Genetically determined differences in the response of alpha1-antitrypsin levels in human serum to typhoid vaccine

Summary Normal individuals and those homozygous and heterozygous for the gene which determines deficiency in serum ₁-antitrypsin (₁-at) were given an intravenous injection of typhoid vaccine. Following this injection the trypsin-inhibiting capacity of serum and the concentration of ₁-at increases markedly in normal individuals. Heterozygotes for the deficiency gene have an elevation of ₁-at and trypsin-inhibiting capacity of their serum but reach only 50 percent of the levels reached by homozygotes for the common gene (normal individuals). Their values are however temporarily in the range of normal men not given typhoid vaccine. Homozygotes for the deficiency gene show only a small elevation of ₁-at under these conditions. These findings demonstrate that (1) the increase in trypsin-inhibiting capacity of serum after an injection of typhoid vaccine is largely due to the elevation of the ₁-at concentration, and (2) the ₁-at deficiency gene inhibits the quantitative response of the ₁-at to such a stimulus.Supported in part by USPHS grant HE-06285 from the National Heart Institute.Some of these studies were carried out in the General Clinical Research Center Ward, U. C., FR-79, supported by the Division of Research Facilities and Resources, U.S. Public Health Service.     

Identification of the heterozygous state for the α1-antitrypsin deficiency gene in man

Alpha₁-antitrypsin (₁-at) of individuals homozygous for a gene determining low serum concentrations of this protein can be distinguished electrophoretically from ₁-at of homozygotes for the more common gene. Heterozygotes possess both electrophoretic species, and they may have ₁-at levels intermediate between those of both homozygotes or may be in the range of the homozygotes for the common gene. The frequency of the gene determining a deficiency of ₁-at in a population sample of 100 individuals was 0.075.This work was supported in part by NIH Program Project Grant HE-06285 from the National Heart Institute.     

α1-antitrypsin (Pi) phenotypes in a village population from the Gambia,West Africa

Summary A total of 701 individuals from a village in The Gambia, West Africa were tested for serum ₁ ^- antitrypsin phenotypes by isoelectric focusing in thinlayer polyacrylamide gels (pH 4-5). A new variant allele, Pi ^GAM , was discovered at a polymorphic frequency (0.0642), and the inheritance of the variant phenotype was confirmed by family studies. The variant was not found to be associated with any decrease in serum ₁ ^- concentration. The only other allele found within this population was the common allele Pi ^M (0.9358).     

1H-filtered correlation experiments for assignment and determination of coupling constants in backbone labelled proteins

The implementation of [¹³C,¹³C,¹⁵N,²H] labelled amino acids into proteins allows the acquisition of high resolution triple resonance experiments. We present for the first time resonance assignments facilitated by this new labelling strategy. The absence of ¹J_C,C couplings enables us to measure ¹J_C,C scalar and ¹D_C,C residual dipolar coupling constants using modified HNCA experiments which do not suffer from sensitivity losses characteristic for ¹³C constant time experiments.     

Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B₁₂ (Co-[-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Co-[-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B₁₂ (Co-[-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

When programmed with yeast prepro--factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp--F_cyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp--F₃, 27.5 kDa). Glycosylation of the membrane specific pp--F₃ species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp--F₀) , whereas the primary translation product pp--Fcyt is not affected. Likewise, only the glycosylated pp--F₃ structure is digested by Endo H yielding a polypeptide with a molecular mass between PP--F₀ and pp--Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp--Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp--F₃ polypeptide indicates that its oligosaccharide chains are processed to presumbly Man₉-GlcNAc₂ structures under thein vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.Abbreviations dNM 1-deoxynojirimycin - N-Me-dNM N-methyl-dNM - dMM 1-deoxymannojirimycin - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Selective down-regulation of the Cqα/G11α G-protein family in tumour necrosis factor-α induced cell death

Investigations into the regulation of heterotrimeric GTP-binding protein a-subunits in models of tumour necrosis factor- (TNF)-induced cell death, revealed the selective down-regulation of the G_q/G₁₁ family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G_q/G₁₁ family protein levels, but not G_s, G_i and G_o protein levels as determined by Western analyses. This effect of TNF was observed in a concentration - and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G_q/G₁₁ family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca²⁺],_i release. In contrast, G_s activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca²⁺-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.     

