Search

Deep observation (~113 hrs) of the Cygnus region at TeV energies using the HEGRA stereoscopic system of air Čerenkov telescopes has serendipitously revealed a signal positionally inside the core of the OB association Cygnus OB2, at the edge of the 95% error circle of the EGRET source 3EG J2033+4118, and ~$0.5^\circ$north of Cyg X-3. The source centre of gravity is RA $\alpha_{\rm J2000}$: $20^{\rm hr} 32^{\rm m} 07^{\rm s}\pm 9.2^{\rm s}_{\rm stat} \pm2.2^{\rm s}_{\rm sys}$, Dec $\delta_{\rm J2000}$: $+41^\circ 30^\prime 30^{\prime\prime}\pm 2.0^\prime_{\rm stat} \pm 0.4^\prime_{\rm sys}$. The source is steady, has a post-trial significance of +4.6σ, indication for extension with radius $5.6^\prime$at the ~$3\sigma$level, and has a differential power-law flux with hard photon index of $-1.9 \pm0.3_{\rm stat}\pm0.3_{\rm sys}$. The integral flux above 1 TeV amounts ~3% that of the Crab. No counterpart for the TeV source at other wavelengths is presently identified, and its extension would disfavour an exclusive pulsar or AGN origin. If associated with Cygnus OB2, this dense concentration of young, massive stars provides an environment conducive to particle acceleration and likely subsequent interaction with a nearby gas cloud. Alternatively, one could envisage γ-ray production via a jet-driven termination shock.

A search for potential point sources of very high energy gamma rays has been carried out on the data taken simultaneously by the HEGRA AIROBICC and Scintillator arrays from August 1994 to March 2000. The list of sought sources includes supernova remnants, pulsars, AGNs and binary systems. The energy threshold is around 15 TeV. For the Crab Nebula, a modest excess of 2.5 standard deviations above the cosmic ray background has been observed. Flux upper limits (at $90\%$c.l.) of around 1.3 times the flux of the Crab Nebula are obtained, in average, for the candidate sources. A different search procedure has been used for an all-sky search which yields absolute flux upper limits between 4 and 9 crabsdepending on declination, in the band from $\delta = 0$to $\delta = 60^\circ$.

Using the HEGRA system of imaging atmospheric Cherenkov telescopes, a region of the Galactic plane ($-10^\circ < b < 5^\circ$, $38^\circ < l < 43^\circ$) was surveyed for TeV gamma-ray emission, both from point sources and of diffuse nature. The region covered includes 15 known pulsars, 6 known supernova remnants (SNR) and one unidentified EGRET source. No evidence for emission from point sources was detected; upper limits are typically below 0.1 Crab units for the flux above 1 TeV. For the diffuse gamma-ray flux from the Galactic plane, an upper limit of $6.1\times10^{-15}$ph cm-2s-1sr-1MeV-1was derived under the assumption that the spatial distribution measured by the EGRET instrument extends to the TeV regime. This upper flux limit is a factor of about 1.5 larger than the flux expected from the ensemble of gamma-ray unresolved Galactic cosmic ray sources.

Tycho's supernova remnant (SNR) was observed during 1997 and 1998 with the HEGRA Čerenkov Telescope System in a search for gamma-ray emission at energies above ~1 TeV. An analysis of these data, ~65 hours in total, resulted in no evidence for TeV gamma-ray emission. The 3σupper limit to the gamma-ray flux (>1 TeV) from Tycho is estimated at $5.78\times 10^{-13}$photons cm-2s-1, or 33 milli-Crab. We interpret our upper limit within the framework of the following scenarios: (1) that the observed hard X-ray tail is due to synchrotron emission. A lower limit on the magnetic field within Tycho may be estimated $B\geq22$μG, assuming that the RXTE-detected X-rays were due to synchrotron emission. However, using results from a detailed model of the ASCA emission, a more conservative lower limit $B\geq6$μG is derived. (2) The hadronic model of Drury and (3) the more recent time-dependent kinetic theory of Berezhko & Völk. Our upper limit lies within the range of predicted values of both hadronic models, according to uncertainties in physical parameters of Tycho, and shock acceleration details. In the latter case, the model was scaled to suit the parameters of Tycho and re-normalised to account for a simplification of the original model. We find that we cannot rule out Tycho as a potential contributor at an average level to the Galactic cosmic-ray flux.

