Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.     

Preparation and purification of γγ enolase (neuron-specific enolase) using high performance anion exchange chromatography

A simple and rapid method, using only two chromatographic steps, is described for the purification and preparation of enolase isoenzymes from human and beef brain extracts. In the first step, a crude enolase was obtained by chromatography on Q-Sepharose Fast Flow column. The crude fraction was then purified by high performance anion exchange chromatography on a Mono-Q column. enolase obtained in this manner was shown to be homogeneous by two dimensional polyacrylamide gel electrophoresis and by high performance gel permeation chromatography. The yield of enolase by this method was 7–8 mg of pure enzyme per 100 g of brain.     

The NGF complex from the African ratMastomys natalensis

The 7S NGF complex from the male mouse submaxillary gland consists of the , and subunits in the ratio ₂₂. The (NGF) subunit contains all the known biolocial activity of 7S NGF. The and subunits are both members of glandular kallikrein gene family, yet only subunit has protease activity. The subunit plays a role in the processing of preproNGF to its mature form, while the role of the subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the or subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the ratMastomys natalensis in order to more fully understand the roles of the and subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio ₂. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.Special issue dedicated to Dr. Lawrence Austin     

γ-Glutamylamine cyclotransferase

Summary -Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of -(L--glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L--glutamylamines. Among its substrates are both the mono- and di--glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of -(L--glutamyl)-L-lysine in which both the -amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most -glutamyl--amino acids, nor is it active toward the -lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of -(L--glutamyl)-L-lysine in which the -amino or the -carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of -glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which -glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.     

Purification and immunocytochemical localization of kafirins inSorghum bicolor (L. Moench) endosperm

Summary Kafirins are the storage proteins of sorghum and are found in protein bodies in the seed endosperm. They have been classified as -, -, and -kafirins according to differences in molecular weight, solubility, and structure. The kafirins were purified, amino acid composition was determined, and immunolocalization methods were used to determine the organization of the protein bodies and distribution of kafirins throughout the endosperm. All three groups of kafirins were low in lysine. -Kafirins and -kafirins were relatively high in cysteine, and -kafirins were relatively high in methionine. Transmission electron microscopy showed that protein bodies in the peripheral endosperm were spheroid with concentric rings and few darkly stained inclusions. In contrast, protein bodies of the central endosperm were irregularly shaped with a higher proportion of darkly stained material. The light staining regions of the protein bodies are composed primarily of -kafirins with minor portions of - and -kafirins. The dark staining regions, however, are composed primarily of - and -kafirins. Immunoelectron microscopy showed that protein bodies in the peripheral endosperm contain predominantly a-kafirin with minor amounts of - and -kafirin. Central endosperm protein bodies are also predominantly -kafirin, but have a higher proportion of -kafirin and -kafirin than the peripheral endosperm protein bodies.Abbreviations GAR-HRP Goat anti-rabbit horseradish peroxidase - IgG immunoglobulin G - 2-ME 2-mercaptoethanol - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffer saline - TBS-T Tris buffer saline with Tween - TBS-T-B Tris buffer saline with Tween and bovine serum albumin - TCA trichloroacetic acid - UV ultraviolet     

AViscum album oligosaccharide activating human natural cytotoxicity is an interferon γ inducer

Summary CommercialViscum album extract Helixor-M contains a dialysable oligosaccharide (HM-BP) that activates natural killer (NK) cytotoxicity against K562 tumour cells when preincubated with human peripheral blood mononuclear cells (PBMC) for 72 h. The activated effector cells were exclusively found in the monocyte/macrophage subpopulation. However, when peripheral non-adherent cells (PNAC) were preincubated with HM-BP for 72 h the NK cytotoxicity of CD56⁺CD3^– NK cells was activated. This discrepancy was found to be due to the release of prostaglandin E₂ from activated monocytes/macrophages, which blocked activation of the cytotoxicity of NK cells. Analysis of the supernatant culture medium after 72 h preincubation demonstrated that HM-BP induced release of interferon (IFN) from T cells (preferentially from CD3⁺CD4⁺ cells) and of tumour necrosis factor (TNF) from monocytes/macrophages. Release of IFN was the crucial step for activation of NK cytotoxicity since enhancement of NK cytotoxicity during pretreatment of PBMC or PNAC with HM-BP was completely blocked in the presence of anti-IFN antibodies. Anti-interleukin-2, anti-TNF or anti-IFN antibodies had no effect on the HM-BP-induced enhancement of NK cytotoxicity. The activation of the NK cytotoxicity of nonadherent cells by interleukin-2 treatment was found to be synergistic to the enhancement of NK cytotoxicity by treatment with HM-BP.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at Gly-149 was suppressed by the second mutations at the outer surface of the subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that / and / subunits interactions are important for the energy coupling between catalysis and H⁺ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

