Intestinal synthesis of 24-keto-1,25-dihydroxyvitamin D3. A metabolite formed in vivo with high affinity for the vitamin D cytosolic receptor

24-Keto-1,25-dihydroxyvitamin D3 has been identified as an intestinal metabolite of 1,25-dihydroxyvitamin D3 by ultraviolet absorbance, mass spectroscopy, and chemical reactivity. The metabolite was produced from 1,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3 in rat intestinal mucosa homogenates. 24-Keto-1,25-dihydroxyvitamin D3 is present in vivo in the plasma and small intestinal mucosa of rats fed a stock diet, receiving no exogenous 1,25-dihydroxyvitamin D3, and in the plasma and small intestinal mucosa of rats dosed chronically with 1,25-dihydroxyvitamin D3. 24-Keto-1,25-dihydroxyvitamin D3 has affinity equivalent to 1,24R,25-trihydroxyvitamin D3 for the 3.7 S cytosolic receptor specific for 1,25-dihydroxyvitamin D3 in the intestine and thymus. In cytosolic preparations contaminated with the 5 S vitamin D-binding protein, both metabolites are about 7-fold less potent than 1,25-dihydroxyvitamin D3. In contrast, in cytosolic preparations largely free of the 5 S binding protein, both metabolites are equipotent with the parent compound. No evidence was obtained supporting a substantial presence of 23-keto-1,25-dihydroxyvitamin D3 in vivo; nor was the latter compound generated in detectable amounts from 1,25-dihydroxyvitamin D3 by intestinal homogenates. Thus, C-24 oxidation is a significant pathway of intestinal 1,25-dihydroxyvitamin D3 metabolism that produces metabolites with high affinity for the cytosolic receptor which mediates vitamin D action.     

Prognosis of non-Hodgkin's lymphomas in the Dresden district with special reference to combined radio-/chemotherapy

The total population affected with non-Hodgkin's lymphoma and treated by means of radiotherapy or combined radio-chemotherapy between 1960 and 1985 at the Medical Academy Dresden was analysed as to prognosis. 247 patients were classified according to the previous German scheme, 79 were subdivided on the basis of the recommendations laid down in the Kiel classification. The remission rates and survival curves achieved will stand out the comparison with international literature (remission rates of the low malignancy group amounted to 85.3 p.c. and those of the high malignancy group to 80.0 p.c.; the 5-years survival rates of the low malignancy group amounted to 61.9 p.c. and those of the high malignancy group to 41.7 p.c.). The influence of histology, clinical stage and involvement of organs is discussed on the basis of our results and informations obtained in literature. Our analysis confirms the high importance which must be attached to a common radiologic-internal outpatient-department for co-ordinating the diagnostic and therapeutical programme.     

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.     

Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: a nuclear-cytoplasmic equilibrium

In order to investigate the subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25-[3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25-dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization.     

Pleiotropic effects of a butyrolactone-type autoregulator on mutants of Streptomyces griseus blocked in cytodifferentiation

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6'-methylheptanol-1'-yl)-3-hydroxymethyl-4-butanolide+ ++. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.     

Evidence for Membrane-Associated Choline Kinase Activity in Rat Striatum

The distribution of choline kinase (EC 2.7.1.32) activity was investigated in subcellular fractions of rat striatum. Enzyme activity in the crude mitochondrial fraction, determined after dissolution in Triton X-100, was 5.90 mumol/g initial wet weight/h. When a crude mitochondrial preparation was hypoosmotically shocked and fractionated, followed by the addition of Triton X-100, choline kinase activity in the soluble and particulate fractions was 4.58 and 1.40 mumol/g initial wet weight/h, respectively. Enzyme activity in the particulate fraction was not detected in the absence of Triton X-100 or in the presence of NaCl (up to 1.5 M). Subcellular enzyme markers indicated that the membrane-associated activity was not attributable to mitochondrial or microsomal contamination. Kinetic analysis of the activity of soluble and membrane-solubilized choline kinase indicated Km values of 0.74 mM and 0.68 mM, respectively. Results indicate that choline kinase activity may be measured in both the soluble and the particulate fractions of rat striatum, the latter most likely involving enzyme associated with membrane through hydrophobic or covalent interactions. The specific function of the membrane-associated enzyme has not yet been determined.     

Morphometrisch-ultrastrukturelle Untersuchungen am Nucleus supraopticus der Ratte nach Dehydratation

Zusammenfassung Der Nucleus supraopticus der Ratte, die einer Dehydratation ausgesetzt war, wurde ultrastrukturell-morphometrisch analysiert. Dabei zeigte sich, daß die relativen Volumenanteile der einzelnen Zellkompartimente während der fünftägigen Durstperiode eine auffallende Konstanz aufweisen. Hingegen läßt sich eine absolute Zunahme der Einzelzellvolumina und somit auch der an der Synthese und Sekretion der Neurohormone beteiligten Zellkompartimente feststellen. Die vorliegenden Befunde sprechen für einen beschleunigten Abtransport des neurosekretorischen Materials bei gesteigerter Synthese. Auf eine optimale Standardisierung der Perfusionsmethode bei Untersuchungen am neurosekretorischen Zwischenhirnsystem wird hingewiesen.

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Morphometric-ultrastructural investigations on the supraoptic nucleus of dehydrated rats

Summary The supraoptic nucleus of the dehydrated rat has been analysed by electron microscopy and morphometry. With that it appears, that the relative volumes of the different cell compartments are striking constant. Otherwise one can see an absolute increase of the cell volume together with the cell compartments which take part at the synthesis and secretion of the neurohormones. These results are expression of an accelerated move of the neurosecretory material during increased synthesis. The importance of an optimal standardization of the perfusion-method in investigations of the neurosecretory system is demonstrated.

Wesentliche Teile der vorliegenden Arbeit werden von H. F. Reinhardt der Medizinischen Fakultät der Universität Basel als Dissertation vorgelegt.     

Autoinduction of bacterial bioluminescence in a carbon limited chemostat

Several strains of four species of luminous marine bacteria were maintained in a chemostat at a constant dilution rate and a variety of steady state densities by carbon (glycerol) limitation in order to study the relationship between culture density and bioluminescence activity. In general, luminescence per cell was constant at high culture density, and decreased dramatically at low culture density. For Vibrio fischeri, luminescence decreased to nondectable levels when the culture was maintained at low density; such dark cells were stimulated to synthesize luciferase and became luminous within minutes when purified autoinducer was added to the chemostat. Two strains, Photobacterium phosphoreum NZ11D and Photobacterium leiognathi S1, did not show the decrease in light intensity at low culture density that was characteristic of all other strains tested; they appeared to be constitutive for bioluminescence.Abbreviations BCM basal salts glycerol medium - BM basal salts medium - BSA bovine serum albumin - D dilution rate - DTT dilitiothreitol - LU light unit=2×10¹⁰ quanta s^-1 - OD optical density - SWC sea water complete medium - specific growth rate     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.