232 hours of data were accumulated from 1997 to 1999, using the HEGRA Stereoscopic Cherenkov Telescope System to observe the supernova remnant Cassiopeia A. TeV γ-ray emission was detected at the $5 \sigma$level, and a flux of $(5.8 \pm 1.2_{\mathrm{stat}} \pm 1.2_{\mathrm{syst}}) 10^{-9}\ {ph} {m}^{-2} {s}^{-1}$above 1 TeV was derived. The spectral distribution is consistent with a power law with a differential spectral index of $-2.5 \pm 0.4_{\mathrm{stat}} \pm 0.1_{\mathrm{syst}}$between 1 and 10 TeV. As this is the first report of the detection of a TeV γ-ray source on the "centi-Crab"scale, we present the analysis in some detail. Implications for the acceleration of cosmic rays depend on the details of the source modeling. We discuss some important aspects in this paper.

For distant extragalactic sources of gamma-rays in the PeV (1015eV) energy range, interactions of the gamma rays with intergalactic diffuse radiation fields will initiate a pair cascade. Depending on the magnetic fields in the vicinity of the source, the cascade can either result in an isotropic halo around an initially beamed source, or remain more or less collimated. Data recorded by the HEGRA system of imaging atmospheric Cherenkov telescopes are used to derive limits on the halo flux from the AGN Mrk 501. This is achieved by comparing the angular distribution of TeV gamma-rays during the 1997 burst phase -where direct photons should dominate -with the distribution during the 1998/99 quiescent state, where a steady-state halo contribution should be most pronounced. The results depend on the assumptions concerning the angular distribution of the halo; limits on the halo flux within $0.5^\circ$to $1^\circ$from the source range between 0.1% and 1% of the peak burst flux.

Data taken with the HEGRA system of imaging atmospheric Cherenkov telescopes during the 1997 flares of Markarian 501 (Mkn 501) are reanalyzed using an algorithm providing improved energy resolution. A resolution of 10% to 12% is obtained by accounting for the variation of the Cherenkov light yield with the height of the shower maximum in the atmosphere. The improved energy resolution is particularly relevant for the study of the high-energy cutoff in the spectrum, which might be caused by interactions with the intergalactic infrared background radiation. The reanalysis presented here confirms the results obtained in the previous analysis, but hints a steeper slope of the spectrum in the region around 20 TeV.

Fracture behaviour of two intermetallic alloys based on FeAl and Fe3Al was studied. On the alloys Fe-40Al-1C (at%) and Fe-29.5Al-2.3Cr-0.63Zr-0.2C (at%) (FA06Z), a basic characterization, the fracture toughness tests and fractographic analysis were carried out. Tensile tests and fracture toughness tests were performed at 20, 200, 400 and 600°C. The fracture toughness values range from 26 MPa.m1/2 at 20°C to 42 MPa.m1/2 at 400°C. In addition, Jintegral dependence on [removed info]a obtained by potential method was measured. The fractographic analysis showed that samples fractured at 20, 200 and 400°C in the tensile or fracture toughness tests exhibit transgranular cleavage fracture, while at 600°C the ductile dimple fracture predominates.

Novel 19-nor analogues of 1α,25-dihydroxyvitamin D3 were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig−Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann's ketones. Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2α- and 2β-ethyl compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety in a trans relationship to the C(6)−C(7) bond (E-isomers) were more potent than the corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D3 and both 2α-ethyl-19-norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than does 1α,25-(OH)2D3. Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin D receptor and the computational docking of four synthesized (20R)-19-norvitamin D3 analogues into its binding pocket are also reported.