The use of 1JCαHα coupling constants as a probe for protein backbone conformation

Summary Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond J_CH coupling constants in proteins that are uniformly enriched with ¹³C. An empirical ,-surface is calculated which describes the deviation of ¹J_CH from its random coil value, using 203 ¹J_CH values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which and are know with good precision from previous X-ray crystallographic studies. Residues in -helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in -sheet are small and, on average, slightly negative. Data indicate that ¹J_CH depends primarily on , and that ¹J_CH may be useful as a qualitative probe for secondary structure. Comparison of ¹J_CH coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the central helix is even more extensive than in free calmodulin. Supplementary material available from the authors: One table listing 352 ¹J_CH and ¹J-values, together with ,-values for 203 residues of known conformation. Two figures showing (a) a Ramachandran plot of the ,-values of 203 residues used in deriving ¹J(,), and (b) the r.m.s.d. ¹J(,) distribution.     

Measurement of15N-13C J couplings in staphylococcal nuclease

Summary ¹⁵N-C and¹⁵N-C J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with¹⁵N and¹³C. It is found that the^IJ_C'N coupling is similar for -sheet, J=14.8 ± 0.5 and for -helix, J = 14.8 ± 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J=15.6 ± 0.5). On average,¹J_NC are smaller for -helical residues (J=9.6 ± 0.3 Hz) compared to -sheet (J=10.9 ± 0.8 Hz) and a substantial difference is observed for²J_NC in -helices (J=6.4 ± 0.4 Hz) and -sheets (J=8.3 ± 0.8 Hz).Dedicated to the memory of Professor V.F. Bystrov     

Evolution of the single copyαA-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3′ non-coding regions

A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat A₂-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3-untranslated regions of A₂-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The A₂-mRNA 3-non-coding regions of reptiles and birds are 300–550 bases longer than those of mammals. Some rodents produce next to the A₂-mRNA another messenger that encodes the A^lns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the A₂-polypeptide chain. A₂ and A^lns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides foundin vivo andin vitro. The size heterogeneity of the A₂-mRNA from most mammals examined is due to the variable size of the poly(A) tail.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

The minimal polymorphism of class II Eα chains is not due to the functional neutrality of mutations

Given the extensive allelic amino acid sequence polymorphism present in the first domain of A, A, and E chains and its profound effects on class II function, the minimal polymorphism in the mouse E chain (and in its human homologue DR) is paradox. Two possible explanations for the lack of polymorphism in E are: (1) the E chain plays such a uniquely critical structural/functional role in antigen presentation, T-cell activation, repertoire selection, and/or pairing with E or other proteins for expression that it cannot vary, and mutations are selected against; (2) the E chain plays a less significant role than the outer domains of other major histocompatibility complex (MHC) proteins in determining the interactions with processed peptides or with T-cell receptor (TCR), so there is no selective pressure to maintain new mutations. To explore this question we compared the ability of transfectants expressing wild type (wt) EE ^d and mutant E wt E ^d proteins to present peptides and bacterial superantigens to T-cell hybridomas. Mutations at the E amino acid positions 31, 52, and 65&66, to residues that represent allelic alternatives in A chains, significantly reduced activation of peptide-specific T hybridomas, and mutations at 71 sometimes enhanced T-cell stimulation. None of the E mutations reduced, and some enhanced, superantigen stimulation of T-cell hybridomas. These results argue against the hypothesis that E chains are minimally polymorphic because mutations in E are functionally neutral.     

Localisation régionale des gènes humains LDHB,TPI, ENO2, PepB,PGK, αGALA,G6PD et HGPRT par l'hybridation cellulaire interspécifique

