Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Glutamate decarboxylase from barley embryos and roots. General properties and the occurrence of three enzymic forms.

Glutamate decarboxylase in extracts of barley has a Km value for L-glutamate of 22 mM and is activated by the addition of pyridoxal phosphate by up to 3.5 times. Sucrose-density-gradient experiments indicate the presence of two enzyme forms with molecular weights 256000 and 120000. The lower-molecular-weight form appears to be relatively inactive and spontaneously associates to the higher-molecular-weight form on storage. The enzyme is inhibited by thiol reagents and the distribution of activity on density gradients is altered in favour of the lower-molecular-weight form by the presence of 2-mercaptoethanol. After removal of the 2-mercaptoethanol spontaneous association to the higher-molecular-weight form occurs. The presence of oxygen in the extraction buffer and in the water during imbibition leads to a relative increase in the higher-molecular-weight form compared with situations where oxygen is excluded. In contrast, glutamate decarboxylase in extracts of 3-day-old barley roots has a Km value for L-glutamate of 3.1 mM and is activated up to 10% by addition of pyridoxal phosphate. The root enzyme occurs as a single species with molecular weight 310000 and this is unaffected by 2-mercaptoethanol although thiol reagents do act as weak inhibitors. The molecular weight is also unaffected by the presence or absence of oxygen in the extraction buffers.     

Purification and properties of Oxidized and reduced forms of Hepatic fructose 1,6-bisphosphatase

D-Fructose 1,6-bisphosphate 1-phosphohydrolase (EC 3.1.3.11) was isolated from rat liver in two forms: "A," isolated in the presence, and "B," isolated in the absence of dithiothreitol. Both forms had an apparently identical molecular weight of approximately 37,000/subunit and the same Km for fructose 1,6-bisphosphate of 2 microM. However, the Ki of the AMP inhibition of form A was 140 microM and of form B, 370 microM. With form B the same inhibition as with form A was reached by incubating the enzyme with dithiothreitol. The two forms of the enzyme differed in their total, as well as in their number of fast reacting thiol groups. Form A was the more reduced form, exhibiting 22.4 thiol groups/molecule, 2.5 of them fast reacting with 5,5'-dithiobis-(2-nitrobenzoic acid). Only 0.5 fast reacting groups and a total of 19.2 were found with form B. The fast reacting thiol groups disappeared when assayed in the presence of AMP. It is suggested that a redox reaction alters a site that influences the inhibitory action of AMP, so as to regulate the activity of fructose 1,6-bisphosphatase.     

An alkaline proteinase which has inflammatory activity isolated from guinea pig lymphoid cells

A phosphate buffer extract of regional lymph node cells (more than 90% lymphocytes) of guinea pigs immunized with bovine immunoglobulin G contained acid and alkaline proteinase activities. The immunized cells contained approximately twice as much proteinase activities than the normal cells. The alkaline proteinase was purified and two forms could be separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one form labeled with [³H]diisopropylfluorophosphate was localized at M_r 28 000 in a single peak in the presence and absence of reducing agent. The enzyme was inhibited by diisopropyl-fluorophosphate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetic acid, and was also partially inhibited by phenylmethylsulfonyl fluoride. It was relatively heat-stable. Intradermal injection of the purified form into normal guinea pigs induced a mixed neutrophil-mononuclear infiltrate. The immune reaction was reduced in intensity with the diisopropylfluorophosphate-inhibited enzyme. These findings suggests that the proteinase probably participates in the inflammatory response of cellular hypersensitivity.     

β-N-Acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia

The spleen from a patient with hairy-cell leukaemia had β-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an ‘extra’ form that accounted for 15% of total activity. The ‘extra’ form hydrolysed the synthetic substrate 4-methylumbelliferyl-β-N-acetylglucosamine 6-sulphate, indicating the presence of α-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the ‘extra’ form is entirely composed of α-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.     