We present exactly simultaneous X-ray and TeV monitoring with RXTEand HEGRA of the TeV blazar Mrk 501 during 15 days in 1998 June. After an initial period of very low flux at both wavelengths, the source underwent a remarkable flare in the TeV and X-ray energy bands, lasting for about 6 days and with a larger amplitude at TeV energies than in the X-ray band. At the peak of the TeV flare, rapid TeV flux variability on subhour timescales is found. Large spectral variations are observed at X-rays, with the 3-20 keV photon index of a pure power-law continuum flattening from ? = 2.3 to ? = 1.8 on a timescale of 2-3 days. This implies that during the maximum of the TeV activity the synchrotron peak shifted to energies 50 keV, a behavior similar to that observed during the longer lasting, more intense flare in 1997 April. The TeV spectrum during the flare is described by a power law with photon index ? = 1.9 and an exponential cutoff at ~4 TeV; an indication for spectral softening during the flare decay is observed in the TeV hardness ratios. Our results generally support a scenario in which the TeV photons are emitted via inverse Compton scattering of ambient seed photons by the same electron population responsible for the synchrotron X-rays. The simultaneous spectral energy distributions can be fit with a one-zone synchrotron self-Compton model assuming a substantial increase of the magnetic field and the electron energy by factors of 3 and 10, respectively.

The sex steroid 17β‐estradiol (17β‐E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3‐month‐old Brown Norway rats was designed to investigate the role of 17β‐E2in the regulation of calcium homeostasis. Rats were divided in four groups, sham‐operated, ovariectomized (OVX), and OVX supplemented with either a 0.025‐mg or 0.05‐mg 17β‐E2pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17β‐E2supplementation. OVX resulted in a nonsignificant increase in serum 1,25‐dihydroxyvitamin D3(1,25(OH)2D3). Supplementation with either one of the 17β‐E2dosages resulted in an 80% reduction of 1,25(OH)2D3and only a 20% reduction in 25‐hydroxyvitamin D3levels. OVX, as well as supplementation with 17β‐E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3levels were also significantly decreased in the 17β‐E2‐supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3levels (18.1 ± 2.1 and 16.4 ± 2.2 pmol/l compared with 143.5 ± 29 pmol/l for the OVX group), the OVX‐induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17β‐E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17β‐E2did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17β‐E2is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17β‐E2exerts this effect independent of 1,25(OH)2D3. In general, 17β‐E2not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption.

The sex steroid 17β‐estradiol (17β‐E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3‐month‐old Brown Norway rats was designed to investigate the role of 17β‐E2in the regulation of calcium homeostasis. Rats were divided in four groups, sham‐operated, ovariectomized (OVX), and OVX supplemented with either a 0.025‐mg or 0.05‐mg 17β‐E2pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17β‐E2supplementation. OVX resulted in a nonsignificant increase in serum 1,25‐dihydroxyvitamin D3(1,25(OH)2D3). Supplementation with either one of the 17β‐E2dosages resulted in an 80% reduction of 1,25(OH)2D3and only a 20% reduction in 25‐hydroxyvitamin D3levels. OVX, as well as supplementation with 17β‐E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3levels were also significantly decreased in the 17β‐E2‐supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3levels (18.1 ± 2.1 and 16.4 ± 2.2 pmol/l compared with 143.5 ± 29 pmol/l for the OVX group), the OVX‐induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17β‐E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17β‐E2did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17β‐E2is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17β‐E2exerts this effect independent of 1,25(OH)2D3. In general, 17β‐E2not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption.

Parched Elasto Hydrodynamic Lubricant (PEHL) film thickness in a large instrument ball bearing is measured by electrical capacitance across its ball set. Correlation is shown between changes in film thickness and changes in Basic Speed Ratio (BSR) measured at the same time. BSR is confirmed as a sensitive, non-intrusive measure of transients in film thickness in a real bearing.

Secreted phosphoprotein 1 (Spp-1; osteopontin) is one of the abundant noncollagenous proteins in bone matrix and is produced by osteoblasts. We examined the promoter region of the mouse Spp-1 gene and identified a sequence responsible for 1,25-dihydroxyvitamin D3 enhancement of the Spp-1 gene expression. This 24-base-pair (bp) sequence (vitamin D response element) is located 761 bp upstream of the transcription start site and consists of two direct repeats of a unique 9-bp motif, AGGTTCACG. The vitamin D response element confers responsiveness of a heterologous promoter to 1,25-dihydroxyvitamin D3 in a position- and orientation-independent and copy-number-dependent manner. The basal level of expression of the reporter constructs containing this sequence and its response to 1,25-dihydroxyvitamin D3 were not affected by cotreatment with transforming growth factor beta or the tumor promoter phorbol 12-myristate 13-acetate or by cotransfection with a JUN expression vector. The vitamin D response element forms DNA-protein complexes, as indicated by gel-retardation assays. The addition of a monoclonal antibody raised against the vitamin D receptor further retarded the mobility of the DNA-protein complex. Another antibody that recognizes the DNA binding region of the vitamin D receptor attenuated its binding to the sequence. These results indicate that this 24-bp sequence containing two 9-bp motifs binds to the vitamin D receptor and mediates the vitamin D3 enhancement of murine Spp-1 gene expression.

