Storage of vaccines in the community: weak link in the cold chain?

OBJECTIVE--To assess quality of storage of vaccines in the community. DESIGN--Questionnaire survey of general practices and child health clinics, and monitoring of storage temperatures of selected refrigerators. SETTING--Central Manchester and Bradford health districts. SUBJECTS--45 general practices and five child health clinics, of which 40 (80%) responded. Eight practices were selected for refrigeration monitoring. MAIN OUTCOME MEASURES--Adherence to Department of Health guidelines for vaccine storage, temperature range to which vaccines were exposed over two weeks. RESULTS--Of the 40 respondents, only 16 were aware of the appropriate storage conditions for the vaccines; eight had minimum and maximum thermometers but only one of these was monitored daily. In six of the eight practices selected for monitoring of refrigeration temperatures the vaccines were exposed to either subzero temperatures (three fridges) or temperatures up to 16 degrees C (three). Two of these were specialised drug storage refrigerators with an incorporated thermostat and external temperature gauges. CONCLUSION--Vaccines were exposed to temperatures that may reduce their potency. Safe storage of vaccines in the clinics cannot be ensured without adhering to the recommended guidelines. Provision of adequate equipment and training for staff in maintaining the "cold chain" and the use and care of equipment are important components of a successful immunisation programme.     

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.     

Modulation of the orientational order profile of the lipid acyl chain in the Lα phase

The orientational order profile along the lipid acyl chain has been characterized under several different conditions of polar headgroup composition, temperature, and cholesterol content. Despite the different nature of these factors, the variation of the order is governed by two common trends. First, the relative change of order induced by the variation of these factors is always more pronounced towards the end of the chain than for the methylene groups near the interface. Second, there is, to a first approximation, a distinct correlation between the magnitude of the order parameters and the shape of the order profile. For example when the chain is highly ordered, the relative width of the order distribution is narrow indicating that the plateau region is longer. These conclusions suggest that the orientational order profile depends on only a small number of parameters and demonstrate clearly that the correlation length for changes in orientational order is much greater than one C-C bond length. Our results also show that the reduced temperature is not related in simple terms to orientational order and probably has little theoretical significance. The orientational order profiles of POPC and POPE bilayers are significantly different even when expressed in terms of reduced temperature. The behavior of POPC/cholesterol systems also indicates that the orientational order of the lipid chain and the gel-to-liquid crystalline phase transition temperature are not related in a straightforward manner.Abbreviations POPC 1-palmitoyl-2-oleoyl-phosphatidylcholine - POPE 1-palmitoyl-2-oleoyl-phosphatidylethanolamine - PC phosphatidylcholine - PE phosphatidylethanolamine - NMR nuclear magnetic resonance - EDTA ethylenediaminetetraacetic acid Offprint requests to: M. Bloom     

Structure and diversity of the T-cell receptor α chain in the Mexican axolotl

 Polymerase chain reaction was used to isolate cDNA clones encoding putative T-cell receptor (TCR) α chains in an amphibian, the Mexican axolotl (Ambystoma mexicanum). Five TCRα-V chain-encoding segments were identified, each belonging to a separate family. The best identity scores for these axolotl TCRα-V segments were all provided by sequences belonging to the human TCRα-V1 family and the mouse TCRα-V3 and TCRα-V8 families. A total of 14 different TCRA-J segments were identified from 44 TCRA-V/TCRA-J regions sequenced, suggesting that a large repertoire of TCRA-J segments is a characteristic of most vertebrates. The structure of the axolotl CDR3 α chain loop is in good agreement with that of mammals, including a majority of small hydrophobic residues at position 92 and of charged, hydrophilic, or polar residues at positions 93 and 94, which are highly variable and correspond to the TCRA-V/J junction. This suggests that some positions of the axolotl CDR3 α chain loop are positively selected during T-cell differentiation, particularly around residue 93 that could be selected for its ability to makes contacts with major histocompatibility complex-associated antigenic peptides, as in mammals. The axolotl Cα domain had the typical structure of mammalian and avian Cα domains, including the charged residues in the TM segment that are thought to interact with other proteins in the membrane, as well as most of the residues forming the conserved antigen receptor transmembrane motif. Received: 12 June 1996 / Revised: 11 September 1996     