Phylogenetic affinities of an enigmatic nannoplankton,Braarudosphaera bigelowii based on the SSU rDNA sequences

The phylogenetic position of an enigmatic calcareous nannoplankton Braarudosphaera bigelowii, whose taxonomic position has long been questioned, was investigated using molecular genetic methods. The SSU rDNA sequences of B. bigelowii were obtained using a single cell PCR technique independently from two single cells isolated from field samples. A BLAST search revealed that the SSU rDNA of B. bigelowii was most similar to those of the division Haptophyta. Subsequent phylogenetic analyses showed that B. bigelowii was included in a clade comprising members of the orders Isochrysidales and Coccolithales, the genus Chrysoculter and two unidentified haptophytes. Morphometric measurements of extant B. bigelowii populations were made to obtain precise information on their intraspecific size variation. The results showed that these populations could be subdivided into a small form (< 2.4 μm), an intermediate form A (4.0–5.3 μm), and an intermediate form B (5.3–7.2 μm), although the borders of the latter two forms overlapped. The SSU rDNA sequences showed the presence of 10-bp substitutions plus six insertions/deletions between specimens of intermediate forms A and B. The levels of DNA variation between them suggested that these two forms are genetically distinct from each other.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

Metabolism of L-beta-lysine in a Pseudomonas: conversion of 6-N-acetyl-L-beta-lysine to 3-keto-6-acetamidohexanoate and of 4-aminobutyrate to succinic semialdehyde by different transaminases.

Some kinetic properties of two new species of transaminase found in extracts of a β-lysine-utilizing Pseudomonas are reported. Transaminase A catalyzes transamination between 6-N-acetyl-l-β-lysine (3-amino-6-acetamidohexanoate) and α-ketoglutarate to form 3-keto-6-acetamidohexanoate and glutamate. Transaminase B catalyzes a reaction between 4-aminobutyrate and pyruvate to form succinic semialdehyde and alanine. The formation of both transaminases is induced by growth of the bacteria on l-β-lysine, although transaminase B is also produced in the absence of this substrate. Transaminase A requires pyridoxal phosphate for activity. The β-keto acid formed from acetyl-β-lysine by transaminase A has been purified and characterized by decarboxylation, conversion to a formazan, reduction to a stable β-hydroxy acid, and conversion of the latter to its methyl ester. Transaminase B, unlike previously reported transaminases utilizing 4-aminobutyrate, cannot use α-ketoglutarate as an amino group acceptor. This enzyme is not stimulated by addition of pyridoxal phosphate, but is inhibited by hydroxylamine or cyanide. Both transaminases appear to function in the main pathway of β-lysine degradation.     

Model complexes for the high spin iron(II) state in the catalytic cycle of cytochrome P450

Studies using ⁵⁷Fe Mössbauer spectroscopy on frozen concentrated solutions of two iron(II) porphyrins in the presence of a large excess of thiophenol or 2-mercaptoethanol are reported. The iron(II) porphyrins used were photoporphyrin IX iron(II) (PPIXFe(II)) and tetra(p-sulphophenyl)porphinato-iron(II) (TTPPSFe(II)). Evidence for high-spin five-coordinate iron(II) complexes were found for both iron(II) porphyrins with thiophenol, but no reaction was found to occur with 2-mecaptoethanol. In contrast to these findings in solutions dilute in TPPSFe(II) evidence was found (from electronic absorption spectroscopy and spectrophotometric titrations) for both thiophenol and 2-mercaptoethanol acting as axial ligands in high-spin five-coordinate TPPSFe(II) species. Mössbauer data for frozen solutions containing TPPSFe(II), carbon monoxide (CO) and either thiol, are consistent with the presence of only low-spin six-coordinate iron(II) complexes. These latter complexes are deduced to contain both a thiol and a CO molecule as axial ligands> These results are discussed in relation to earlier work on PPIXFe(II)-thiol solutions and also in relation to the high-spin iron(II) state in the catalytic cycle of cytochrome P450.The reactions of TPPSFe(II) with excess of either 2-mercaptoethanol or ethyl 2-mercaptoacetate in air are also reported and their complex nature discussed.     

Stabilization of neurotoxic soluble beta-sheet-rich conformations of the Alzheimer's disease amyloid-beta peptide

An emerging paradigm for degenerative diseases associated with protein misfolding, such as Alzheimer's disease, is the formation of a toxic species due to structural transitions accompanied by oligomerization. Increasingly, the focus in Alzheimer's disease is on soluble oligomeric forms of the amyloid-β peptide (Aβ) as the potential toxic species. Using a variety of methods, we have analyzed how sodium dodecyl sulphate (SDS) modulates the folding of Aβ40 and 42 and found that submicellar concentrations of SDS solubilize Aβ and induce structural transitions. Under these conditions, Aβ40 and 42 are interconverting oligomeric ensembles with a predominantly β-sheet structure. The Aβ42 soluble oligomers form β-sheet structures more readily and have increased stability compared with Aβ40 under identical conditions. The presence of added Cu²⁺ significantly promotes and stabilizes the formation of the soluble oligomeric β-sheet structures but these structures are nonamyloidogenic. In contrast, in the absence of added Cu²⁺, these β-sheet oligomers possess the hallmarks of amyloidogenic structures. These SDS-induced β-sheet forms of Aβ, both in the presence and absence of Cu²⁺, are toxic to neuronal cells.     