The levels of ζ, γ, and δ chains in patients with Hb H disease

Summary Details are given of a study of blood samples from 24 patients with Hb H disease from different Mediterranean countries and from the Far East. Four different types of -thal-1 (--) were observed, namely-() ( 20.5-kb deletion);--MED-I ( 17.5-kb deletion);--MED-II (>26.5-kb deletion); and--SEA ( 18-kb deletion, in Orientals only). The -thal-2 was mainly of the deletion type (16 with the 3.7-kb deletion; 1 with the 4.2-kb deletion), while 4 of the 7 patients with a nondeletional type had the five-nucleotide deletion at the donor splice site of the first intron of the 2 gene. All patients had a mild-to-moderate hemolytic anemia; no significant differences in hematology were observed between the groups. Hb A₂ was decreased to about one-third of the normal level. The Hb H formation varied considerably and its quantitation was not always satisfactory. Patients with Hb H disease due to any -thal-1 combined with a nondeletional -thal-2 had the highest Hb H levels and a more marked anemia. The chain production was small and absent in patients with the MED-II type of -thal-1 because this deletion included the and genes. The highest chain levels were present in the four patients with the SEA type of -thal-1. The chain production was increased, particularly in patients with a mutation of C T at position-158 to the ^G globin gene. This chain was primarily present as Hb Bart's (or ₄) and only about 15% was recovered as Hb F or ₂₂. The evaluation of the rate of chains produced in these patients was greatly facilitated by data from one patient who had Hb H disease and a heterozygosity for the ^A-⁺. The low levels of Hb A₂ and of Hb F (relative to Hb Bart's) can be explained by a decreased affinity of chains for and chains as compared with chains in conditions of severe chain deficiency.     

Theoretical studies on the necessary number of components in mixtures

Summary Theoretical studies on the necessary number of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to yielding ability. All theoretical investigations are based upon a Gram-Charlier frequency distribution of the component means with skewness ₁ and kurtosis ₂. The selected fraction p of the best components constitutes the mixture under consideration. The same selection differential S = S (p, ₁, ₂) can be realized by different parameter values of p, ₁ and ₂. Therefore, equal yield levels of the mixture can be achieved by different selected fractions p which implies different numbers of components in the mixture. Numerical results of S = S(p) for different values of ₁ and ₂ are presented and discussed. Of particular interest are the selected fractions p which lead to a maximal selection differential S. These results on S for large populations must be reduced in the case of finite population size. For this correction term we used an approximation B = B (p, n, ₁, ₂) given by Burrows (1972) where n = number of selected components. For given parameter values of ₁, ₂ and p, the necessary number n of components can be calculated by using the condition: Burrows-correction less than a certain percentage g of S — for example with g = 0.05 or g = 0.01. For given ₁ and ₂, the number n leading to a maximal selection differential S can be regarded as necessary number of components (necessary = maximum gain of selection under the given conditions). Numerical results are given for ₂ = 0 and for eight situations which are defined by linear relations ₂ = c ₁ between skewness and kurtosis. These cases will contain all possible numerical situations for ₁ and ₂, which may be relevant for practical applications. The necessary number of components turns out to be nearly independent of the numerical value of the kurtosis ₂. The n-intervals leading to selected fractions p from 0.01 to 0.20 approximately are: 2 n 4 for g = 0.05, 6 n 20 for g = 0.01 and 11 n 40 for g = 0.005, respectively. However, percentages g less than 0.01 would be unrealistically excessive. Therefore, following the assumptions and restrictions given in this paper one may conclude that n = 20 seems to be an appropriate upper bound for the necessary number of components in mixtures.     

Syntheses ofγ-fluoro-α-amino acids

Summary Methods for the synthesis of racemic and optically active title compounds are presented. Key step of these four-step procedures is the alkylation with 1-bromo-2-fluoroalkanes of glycine-ester-derived imines in anhydrous medium using lithium diisopropylamide as a base at low temperature or phase transfer catalyzed alkylation with 50% NaOH and triethylbenzylammoniumchloride as the phase transfer catalyst, respectively. Subsequent three-step deprotection gave the free acids in 13–33% overall yield. Deracemization of-fluoro--aminobutyric acid methyl and ethyl esters with-chymotrypsin was shown to give the (–)-enantiomers of the esters and (+)--fluoro--aminobutyric acid in >98% ee, while from thetert-butylester the opposite stereochemical result was observed giving the (–)-acid with 88% ee. Optically active-fluoro--amino acids were synthesized alternatively by phase transfer catalysis with N-benzyl-cinchonium chloride or using an auxiliary-directed asymmetric alkylation of the imine derived from (R)-(+)-camphor or (R)-(+)-2-hydroxypinan-3-one. These processes gave different enantiomers of-fluoro--aminobutyric acid via a monomeric lithium enolate in the first or a dimeric lithium enolate in the second case, respectively. The enantiomeric excess can be improved by lithium/magnesium exchange.     