1α,25-Dihydroxy-18-norvitamin D3 and 1α,25-dihydroxy-18,19-dinorvitamin D3 were prepared via Wittig−Horner coupling of 25-hydroxy-18-nor Grundmann type ketone with the corresponding A-ring phosphine oxides. Configuration at C-13 in the 18-nor Grundmann type alcohol (C,D-ring synthon), obtained by oxidative degradation of vitamin D3, was determined by ¹H NMR spectroscopy and molecular mechanics calculations. Additional proof of the assigned trans-C/D-junction of the key intermediate 18-nor Grundmann type ketone follows from its chiroptical properties (circular dichroism data) and further chemical transformations. 1α,25-Dihydroxy-18-norvitamin D3 was found more potent than 1α,25-dihydroxyvitamin D3 in binding to the porcine intestinal vitamin D receptor (5−10×), in differentiation of HL-60 cells (5−10×), and in inhibition of HL-60 proliferation. 1α,25-Dihydroxy-18,19-dinorvitamin D3 appeared equally active as 1α,25-dihydroxyvitamin D3 in these activities. In vivo, 1α,25-dihydroxy-18-norvitamin D3 was only slightly less active than 1α,25-dihydroxyvitamin D3 in intestinal calcium transport and bone calcium mobilization, while 1α,25-dihydroxy-18,19-dinorvitamin D3 showed activities 10 times lower. These studies imply that deletion of C-18 does not impair activity of analogs of 1α,25-dihydroxyvitamin D3.

Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59- to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by size-exclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.

Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.

New highly active isomers of the natural hormone 1α,25-dihydroxyvitamin D3 possessing an exomethylene group at the 2-position were prepared in a convergent manner, starting with (−)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundmann ketones. These 2-methylene-19-norvitamins were efficiently converted to the 2-methyl and 2-hydroxymethyl derivatives, some of which exhibited pronounced in vivo biological activity. Configurations of the A-ring substituents were determined by ¹H NOE difference spectroscopy as well as by spin decoupling experiments. It was established that the bulky methyl and hydroxymethyl substituents at C-2, due to their large conformational free energies, occupy mainly equatorial positions. Additionally, hydroxylation of the C(10)−C(19) double bond in 1α,25-(OH)2D3 was performed, resulting in 1α,19,25-trihydroxy-10,19-dihydrovitamin D3 derivatives in which the hydroxymethyl substituent at C-10, for steric reasons, is forced to occupy an axial position. In consequence, the vitamin D3 analogues were synthesized in which the 1α-hydroxy group, required for biological activity, is almost exclusively axially or equatorially oriented because of stabilization of the single A-ring chair conformations. The relative ability of the synthesized analogues to bind the porcine intestinal vitamin D receptor was assessed and compared with that of the natural hormone. It was established that vitamins possessing the axial orientation of the 1α-hydroxy substituent exhibit a significantly increased receptor binding affinity. Compounds with a 2-methylene substituent showed selective calcemic activity profiles, being extremely effective on bone calcium mobilization. 2α-Methyl-substituted vitamins proved to be much more active in vivo than the corresponding epimers with 2β-configuration. All of the 2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2α-substituted in the 20S-series being especially potent. The present studies imply that the axial orientation of the 1α-hydroxy group is necessary for biological activity of vitamin D compounds.

The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.

