Triggering of affinity-enriched B cells. II. Composition and B cell triggering properties of affinity-purified, antigen-specific, T cell-derived helper factor

Antigen-specific, T cell-derived helper factor (ASHF), recognizing alloantigens on chicken red blood cells (CRBC), had previously been shown to trigger an IgM anti-CRBC response in vitro by 2 X 10(3) affinity-enriched B cells. Triggering signals for B cell proliferation were shown to be substituted by ASHF or lipopolysaccharide, and signals for differentiation to IgM production by Thy-1+ cells. In an effort to demonstrate unequivocally antigen specificity of helper factor, secretory products and cell extracts from helper T cells that had been primed by either the B2 or the B13 chicken alloantigens, were purified by antigen affinity and ion exchange chromatography. With each step of purification, antigen-specific biologic activity of ASHF-containing material increased such that ASHF from B2, but not B13, primed helper T cells would help trigger a B2, but not a B13, -specific response and vice versa. Gel filtration of ASHF suggests it to have a m.w. of at least 50,000. Absorption studies showed that ASHF binds to B cells in the absence of antigen. Delivery of the triggering signal requires ASHF to bind simultaneously to both, the cell surface and the antigen. ASHF consists of at least two subunits that may be separated from each other by chelating agents. This yields two biologically inactive fractions, only one of which binds to the nominal antigen. Upon recombining them in the presence of Ca++, full biologic activity is restored.     

Localization of gamma-glutamyl transpeptidase in human sweat glands: an immunohistochemical study using a monoclonal antibody

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.     

Selectin GMP-140 (CD62; PADGEM) binds to sialosyl-Le(a) and sialosyl-Le(x), and sulfated glycans modulate this binding.

GMP-140 (CD62; PADGEM) is a member of the selectin family expressed highly at the surface of platelets and endothelial cells by agonists such as thrombin or phorbol esters. Previous studies indicate that the lectin domain of GMP-140 recognizes sialosyl-Le(x) (SLex) and to a lesser extent Le(x) (Polley MJ, et al., Proc Natl Acad Sci USA 88:6224, 1991). We now report that GMP-140 binds to sialosyl Lea (SLea) and to SLex, and that degree of binding to SLea is greater than that to SLex under our experimental conditions. Binding of activated platelets to SLea or SLex was inhibited to various degrees in the presence of sulfated glycans, suggesting that sulfated glycans induce conformational change in the lectin domain of GMP-140 and modulates its binding affinity to SLea and SLex.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.     

A study of the double mutation of dnaJ and cbpA, whose gene products function as molecular chaperones in Escherichia coli.

The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli. To gain insight into the function of CbpA, we examined the nature of a cbpA null mutation with special reference to those of dnaK and dnaJ null mutations. In particular, the cbpA dnaJ double-null mutant was found to exhibit severe defects in cell growth, namely, a very narrow temperature range for growth, a defect in cell division, and susceptibility to killing by carbon starvation. These phenotypes are very similar to those reported for dnaK null mutants but not to those of dnaJ null mutants. Our results are best interpreted by assuming that CbpA is capable of compensating for DnaJ for cell growth and thus that the function(s) of CbpA is closely related to that of DnaJ.     

Dual metabolic pathway of 25-hydroxyvitamin D3 catalyzed by human CYP24.

Human 25-hydroxyvitamin D3 (25(OH)D3) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D3, 1alpha,25(OH)2D3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D3 and 1alpha,25(OH)2D3, whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada, N. Nonaka, Y. Ohyama, Y. & Inouye, K. (1999) Eur. J. Biochem. 262, 43-48]. HPLC analysis and mass spectrometric analysis revealed that human CYP24 catalyzed all the steps of the C-23 hydroxylation pathway from 25(OH)D3 via 23S, 25(OH)2D3, 23S,25,26(OH)3D3 and 25(OH)D3-26,23-lactol to 25(OH)D3-26, 23-lactone in addition to the C-24 hydroxylation pathway from 25(OH)D3 via 24R,25(OH)2D3, 24-oxo-25(OH)D3, 24-oxo-23S,25(OH)2D3 to 24,25,26,27-tetranor-23(OH)D3. On 1alpha,25(OH)2D3 metabolism, similar results were observed. These results strongly suggest that the single enzyme human CYP24 is greatly responsible for the metabolism of both 25(OH)D3 and 1alpha,25(OH)2D3. We also succeeded in the coexpression of CYP24, adrenodoxin and NADPH-adrenodoxin reductase in E. coli. Addition of 25(OH)D3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs.     

Age-related variations in glycosylation of hydroxylysine in human and rat skin collagens.

The extent of glycosylation of hydroxylysine in human skin collagen rapidly decreased during maturation and then gradually increased in proportion to the age. This decrease of glycosylation observed during maturation was also confirmed in whole, soluble and insoluble collagens from rat skin. These findings may contribute to the investigations on the functional role of glycosylation and also on the mechanism of maturational as well as senile processes.     

Induction of cytotoxic lymphocyte responses by antigen-specific helper factors

Antigen-specific helper factors (ASHF) were purified by antigen-affinity chromatography from supernatants of long-term helper T lymphocyte (TH) lines. We have modified an established helper-dependent assay system to demonstrate the antigen specificity and H-2 restriction properties of ASHF in the induction of cytotoxic T lymphocyte precursors (CTLp). Antigen specificity is demonstrated by the binding of ASHF molecules only to nominal antigen, both during purification and in tests of functional activity. Our ASHF preparations do not contain any interleukin 2 (IL 2) activity. The ASHF, purified by antigen-affinity chromatography in the presence of Ca++, is defined as Ca++-sufficient ASHF, whereas ASHF purified on antigen-affinity columns in the absence of Ca++ is defined to be Ca++ deficient. Ca++-sufficient ASHF is not H-2 restricted (as defined by the phenotype of the ASHF-producing cells) in the recognition of nominal antigen or in its interactions with CTLp or adherent stimulator cells. In contrast, when the "complete" (Ca++-sufficient) ASHF is functionally dissociated into subunits by removal of Ca++, the "incomplete" antigen-specific subunit of ASHF (Ca++-deficient ASHF) is still H-2-unrestricted in its ability to bind nominal antigen, but requires products from syngeneic adherent cells to trigger CTLp. When adherent cells that are H-2 identical to the ASHF are provided in culture, the "incomplete" ASHF is able to trigger either syngeneic or allogeneic CTLp in an antigen-specific manner. We interpret the results of our experiments to suggest that an H-2-restricted molecular interaction occurs in CTLp induction by ASHF. An antigen-specific, TH-derived receptor appears to require association with Ca++ and self major histocompatibility complex (MHC)-encoded molecules to form a "complete" ASHF that is able to trigger CTLp in an apparently H-2-unrestricted manner.