Unique 24-hydroxylated metabolites represent a significant pathway of metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 and 1,24-dihydroxyvitamin D2 detectable in human serum.

We have produced evidence for a new metabolic pathway for vitamin D2 in humans involving the production of 24-hydroxyvitamin D2 (24OHD2) and 1,24-dihydroxyvitamin D2 [1,24-(OH)2D2]. These metabolites were produced after either a single large dose (10(6) IU) of vitamin D2 or repeated daily doses between 10(3) and 5 x 10(4) IU. We developed assay systems for the metabolites in human serum and showed that in some chronically treated patients, the concentration of 1,24-(OH)2D2 equalled that of 1,25-(OH)2D2 at about 100 pmol/L. The metabolites were identified by high performance liquid chromatography with diode array spectrophotometry for 24OHD2 and by high resolution gas chromatography-mass spectrometry for 1,24-(OH)2D2. We show that 1,24-(OH)2D2 synthesis can be stimulated by PTH, indicating a renal origin for this metabolite and postulate that it is formed from 24OHD2, which may be synthesized in liver. We conclude from this study that vitamin D2 gives rise to two biologically active products, 1,24-(OH)2D2 and 1,25-(OH)2D2, and that 1,24-(OH)2D2 could be an attractive naturally occurring analog of 1,25-(OH)2D3 for clinical use.

Evidence for nonrenal synthesis of 1,25-dihydroxyvitamin D in patients with inflammatory arthritis.

The extrarenal synthesis of 1,25-dihydroxyvitamin D [1,25-(OH)2D] is a characteristic of activated macrophages and has been demonstrated to occur in vitro in synovial fluid macrophages from patients with inflammatory arthritis. To examine whether such synthesis occurs in vivo, 19 patients with rheumatoid arthritis, 5 patient controls, and 5 healthy controls received a challenge oral dose of 250 micrograms 25-hydroxyvitamin D3 (25-OHD3) and the serum 1,25-(OH)2D3 response was measured. The median rise in serum 1,25-(OH)2D3 was significantly greater (22 pg/ml) in the rheumatoid patients compared to either of the control groups (8 pg/ml), although the increase in precursor 25-OHD3 was similar in all groups. The serum 1,25-(OH)2D concentration did not rise above the normal upper limit in any of the control subjects but exceeded the normal range in 8 of the rheumatoid patients. Extrarenal 1,25-(OH)2D synthesis is substrate dependent, unlike renal 1 alpha-hydroxylation, which is homeostatically controlled. Excessive 1,25-(OH)2D3 synthesis in the rheumatoid group on raising the 25-OHD3 concentration is indicative of nonrenal production of the hormonal metabolite. Further evidence for substrate-dependent extrarenal synthesis came from measurements of 25-OHD and 1,25-(OH)2D in paired serum and synovial fluid samples from 19 patients with inflammatory arthritis, including 15 with rheumatoid arthritis. Synovial fluid 1,25-(OH)2D was usually present at a lower concentration than serum 1,25(OH)2D, with which it was strongly correlated (Kendall's R = 0.46, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive radioimmunoassay using a monoclonal antibody that is equipotent for ercalcitriol and calcitriol (1,25-dihydroxy vitamin D2 and D3).

A monoclonal antibody has been used in a sensitive radioimmunoassay that measures 1,25-dihydroxyvitamin D2 (ercalcitriol) and 1,25-dihydroxyvitamin D3 (calcitriol) with equal potency. This important characteristic has not been reported for any other radioimmunoassay for 1,25-dihydroxyvitamin D. The two forms can be assayed in human serum together or individually after HPLC separation. Sample preparation entails acetonitrile extraction followed by C18-Sep-pak chromatography and HPLC. The assay measures 98% of added analyte, and achieves inter- and intra-assay coefficients of variation of 10.7% at 34 pg/ml and 7.8% at 81 pg/ml respectively. The limit of detection is 1.25 pg/tube and 50% displacement of bound ligand is achieved at 14 pg/tube. The reference interval is 20-50 pg/ml, mean 35. The correlation between results from the monoclonal radioimmunoassay and an established polyclonal antibody method was r = 0.98, slope 0.99. The assay has particular application in patients treated with vitamin D2 since 1,25-dihydroxyvitamin D2 can now be measured accurately in the presence of 1,25-dihydroxyvitamin D3.

cDNAs of two non-allelic sucrose synthase genes in maize: cloning, expression, characterization and molecular mapping of the sucrose synthase-2 gene.

cDNA clones of the two non-allelic sucrose synthase (Ss) genes, Ss2 and Sh, have been isolated from λgt11 expression libraries derived from immature kernel poly(A)(+) RNA of sh-deletion and Sh/Sh genotypes of maize respectively. Recombinant clones containing the longest Ss2 and Sh cDNA inserts, each of approximately 2.5 kb size, were characterized and comparatively analyzed. Although the Sh cDNA insert expresses as a sucrose synthase-1 (SS1) ß-galactosidase fusion protein (∼ 200 kD) in λ lysogens, the Ss2 cDNA failed to form such a chimeric protein and instead showed a ∼ 70 kD SS2 polypeptide. The Ss2 and Sh cDNAs as hybridization probes on RNA blots of immature kernels detected a larger Ss2 transcript (∼ 2900 b) than the Sh transcript (∼ 2750 b). Because SS1 and SS2 protein subunits are known to be of identical size, the significance of difference in transcript size is not apparent. A comparative restriction enzyme mapping of the two cDNA clones and a genomic Ss2 clone show sequence diversity over the entire lengths of Ss2 and Sh clones. Interestingly, restriction endonuclease sites around the 3' ends are more conserved than the 5' ends of these two genes. Genetic data indicate that the Ss2 locus is on chromosome 9 and molecular mapping using the Ss2 cDNA clone on recombinant inbred lines and B-A translocations stocks suggest that Ss2 is about 20 map units away from the Wx locus on 9L.