The HEGRA experiment is a hybrid installation characterized by four different types of detectors (scintillator counters, open Cherenkov counters, Cherenkov telescopes and Geiger towers) located within one detector array. The Geiger towers are built with the objective to measure the local energy deposition and to reconstruct and identify muon tracks. In addition each tower may serve as an independent high resolution muon flux monitor. With these properties the Geiger towers are operating as geometric and energetic calibration instruments for the other detector components and as additional independent measurement instruments with respect to the γ-hadron separation and the measurement of the chemical composition. In this paper the design, the performance, and the basic principles of the data analysis of the lead-concrete Geiger towers are described.

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)2D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25–35 years and 43 elderly women aged 65–83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1,25(OH)2D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism.

The rat 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor has been expressed at elevated levels in Spodoptera frugiperda cells using the baculovirus expression vector system. The recombinant 1,25-(OH)2D3 receptor is full-length, binds 1,25-(OH)2D3, and is recognized by a monoclonal antibody specific for 1,25-(OH)2D3 receptor. Densitometric scanning of Coomassie brilliant blue-stained SDS/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein from the insect cells. The hydroxylapatite binding assay revealed average levels of 2 nmol of unoccupied 1,25-(OH)2D3 receptor per mg of protein in insect cells at 72 hr after infection with recombinant baculovirus. A measure of total 1,25-(OH)2D3 receptor using a ligand-independent, immunoradiometric assay disclosed average levels of 2.3 nmol of receptor per mg of protein produced by these same cells. A monoclonal antibody directed against the 1,25-(OH)2D3 receptor, and reported to cross-react with this receptor derived from several species, recognized the recombinant rat 1,25-(OH)2D3 receptor upon Western analysis. A monoclonal antibody directed specifically against the porcine receptor failed to recognize the recombinant rat 1,25-(OH)2D3 receptor protein. The cytosolic preparation of insect cells infected with recombinant baculovirus exhibited an equilibrium dissociation constant of 1 x 10(-11) M as determined by a 1,25-(OH)2D3 saturation analysis plotted by the method of Scatchard. This expression system provides an adequate source from which abundant quantities of 1,25-(OH)2D3 receptor can be purified for subsequent x-ray crystallographic analyses.

Human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA clones were isolated from an HL-60 cell cDNA library by using a reverse transcription/polymerase chain reaction-generated human cDNA probe. The 24-hydroxylase cDNA consists of a 1539-bp open reading frame encoding a 513-amino acid polypeptide. Protein sequence analysis shows that the human 24-hydroxylase is 90% homologous (82% identical) to that of the rat, with 100% homology in the 21-amino acid heme-binding region. Northern blot analysis showed that the 24-hydroxylase cDNA probe hybridized to a 3.4-kb mRNA species. Treatment of HL-60 cells with 0.1 microM 1,25-dihydroxyvitamin D3 for 24 hr produced a 30-fold increase in the 24-hydroxylase mRNA level. This result is consistent with previous studies in the same cell line, in which 24-hydroxylase activity was elevated to a maximum in 24 hr by a similar treatment with 1,25-dihydroxyvitamin D3. To verify the identity of these isolated cDNA clones, two polymerase chain reaction-amplified human 24-hydroxylase cDNA fragments containing the entire coding region were used to produce 24-hydroxylase enzyme activity in two genetic expression systems. Transient levels of 24-hydroxylase activity were measured in transfected mammalian COS-1 cells and in recombinant baculovirus-infected Spodoptera frugiperda (Sf21) insect cells.

cDNA clones encoding Japanese quail chorioallantoic membrane and chicken kidney 1,25-dihydroxyvitamin D3 receptors were isolated and the total 448-amino acid (aa) sequence was deduced. The sequences of the chicken and quail receptors are identical. The nucleotide and deduced amino acid sequences of the avian receptors are similar but not identical to the reported rat or human receptor sequences. There is a 78% similarity in the nucleotide sequences and 98.5% and 87.5% similarities in the amino acid sequences of the DNA-binding and ligand-binding domains, respectively. Two avian receptor proteins (58 and 60 kDa) arise from a single mRNA transcript by alternate initiation of translation. The avian 1,25-dihydroxyvitamin D3 receptors were produced using a bacterial expression system. Form A receptor was expressed from a cloned cDNA that contains the first translation signal (ATG) and corresponds with the 60-kDa avian receptor protein, and form B receptor was initiated from the third ATG on the same mRNA transcript to give rise to the 58-kDa protein. The cysteine-rich DNA-binding domain is almost conserved among human, rat, and avian receptors. The position of the nine cysteines was conserved in all three sequences. The avian receptor differs in the second zinc finger domain, where a methionine replaces a leucine, a serine replaces an asparagine, and a lysine replaces an arginine at aa 77, 83, and 87, respectively, of the avian sequence. The increased length of the avian receptor results from a 20-aa extension of the N-terminal region. RNA hybridization indicates there is a single mRNA species of approximately 2700 bp for both the chicken and quail receptors compared to 4400 bp for the rat transcript. Surprisingly, the translated avian sequence is larger (448 aa) than the 423-aa rat receptor protein. Therefore, our results confirm that despite the difference in molecular mass between different receptor proteins, there is a similarity in gene organization such that the DNA-binding and hormone-binding domains are positionally conserved from the C terminus.