Lindane degradation by cell-free extracts of Clostridium rectum

For lindane degradation, a cell suspension of Clostridium rectum strain S-17 demands the addition of substrates such as leucine, alanine, pyruvate, a leucine-proline mixture, and molecular hydrogen. In the presence of leucine-proline mixture, lindane decomposed in parallel with isovaleric acid formation, and both lindane degradation and isovaleric acid formation were inhibited by monoiodoacetic acid, suggesting a close relation between lindane degradation and the Stickland reaction. Lindane was degraded by cell-free extracts of C. rectum in the presence of dithiothreitol (DTT). Radiogaschromatograms of n-hexane soluble metabolites from [¹⁴C] lindane showed the presence of monochlorobenzene and -3,4,5,6-tetrachlorocyclohexene, Leucine, NADH, and NADPH were somewhat less active than DTT for lindane degradation in cell-free extracts. Reductive dechlorination seemed the major route of lindane degradation in cell-free extracts as well as in the intact cells of C. rectum.Abbreviations Lindane (-HCH) -1,2,3,4,5,6-hexachlorocyclohexane - -HCH -1,2,3,4,5,6-hexachlorocyclohexane - -TCH -3,4,5,6-tetrachlorocyclohexene - -PCH -1,3,4,5,6-pentachlorocyclohexene - DTT 1,4-dithiothreitol     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

Phosphorylation of protein Phosphatase-1 isoforms by cdc2-cyclin B in vitro

Protein Phosphatase-1 is phosphorylated in vitro by cdc2-cyclin B (Villa-Moruzzi, FEBS Lett 304: 211-215, 1992). In the present study we show that all the three Phosphatase-1 isoforms, , 1, , are phosphorylated by cdc2-cyclin B. Phosphorylation is specific for this kinase and involves a C-terminal Thr. This site is most likely Thr 320 in a (shown by others to be phosphorylated also by cdc2-cyclin A). Such Thr is conserved in 1, and in the testis-specific 2, and is the only Thr that fits the cdc2-consensus sequence in the C-terminal region. Phosphorylation of Phosphatase-1 purified from skeletal muscle, which is a mixture of the a, 1 and isoforms, is up to 0.4 mol/mol and induces 30-35% enzyme inactivation. Following tryptic proteolysis each isoform yields a distinct phosphopeptide map. This is in agreement with the different sequences of the isoforms in the C-terminal regions and may be useful to distinguish the isoforms lts suggest that all the Phosphatase-1 isoforms may be potentially regulated at M-phase.     

Theoretical studies of biliverdin: energetics of the reduction pathways to bilirubin

Geometries and energies of formation of bilirubin formed by reduction of biliverdin via three meso carbon sites, the , and positions, have been calculated using semiempirical methods. It has been shown that -bilirubin with a ridge-tile conformation forms six intramolecular hydrogen bonds and is the most stable of the three above mentioned positions by at least 22 kcal mol^–1. Reduction pathways for -, - and -bilirubin formations from biliverdin are studied in detail. The roles of loss of conjugation and hydrogen bond formations in stability of different conformers have been discussed. -Bilirubin was fully optimized by using ab initio methods. Fine refinements of calculated results show excellent agreement with experimental results. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0078-9.Electronic Supplementary Material available.     

Induction of TH1- and TH2-associated cytokine mRNA in mouse bladder following intravesical growth of the murine bladder tumor MB49 and BCG immunotherapy

Productive immunity to murine and human parasites is associated with the development of a type I T cell response (interferon--producing) while type II responses (interleukin-4-producing) suppress the development of delayed-type hypersensitivity (DTH) and the elimination of the parasite. To determine if a similar regulatory pathway might exist in tumor systems and may be effected by immunotherapeutic manipulation, we have studied the localized cytokine response to the murine bladder tumor MB49 growing intravesically in syngeneic mice. Intravesical growth of MB49 results in the host-derived expression of mRNA for both interleukin-4 (IL-4) (TH2) and interferon (IFN) (TH1), as well as tumor necrosis factor (TNF) expression of indeterminate origin. Intravesical instillation of bacillus Calmette-Guérin (BCG), highly effective in eliminating bladder tumors clinically and in experimental systems, results in IFN and TNF mRNa production in the bladder wall, but no IL-4. Following BCG treatment of intravesical MB49, the number bladders expressing IL-4 mRNA decreases, while IFN and TNF expression remains constant. These results are consistent with the mechanism of action of BCG involving the generation of an enhanced TH1 immune milieu in the bladder wall, which may contribute to the generation of productive tumor-specific immunity.Supported by USPHS grant CA-42908. K.M.M. is the recipient of Foerderer and N. S. E. Predoctoral Fellowships. Presented in part at the American Association for Cancer Research, April 1993 and May 1994     

Multiple Phosphorylation Sites and Quaternary Organization of Guanine-Nucleotide Exchange Complex of Elongation Factor-1 (EF-1βγδ/ValRS) Control the Various Functions of EF-1α

The eukaryotic guanine-nucleotide exchange factor commonly called elongation factor-1 (EF-1), comprises four different subunits including valyl-tRNA synthetase (EF-1/ValRS). The factor is multiply-phosphorylated by three different protein kinases, protein kinase C, casein kinase II and cyclin dependent kinase 1 (CDK1). EF-1/ValRS is organized as a macromolecular complex for which we propose a new structural model. Evidence that EF-1/ValRS is a sophisticated supramolecular complex containing many phosphorylation sites, makes it a potential regulator of any of the functions of its partner EF-1, not only involved in protein synthesis elongation, but also in many other cellular functions.