Structure–activity relationships of memoquin: Influence of the chain chirality in the multi-target mechanism of action

The present article expands on the study of structure–activity relationships of the novel class of quinone-bearing polyamines, as multi-target-directed ligands against Alzheimer’s disease. Namely, the effect of inserting a methyl substituent at the α position of the terminal benzyl amine moieties of lead candidate 1 (memoquin) was evaluated at the multiple targets involved in the multifunctional mechanism of action. The RR stereoisomer 2 resulted more effective than 1 in reverting two important effects mediated by acetylcholinesterase (AChE), that is, acetylcholine hydrolysis and AChE-induced amyloid-β aggregation.     

Comprehensive analysis and characterization of the TCR α chain sequences in the common marmoset

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR α constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68–100%, and 79–98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRβ chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d _S ) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d _N ) were significantly lower in the FRs than in CDRs. Interestingly, the d _N values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRβ than in CDRα by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.     

A model of O·2 - generation in the complex III of the electron transport chain

Oxidation of semiquinone by O₂ in the Q cycle is known to be one of the sources of superoxide anion (O·₂ ^-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeS_III and cytochromes b₁, b_h and c₁. Electron transfers from QH₂ to FeS_III and cytochrome b₁ were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS^red _III -Q·^- and Q·^--cytochrome b₁ complexes. For oxidation/reduction reactions involving cytochromes b_h and b₁, the dependence of the equilibrium and elementary rate constants on the membrane potential () was taken into consideration. The rate of O·₂ ^- generation was found to increase dramatically with increase in above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O·2^- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in below the above mentioned critical level. pH appears to be equally effective as in stimulation of superoxide production which depends, in fact, upon the ^- _H + level.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

FT-ICR-MS characterization of intermediates in the biosynthesis of the α-methylbutyrate side chain of lovastatin by the 277 kDa polyketide synthase LovF

There are very few fungal polyketide synthases that have been characterized by mass spectrometry. In this paper we describe the in vitro reconstitution and FT-ICR-MS verification of the full activity of an intact 277 kDa fungal polyketide synthase LovF of the lovastatin biosynthetic pathway. We report here both the verification of the reconstitution of fully functional holo-LovF by using (13)C-labeled malonyl-CoA to form α-methylbutyrate functionality and also detection of five predicted intermediates covalently bound to the 4'-phosphopantetheine at the acyl carrier protein (ACP) active site utilizing the phosphopantetheine ejection assay and high-resolution mass spectrometry. Under in vitro conditions, the diketide acetoacetyl intermediate did not accumulate on the ACP active site of holo-LovF following incubation with malonyl-CoA substrate. We found that incubation of holo-LovF with acetoacetyl-CoA served as an effective means of loading the diketide intermediate onto the ACP active site of LovF. Our results demonstrate that subsequent α-methylation of the acetoacetyl intermediate stabilizes the intermediate onto the ACP active site and facilitates the formation and mass spectrometric detection of additional intermediates en route to the formation of α-methylbutyrate.     

Where is the weak linkage in the globin chain?

Hemoglobinopathies are important inherited disorders with high prevalence in many tropical countries. Prediction of protein nanostructure and function is a great challenge in proteomics and structural genomics. Identifying the point vulnerable to mutation is a new trend in research on disorders at the genomic and proteomic level. A bioinformatics analysis was performed to determine the positions that tend to correspond with peptide motifs in the amino acid sequence of alpha and beta globin chains. To identify the weak linkage in alpha globin and beta globin chains, a new bioinformatics tool, GlobPlot, was used. For the alpha globin chain, 22 positions were identified: the disorders were found at positions 3-8, 38-42, 46-51, and 75-79. For the beta globin chain, 46 positions were identified: the disorders were found at positions 61-146. The study showed that weak linkages in alpha globin and beta globin chains can be identified and provide good information for predicting possible new mutations that could lead to new hemoglobinopathies.     

