The cellular and molecular regulation of 1,25(OH)2D3 production

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Features of target cell lysis by class I and class II MHC-restricted cytolytic T lymphocytes

The lytic activity of influenza virus-specific murine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), we found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism.     

Characterization of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Modulation of beta-adrenergic response in rat brain astrocytes by serum and hormones

Purified astrocyte cultures from neonatal rat cerebrum respond to isoproterenol, a beta-adrenergic agonist, with a transient rise in cAMP production. This astroglial property was regulated by serum, a chemically defined medium (serum-free medium plus hydrocortisone, putrescine, prostaglandin F2 alpha, insulin, and fibroblast growth factor) and epidermal growth factor. Compared to astrocytes grown in serum-supplemented medium, astrocytes grown in the chemically defined medium were nonresponsive to isoproterenol stimulation, and this difference did not appear to be due to selection of a subpopulation of cells by either medium. The data suggest that a decreased beta-adrenergic receptor number and an increased degradation of cAMP may account for the reduced response to beta-adrenergic stimulation. The nonresponsive state of astrocytes in the defined medium was reversible when the medium was replaced with serum-supplemented medium. An active substance(s) in serum was responsible for restoring the responsiveness of astrocytes. Each of the five components of the defined medium had little effect by itself; however, together they acted synergistically to desensitize astrocytes to beta-adrenergic stimulation. On the other hand, epidermal growth factor, a potent mitogen for astrocytes, was very competent by itself in reducing the cAMP response of astrocytes to beta-adrenergic stimulation. Thus purified astrocytes grown in the chemically defined medium appear to be a good model for the study of hormonal interactions and of serum factors which may modulate the beta-adrenergic response.     

Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.     

The metabolic transformations of columbinic acid and the effect of topical application of the major metabolites on rat skin

The metabolism of columbinic acid by various fatty acid oxidizing enzyme systems was studied. A cyclooxygenase product, 9-hydroxy-(5E,10E,12Z)-octadecatrienoic acid, was formed nearly quantitatively by ram seminal vesicle microsomes and in small amounts by washed human platelets. The major lipoxygenase product from washed human platelets, soybean lipoxygenase, and neonatal rat epidermal homogenate was 13-hydroxy-(5E,9Z,11E)-octadecatrienoic acid, although lesser quantities of other isomers differing in the double bond configurations were also identified by ultraviolet spectrophotometry and gas chromatography-mass spectroscopy. Topical application of the major lipoxygenase product to paws of essential fatty acid-deficient rats resulted in nearly as complete resolution of the scaly dermatitis as did the application of columbinic acid itself; the cyclooxygenase product was not at all effective.     

Arachidonic acid metabolic pathway of the rabbit placenta

Placenta microsomes prepared from animals late in gestation (29 days) efficiently metabolize arachidonic acid into PGE2, PGF2 alpha, PGD2, TxA2 and little or no prostacyclin. In contrast to the late gestation placenta, the early (17 day) placental microsomes synthesize primarily PGE2. The cytosolic (100,000 X g supernatant) fraction from early or late gestation placentae converted arachidonic acid, with a calcium dependent enzyme, into non-polar metabolites whose synthesis was inhibited by ETYA but not indomethacin. These metabolites were purified by HPLC and GC-MS analysis indicated the presence of 12-hydroxy-, 15-hydroxy-, and 11-hydroxy-eicosatetraenoic acid. The mitochondrial (8,000 X g pellet) produced PGE2; PGF2 alpha; 12-, 11-, 15-HETE; the C-17 fragment HHT; and the unusual cyclooxygenase metabolite 15-keto-PGE2. These biologically active metabolites may play a vital role in the reproductive function of the placenta.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.