Thiophosphate analogs of ADP and ATP as substrates in partial reactions of energy conversion in chloroplasts

Thiophosphate analogs of ADP and ATP have been employed in partial reactions of photosynthetic energy conversion in chloroplasts. Substitution of oxygen by sulfur at the α-phosphate yields a pair of diastereomers (ADPαS, ATPαS, A and B forms). Two diastereomeric compounds are also obtained by replacement of oxygen by sulfur in the β-phosphate group of ATP (ATPβS, A and B form) (Eckstein, F. and Goody, R.S. (1976) Biochemistry 15, 1685–1691).The A form of ADPαS is phosphorylated by chloroplasts with a K_m comparable to that of ADP but with a lower V. The B form of ADPαS as well as ADPβS is not a substrate in photophosphorylation and only weakly competes with ADP.The A forms of ADPαS and ATPαS strongly compete with ADP for the tight nucleotide binding site of CF₁ in the light-induced exchange reaction, whereas the B forms display a much smaller competitive effect. The efficiencies of ADPβS and the A isomer of ATPβS are intermediate, and the B form of ATPβS is a weaker competitor.The A forms of ATPαS and ATPβS are hydrolyzed by light-triggered ATPase, whereas the B forms are not. The efficiency of the A isomer of ATPαS is comparable to that of normal ATP, and the A form of ATPβS is cleaved at a lower rate. In trypsin-activated Ca²⁺-dependent ATPase the A form of ATPαS is the only thiophosphate analog to be hydrolyzed.The results indicate a stereospecific interaction of ADP and ATP at the catalytic sites as well as the tight nucleotide binding site of coupling ATPase of chloroplasts.     

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2?cm^?1, that are about 40% smaller than the D-value of the wild-type protein. The ⁵⁷Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01?mm/s at 4.2?K rather than δ=0.32?mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79?mm/s at 4.2?K, consistent with tetrahedral Fe²⁺(Cys)₃(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=?7±1?cm^?1, E/D=0.09 for form A and D=+6.2±1.3?cm^?1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g _z =2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77?K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe²⁺ site. Studies in different buffers in the pH?6–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700?cm^?1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300?cm^?1; a third d-d transition, if present, must occur with energy lower than 3300?cm^?1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100?cm^?1 in form A and 5350, 6380?cm^?1 in form B.     

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma capsulatum

Chemical and ultrastructural studies of the cell walls of the yeastlike (Y) and mycelial (M) forms ofHistoplasma capsulatum G-184B revealed that the Y form contained about 46.5% ofα-glucan, 31.0% ofβ-glucan, 7.7% of galactomannan and 11.5% of chitin, whereas the M form cell wall contained about 18.8% ofβ-glucan, 24.7% of galactomannan, 25.8% of chitin, and essentially noα-glucan. Theα-glucan of the Y form contained mainly anα-(1 → 3)-linkage. Theβ-glucans of both forms may have mainly aβ-(1 → 3)-linkage. Chitin microfibrils were located mainly in the inner portion of the cell walls of the Y and M forms, whereas theα-glucan fibers were observed only in the outer portion of the Y form cell wall.     

Thiols liberate covalently bonded flavin from monoamine oxidase

Although it was recommended that 2-mercaptoethanol should be added to all buffers used in monoamine oxidase purification, purification of the enzyme in the absence of thiols has yielded double the amount of enzyme. In fact the presence of 0.1 M 2-mercaptoethanol or dithioerythritol-SDS¹ was found to liberate about 50% of the covalently bonded FAD. This observation is surprising since the covalently bonded flavin has been reported to be 8-α-cysteinyl-FAD. There is a thioether linkage between the flavin and the protein and mild reducing agents such as mercaptoethanol would not normally cleave the thioether linkage. The importance of the present finding is that the thiols may be used possibly to liberate suicide substrate-flavin adducts from the enzyme for structural studies without isolating pure flavin peptides. The flavin can be removed simply by treatment of the enzyme with 0.1 M thiol solution containing 1% sodium dodecylsulfate followed by chromatography on Sephadex G-25.     