Mycoviruses of Penicillium stoloniferum: influence of carbon-nitrogen nutrition upon replication.

Carbon-nitrogen ratio experiments indicate that limiting nutrition not only hinders Penicillium stoloniferum host proliferation but reduces total PsV-F and PsV-S virus replication. Results of C-N experiments show a pH-induced autolysis and virus release at minimal C levels. Maximal PsV-F levels and biomass were obtained with glucose and sucrose as C sources. Oleic acid also yielded high biomass and PsV-F yields. Yeast extract was an excellent N source; 2.83 g dry weight biomass and 87 A260 units PsV-F after 96 h of growth. Other nitrogen sources, including amino acids, supported only minimal growth and virus replication. The autolysis phenomenon is pH, not viral-induced. High C and N will support maximal growth and unrestricted virus replication with no cellular lysis. Under low C growth conditions, the replication of PsV-S is favored coupled with high pH and autolysis.

The toxicity of ochratoxin to ruminants.

Among the mold toxins the most toxic ochratoxin, ochratoxin A, commonly occurs in many grains, other feedstuffs, and in soil but in low concentrations. The amount required to produce acute toxicity in ruminants makes such occurrences unlikely. Toxic effects are more likely to occur in chronic low-level intoxication. The lethal single oral dose in cattle is high, probably being a few milligrams more than 13 mg/kg. The lethal level produced by repeated feeding to goats was 3 mg/kg. Ochratoxin A occurred in cows milk and urine but only when massive doses were ingested. Abortion or fetal death, though occurring in rodents, are unlikely to be induced in cattle.

Patulin inhibition of mycovirus replication in Penicillium stoloniferum.

Penicillium stoloniferum NRRL5267 contains two electrophoretically distinct viruses (PsV-F and PsV-S). An in vivo system was developed to test whether a number of fungal metabolites had antiviral properties on PsV-F replication in O.erties on PsV-F replication in P. stoloniferum. Preliminary results indicated that the mycotoxin patulin can block mycovirus replication. Portions of 48 h mycelium were incubated in the presence of varying levels of patulin, and after an additional 48 h incubation, PsV-F content was measured in E260 units by polyacrylamide gel electrophoresis. Patulin at 11, 16 and 20 mug/mg dry wt mycelia blocked PsV-F replication 26, 61 and 71%, respectively, compared with untreated controls. At these levels, host biomass RNA and protein synthesis were minimally affected. No-proliferating fungal mycelium is capable of continued support of PsV-F replication, which is sensitive to patulin. Apparently, inhibitory doses of patulin stimulated PsV-S replication during this 48 h incubation. The preferential action of patulin may arise from metabolite binding to functional enzymes required for virus replication.

Penicillium stoloniferum virus: altered replication in ultraviolet-derived mutants.

Phenotypic mutants of the wild type of Penicillium stoloniferum NRRL5267 were obtained from conidia exposed to ultraviolet light for 60 min (10% survival). Virus content of the wild type and of nine phenotypic mutants was determined by polyacrylamide gel electrophoresis. Four mutants had no detectable Penicillium stoloniferum virus F (PsV-F), whereas the other five had levels of PsV-F in the mycelium similar to the wild-type strain. All nine mutants and the wild type had comparable levels of Penicillium stoloniferum virus S (PsV-S). Maximum virus levels occurred after 9 days of submerged culture in a 2% yeast extract-15% sucrose medium. Virus replication in the fungal host continued after protein, RNA and DNA synthesis levelled off. Virus levels ranged from 85 to 150 E-260 units (extinction units at 260 nm in I cm cell) per 4-7 to 5-3 g dry weight of mycelium for the mutant strains compared to 106 E-260 units per 4-2 g dry weight of the wild-type strain.

Penicillium stoloniferum virus: large-scale concentration and purification by polyethylene glycol.

An experimental procedure developed to concentrate fungal viruses from large volumes of homogenized mycelia with water-soluble polymers, such as polyethylene glycol, possesses advantages over more conventional methods. Concentration of the Penicillium stoloniferum fast-moving virus from mycelial homogenates after addition of polyethylene glycol was rapid and produced large quantities of pure virus.

Isolation and partial characterization of a mycotoxin from Penicillium roqueforti.

Extracts of pure cultures of Penicillium roqueforti isolated from toxic feed samples and of P. roqueforti NRRL 849 were lethal to rats by either intraperitoneal or oral administration. Purification studies guided by this test led to the isolation of a major toxin which showed intraperitoneal and oral median lethal dose values in weanling rats of 11 and 115 mg/kg, respectively. Partial characterization of the crystalline compound, C(17)H(20)O(6), by infrared, ultra violet, PMR, and mass spectroscopy, and by several chemical transformations indicated the presence of three C-methyl substituents plus one acetoxy, one aldehyde, and one alpha,beta-unsaturated ketone group. Two oxygen atoms are present either in epoxide or ether form.