Summary Twenty-two independent man-mouse (A9, HGPRT^-) and manhamster (CH, HGPRT)-hybrids using male human with balanced reciprocal translocation XY t(X;12)(q2,3;q1,2) were analysed for human genes localized on chromosome 12 (LDH_B, PepB, ENO₂, TPI), on chromosome X (PGK, GAL_A, G6PD) and for human chromosomes in relation with the balanced reciprocal translocation (chromosome 12, 12q^-, Xq⁺). The human chromosome 12q^- was not analysed because of its morphology is similar to some rodent chromosomes.The following results were obtained:1.Man-mouse hybrids (12 independent hybrids):The chromosome 12 is absent. A positive correlation is observed between Xq⁺, PGK, and PepB. Four hybrids are Xq⁺+PGK+PepB+ and eight hybrids are Xq⁺-PGK-PepB-This result indicate that the genes for human PGK, PepB are on the chromosome Xq+. 2.Man-hamster hybrids (10 independent hybrids):A positive correlation is observed between Xq⁺, PGK, GAL_A. Two hybrids are Xq⁺+PGK+GAL_A+ and eight hybrids are Xq⁺-PGK-GAL_A-.A positive correlation is observed between Xq⁺, PGK, GAL_A, PepB with the seven hybrids missing the normal human chromosome 12. One hybrid is Xq⁺+PGK+GAL_A+PepB+ and six hybrids are Xq⁺-PGK-GAL_A-PepB-. These results indicate that the genes for human PGK, GAL_A, PepB are on the chromosome Xq⁺. 3.Man-mouse and man-hamster hybrids:The highest percentage of presence in the hybrids is observed for the following markers: G6PD (100%), LDH_B, TPI, ENO₂ (90%). This is explained by the fact that these hybrids selected in HAT medium, had to retain a segment of X (Xq⁺ or 12q^-) bearing the human HGPRT gene. The different results indicated that the segment of X retained in these hybrids must be on the 12q^- and not on the Xq⁺, for Xq⁺ is rarely present in man rodent hybrids.The different results implicate finally the following localisations:LDH_B, TPI, ENO₂ on 12 q12 12pter,PepB on 12 q12 12qter;PGK, GAL_A on X q23 Xpter;HGPRT, G6PD on X q23 Xqter.     

Functional Coupling of G Proteins to Endothelin Receptors Is Ligand and Receptor Subtype Specific

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins.2. Coupling between endothelin A (ET_A) or endothelin B (ET_B) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein -subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein -subunits.3. Unstimulated ET_A and ET_B receptors (ET_AR and ET_BR, respectively) were barely coupled to G_o. The unstimulated ET_AR coimmunoprecipitated with G_i3, whereas the unstimulated ET_BR was much less strongly coupled to G_i3. The coupling of ET_BR to G_i1G_i2 -subunits was much stronger than the coupling of ET_AR to these -subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET_BR to G_i3, whereas coupling of the ET_AR to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET_AR to G_q/G₁₁ than in the coupling of ET_BR to these -subunits. Ligand-induced coupling between the receptors and the G_i1 and G_i2 -subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to G_o, except that ET-3 increased the coupling of this -subunit to ET_BR and decreased the coupling to ET_AR. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific.4. It remains to be established whether this diversity of receptor–G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.     

Parameters involved in the enhancement of monoclonal antibody targeting in vivo with recombinant interferon

Summary The effects of recombinant human leukocyte (clone A) interferon (rHu-IFN-A) were investigated on the expression of monoclonal antibody (MAb)-defined tumor antigens expressed on human mammary and colon carcinomas. The rHu-IFN-A treatment substantially increased the localization of radiolabeled MAb B6.2-F(ab)₂ to the transplantable Clouser human mammary carcinoma, as well as to the moderately differentiated human colon xenograft WiDr, when grown as s.c. tumors in athymic mice. In contrast, human tumor cell lines (i.e., LS174T, A375, etc.) that were unresponsive to the antigen-augmenting ability of rHu-IFN-A in vitro were also unresponsive in vivo, indicating a possible method of screening carcinoma cell populations for subsequent rHu-IFN-A adjuvant therapy prior to MAb administration. The method of delivery of rHu-IFN-A was also studied. The i.m. route resulted in a 3–4 h plasma half-life for rHu-IFN-A. The administration of rHu-IFN-A via an osmotic pump resulted in a stable circulating plasma titer of 400–800 antiviral units/ml for 7 days. Utilizing delivery of rHu-IFN-A by the constant infusion route, it was found that the increase in localization of ¹²⁵I-B6.2-F(ab)₂ was dependent on (1) the length of time of treatment and (2) the circulating plasma rHu-IFN-A levels. These results thus provide information useful for subsequent studies to determine the potential efficacy of adjuvant rHu-IFN-A treatment for MAb-targeted tumor diagnosis and treatment.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (³J_HN) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CH region of ¹H 2D NMR spectra. Quantitative determination of all possible ³J_HN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining ³J_HN from NOESY spectra. Because ³J_HN may be determined from 2D peaks outside of the CH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that ³J_HN can be obtained for every residue in the helical peptide Ac-(AAAAK)₃A-NH₂. The resulting ³J_HN profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.Abbreviations ³J_HN three-bond CH-NH scalar coupling constant - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser spectroscopy - COSY two-dimensional correlated spectroscopy - DQF-COSY two-dimensional double-quantum-filtered correlated spectroscopy - TOCSY two-dimensional total correlation spectroscopy To whom correspondence should be addressed.Deceased March 5, 1996.