Liver betaine-homocysteine S-methyltransferase activity undergoes a redox switch at the active site zinc

Using a redox-inert methyl acceptor, we show that betaine-homocysteine S-methyltransferase (BHMT) requires a thiol reducing agent for activity. Short-term exposure of BHMT to reducing agent-free buffer inactivates the enzyme without causing any loss of its catalytic zinc. Activity can be completely restored by the re-addition of a thiol reducing agent. The catalytic zinc of BHMT is bound by three thiolates and one hydroxyl group. Thiol modification experiments indicate that a disulfide bond is formed between two of the three zinc-binding ligands when BHMT is inactive in a reducing agent-free buffer, and that this disulfide can be readily reduced with the concomitant restoration of activity by re-establishing reducing conditions. Long-term exposure of BHMT to reducing agent-free buffer results in the slow, irreversible loss of its catalytic Zn and a corresponding loss of activity. Experiments using the glutamate-cysteine ligase modifier subunit knockout mice Gclm(−/−), which are severely impaired in glutathione synthesis, show that BHMT activity is reduced about 75% in Gclm(−/−) compared to Gclm(+/+) mice.     

Effects of the N-Substitution and Physical Form of Chitosan and the Modification of its Nonreducing End Groups on the Rates of Hydrolysis by Chitinase from Streptomyces griseus

Zymolyase was purified from Zymolyase-60,000 by ion-exchange chromatography with sugar and gel filtration. Zymolyase was separated into the two protein fractions A and B. Neither alone could lyse yeast cells, but together showed high lytic activity. Zymolyase A and B were β-1,3-glucanase and alkaline protease, respectively. On stepwise treatment of yeast cells with the enzymes, yeast cells were lysed only by treatment with Zymolyase A after pretreatment with Zymolyase B. Zymolyase A lysed yeast cells in the presence of 2-mercaptoethanol, but B could not even at high concentration. Zymolyase B decreased the turbidity of a yeast cell suspension by about 13%.     

Purification and Separation of A and B Forms of N-Acetyl-β-D-Hexosaminidase from Human Aorta

Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B₁) and 4 bands (B₂), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B form of the human enzyme, By heat inactivation we found that the aortic enzyme consisted of 51% of A and 49% of B (B₁ + B₂ .). Neuraminidase had no effect on either form of the aortic preparation. Both forms were partially purified and separated by conventional methods. They required BSA for their maximal activity; the A form being more dependent BSA than the B form, With PNP-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide, Km of 1.04 mH and 0.54 mM, respectively, for A form and of 1.74 and 1.48 mM, respectively, for B form were obtained. While the purified B form was stable and did not transform into other species, the purified A form gradually transformed into B form as well as into other new forms during storage at -20°.     

Isolation and partial characterization of carbonic anhydrase from erythrocytes of Meleagris gallopavo

Summary A soluble enzyme with carbonic anhydrase activity has been isolated from domestic turkey(Meleagris gallopavo) erythrocytes and purified by chloroformethanol precipitation, ammonium sulfate fractionation and gel filtration on a Sephadex G-75 column. Analytical polyacrylamide gel disc electrophoresis showed one major and two minor bands. The specific activity for the CO₂ hydration reaction was approximately 2000 Wilbur-Anderson units/mg protein at 0°C. The presence of the reducing agents 2-mercaptoethanol or dithioerythritol was required throughout the procedure. Upon removal of the 2-mercaptoethanol by dialysis the activity was lost but could be restored by addition of the reducing agent. The enzymatic activity was inhibited by acetazolamide,p-chloromercuribenzoate ando-iodosobenzoate. Esterase activity was detected withp-nitrophenylacetate as the substrate. The molecular weight of the enzyme was determined as 31,000 by gel filtration and 34,000 ± 2000 with analytical ultracentrifugation. Atomic absorption spectroscopy indicated the presence of zinc in the ratio of one mole of zinc per one mole of enzyme.     

Determination of d- and l-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde

A sensitive and convenient method for the simultaneous determination of d- and l-aspartic acid in amino acid mixtures is described. The method involves derivatization of the mixture with a chiral fluorogen, followed by high-performance liquid chromatography on a reverse-phase column. The fluorogen used is an adduct of o-phthaldialdehyde with an optically active thiol, N-acetyl-l-cysteine. The sensitivity and accuracy of this method is similar to that using adducts of o-pthaldialdehyde with the achiral thiol, 2-mercaptoethanol. Five picomoles of d-aspartate can be accurately detected in the presence of a 100-fold excess of l-aspartate with a total analysis time (including derivatization) of 10 min.