Cysteine in the triple helical domain of the proα2(I) chain of type-I collagen in nonlethal forms of osteogenesis imperfecta

Summary To determine if some individuals with deforming varieties of osteogenesis imperfecta (OI) carry point mutations in the COL1A2 gene of type-I collagen, we examined collagens synthesized by cell strains from affected individuals for the presence of cysteine in the triple helical domain of the 2(I) chain, a domain from which it is normally excluded. We identified 4 individuals out of 60 whose cells synthesized a population of 2(I) chains with a cysteine residue in the triple helix. The clinical differences among the affected individuals and the heterogeneity in the locations of the cysteine residues suggest that the position of the substitution within the chain is important in determining the clinical phenotype. These data confirm that individuals with nonlethal OI may commonly harbor defects in the COL1A2 gene, and suggest that many of the defects are substitutions for glycine residues in the 2(I) triple helical domain.     

The vγ chain of fetal hemoglobin of the orangutan

Previous studies suggested that orangutans have nonallelic structural genes for their γ chains, because either a threonyl or an alanyl residue may occupy position 135. Further investigation has now detected that position 75 may have either an isoleucyl or a valyl residue. From available evidence, the isoleucyl and threonyl residues are in one chain and the valyl and alanyl in the other. Orangutans appear to be homozygous for the two types of nonallelic genes.     

Separation and characterization of the A chain and B chain in β1-bungarotoxin fromBungarus multicinctus (Taiwan banded krait) venom

The interchain disulfide bond between A chain and B chain of ₁-bungarotoxin (₁-Bgt) was selectively cleaved by dithiothreitol, and the A and B chains were separated by HPLC. The separated A and B chains did not show detectable enzymatic activity and lethal toxicity, but exhibited an immunoreactivity with anti-₁-Bgt antibody. Analytical isoelectrofocusing revealed that the A chain is a neutral subunit with pI=7.4, and the B chain is a basic one with pI=9.6. The A chain exhibited a Ca²⁺-binding ability as revealed by fluorescence measurement. Moreover, fluorescence studies showed that the intact interchain disulfide bond is essential for maintaining the hydrophobic character of substrate binding site in ₁-Bgt and stabilizing the architectural environment of Trp-19 in the A chain. However, combination of the A chain and B chain failed to restore the biological activities and physicochemical properties which the intact ₁-Bgt possessed. These, together with our previous result that the Trp-19 of the A chain is involved in substrate binding, suggest that the integrity of the interchain disulfide bond favors the maintenance of the active conformation of ₁-Bgt.     

NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

Evolution of the rabbit immunoglobulin κ chain genes

We have analyzed the organization and the structure of rabbit chain genes encoding b allotypes in wild rabbits. The 1 gene of the b95 allotype was cloned and its structure determined. The J region is composed of five segments but only J2 appears to be functional and is identical to the J2 segment of the b4 allotype. The J region is highly conserved among the various b allotypes, whereas the constant region exon displays a high level of differences when compared with other allotypes (9%–30% of different amino acids). The b95 J region is closer to that of b4^var and the constant region to b5 allotype constant region. Alignment of nucleotide sequences revealed that the constant region exon displays segmental similarities with b4 and bas constant regions. The mosaic structure of b95 allotype gene indicates that complex allotypes of 1 genes may result from genetic exchanges of gene conversion between the different genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession number M22542. Address correspondence and offprint requests to: P.-A. Cazenave.     

High levels of soluble IFN γ receptor α chain in the plasma of rheumatoid arthritis patients

Soluble receptors for hormones and cytokines have beendescribed. They can serve as natural blockers of theirrespective ligands. The natural soluble interferongamma receptor (sIFNR) has been isolated andcharacterized only in urine. Chromatography of human(hu) plasma from rheumatoid arthritis (RA) patientsand controls on immobilized hu IFN orantibodies against IFN R chainpermitted us to isolate the sIFNR. Thereceptor isolated from one control is a protein witha molecular weight between 60-67 kDa depending on thepresence of reducing agents. We detected asignificantly higher level of plasma sIFNR inpatients with rheumatoid arthritis than in apparentlyhealthy subjects.