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D3, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. We have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 and their delta 22 analogues are 10-fold more potent than 1,25-dihydroxyvitamin D3 in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxyvitamin D3 in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxyvitamin D3 intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 have the same effectiveness as 1,25-dihydroxyvitamin D3 in displacing the tritiated hormone from its receptor in rat intestine or HL-60 cells. These analogues of 1,25-dihydroxyvitamin D3 may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D3 in inducing differentiation.

Monoclonal antibodies against the porcine 1,25-dihydroxyvitamin D3 receptor were immobilized on Sepharose CL-4B and used to obtain a highly purified 1,25-dihydroxyvitamin D3 receptor fraction with a 45% recovery of the 1,25-dihydroxyvitamin D3 binding capacity. The porcine receptor was purified to homogeneity by preparative electrophoresis and digested in sodium dodecyl sulfate/polyacrylamide gels with Staphylococcus aureus strain V8 protease. The resulting peptides were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, electrophoretically transferred to polyvinylidene difluoride membranes, and directly sequenced. The generation and isolation of peptides by this method allows sequencing of proteins present in trace amounts as well as those whose amino termini have been modified. The 1,25-dihydroxyvitamin D3 receptor amino acid sequence corresponded to the sequence predicted from a recently cloned receptor cDNA obtained from rat kidney mRNAs.

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.

The sequences of the C-terminal octadecapeptides obtained by cyanogen bromide cleavage of the γ-chains of myeloma proteins of the four subclasses, and a urinary heavy-chain-disease protein, have been determined. Although the sequences were markedly homologous, unique replacements were identified that distinguished between the γ2b, γ2c and γ2d subclasses. The data are in accord with the postulated existence of four genetic loci or cistrons, these having arisen by the process of gene duplication.

The heavy chain of rabbit immunoglobulin G exists in three major allotypic patterns, Aa1–Aa3. A comparison of the amino acid compositions of the heavy chains isolated from immunoglobulin IgG homozygous for each allotypic determinant revealed the presence of an additional methionine residue per chain in the Aa3 allotype relative to the Aa1 and Aa2 allotypes. The position of the additional methionine residue was determined by cyanogen bromide cleavage and by tryptic digestion of the γ-chains; it coincided with the inter-Fd–Fc area of the chain. Isolation and characterization of the corresponding tryptic peptides of 31 amino acid residues from each of the allotypes showed the presence of a methionine-for-threonine replacement in the Aa3 allotype, but only in about 70–80% of the molecules. No other allotypic variations were seen in this tryptic peptide. Allotypically related variations in composition were also detected in the N-terminal cyanogen bromide-cleavage peptide.

A VARIANT of myeloma has been described1,2; it is known as heavy chain disease, and is characterized by the presence of a homogeneous serum and urinary protein bearing only the antigenic determinants associated with the heavy or γ-chain of human IgG immunoglobulin. Five such cases have been described, but the protein of two patients, Zu1and Cr2,3, have been more extensively investigated because of the availability of material and appear to share common structural features. The proteins are symmetrical dimers, stabilized by disulphide bonds, with molecular weights in the range of 50,000–55,000. Peptide maps reveal they contain the Fc portion of the heavy chain as a chief structural unit3. This conclusion is supported by the content of carbohydrate3, the presence of Gm specificities4, and biological activities5,6of these proteins, properties also attributed to the C-terminal half of the heavy chain.