaqI fragment. Restriction analysis showed that the three affected children are homozygous for the 8.5-kb allele and that the four parents and two unaffected children are heterozygous. This mutation gives rise to a truncated peptide which includes only the first 11 of 118 amino acids of the mature TSHB peptide.     

Characterization of the internal motions of a chimeric protein by 13C NMR highlights the important dynamic consequences of the engineering on a millisecond time scale.

By transferring the central curaremimetic beta hairpin of the snake toxin alpha into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent beta hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin alpha, in order to examine the beta hairpin motions in both structural contexts. Thus, 13C R1, R1rho and 1H-->13C nOe were measured for all the CalphaHalpha and threonine CbetaHbeta vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified beta sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its beta structure is less stable on a minute to hour time scale. On the other hand, when the beta hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin alpha, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the beta hairpin sequence, and the beta sheet seems more stable in toxin alpha than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another.     

Simultaneous single-tube PCR-based assay for the direct identification of the five most common meningococcal serogroups from clinical samples

This study presents a stepdown multiplex PCR assay for the simultaneous detection of the five most common Neisseria meningitidis serogroups (A, B, C, W-135 and Y) in 530 clinical samples obtained from 428 patients (271 blood and 259 cerebrospinal fluid). The sensitivity and the specificity was calculated to 100% [positive predictive value 100% (95%, CI 99.0-100%) and negative predictive value 100% (95% CI 99.0-100%)]. The overall effectiveness permits the rapid, accurate and inexpensive detection of the five most prevalent meningococcal serogroups in clinical samples. It is potentially a valuable tool for diagnosis and epidemiological monitoring of disease due to N. meningitidis.     

Engineering a CD4 mimetic inhibiting the binding of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 to human lymphocyte CD4 by the transfer of a CD4 functional site to a small natural scaffold

The solvent-exposed CDR2-like region of human CD4 was transferred to the structural scaffold of scorpion charybdotoxin, as a means to reproduce that site in a native-like conformation. The chimeric mini-protein (33 amino acids long), obtained by solid-phase synthesis, is able to specifically prevent the interaction of HIV-1 gp120 with CD4. This CD4 mimetic may represent a valuable tool in the study of the HIV–cell interactions and as a lead in the development of antiviral drugs.     

Ichthyosis and hypogonadism in two brothers with deletion of the short arm of the X chromosome

Two brothers were examined because of ichthyosis and hypogonadism. Their testes were small. There was no response of plasma testosterone to human chorionic gonadotropin and no response of plasma luteinizing hormone and follicle-stimulating hormone to intravenous luteinizing-hormone-releasing hormone. They both had hyposmia. Steroid sulfatase activity in white blood cells was zero. Flow cytometry and the use of special probes indicated that these two brothers had a large deletion of the short arm of the X chromosome which included the STS locus, the closely linked locus DXS237 and probably the gene for hypogonadism, findings which offer the opportunity for speculations on the locus of control of normal testicular development and function.