Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin.

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.

[Inflammation mediated osteopenia (IMO): therapeutic effect of D-hormone and the role of cytokines]. / Inflammationsmediierte Osteopenie (IMO): Therapeutischer Effekt von D-Hormon und die Rolle der Zytokine.

Inflammation-mediated osteopenia is an animal model for bone mass reduction following chronic inflammation. We found a reduction of calcitriol serum concentrations during the inflammatory process, in addition cytokines are increased. Treatment of the animals with calcitriol is able to prevent bone mass reduction and to preserve bone formation.

Prevalence of low bone mass and endocrine disorders in hip fracture patients in Southern Germany.

UNLABELLED: Low bone mass and conditions leading to falls are considered to determine the risk of hip fracture. Bone mass is influenced by underlying diseases and risk factors such as malnutrition and life style habits. In order to evaluate the clinical significance of osteopenia and risk factors in the pathogenesis of hip fracture, we have studied 146 consecutive patients (27 males, 119 females, aged 53-95 years) undergoing rehabilitation shortly after hip fracture (4 cases of hip fracture resulting from traffic accidents had been excluded). We measured bone mineral density (BMD) at the hip (DXA, Hologic QDR 2,000+), evaluated vitamin D status (serum 25-OH-Vitamin D) and nutritional calcium intake (clinical dietician), and searched for endocrine disorders (physical examination and serum hormones). Femoral neck BMD was 0.543 +/- 0.084 g/cm2 in females and 0.635 +/- 0.087 g/cm2 in males (p < 0.01). For both sexes, the values were significantly lower compared to age-matched controls. 88% of the females and 69% of the males had T-Scores below -2.5, thus fulfilling the densitometric WHO criteria of osteoporosis. Nutritional calcium intake was similar in both sexes (804 +/- 330 mg/day in women, 738 +/- 295 mg/day in men), but lower compared to coxarthrosis patients (1080 +/- 436 mg/day). Vitamin D deficiency was prevalent in 69% of the women, and in 55%, of the men with hip fracture. In female hip fracture patients, the serum alkaline phosphatase was significantly higher (194 +/- 82 U/I) as compared to patients with surgery because of coxarthrosis (142 +/- 46 U/I), supporting the view that some degree of osteomalacia and high turnover was present. Primary hyperparathyroidism (pHPT) was newly detected in 1% and hyperthyroidism in 4.3% of the cases. CONCLUSIONS: Hip fracture occurs at higher BMD values in men compared to women suggesting different fracture thresholds. Vitamin D deficiency and low calcium intake are common in hip fracture patients. However, before initiation of vitamin D treatment pHPT should be excluded. Determination of TSH is recommendable in all hip fracture patients.

Bone mineral density and calcium regulating hormones in patients with inflammatory bowel disease (Crohn's disease and ulcerative colitis).

Inflammatory bowel disease (Crohn's disease and ulcerative colitis) is associated with decreased bone mineral density and increased risk of osteoporosis. However, the pathogenesis of this bone loss is not yet fully understood. In the present study we measured lumbar bone mineral density (by dual photon absorptiometry), serum levels of parathyroid hormone (PTH) and vitamin D metabolites, and serum markers of bone turnover (alkaline phosphatase and osteocalcin) in 15 patients with Crohn's disease and in 4 patients with ulcerative colitis. The median duration of the disease was 4 years and the median lifetime steroid dose was 10g of prednisone. We compared our results to a control group of 19 normal persons, who were matched for age and sex to the patients. We found that lumbar bone density was reduced by 11% in patients compared with control persons (Z-score -0.6 +/- 0.6 versus -0.1 +/- 0.8; p < 0.05). In patients, the serum levels of PTH, 25-hydroxyvitamin D3, and calcitriol (1,25(OH)2D3) were significantly reduced compared with control persons. Serum alkaline phosphatase activity (AP) was significantly higher in the patients and was inversely related to lumbar bone density. Osteocalcin values were not different between patients and control persons. There was also no difference in serum levels of calcium between the two groups, whereas phosphorus levels were higher in patients. We conclude that malabsorption of calcium was not a primary cause of bone loss in our patients, because we did not find secondary hyperparathyroidism. Accordingly, we did not find a severe vitamin D deficiency, since 25-hydroxyvitamin D3 levels were within the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll synthetase cannot synthesize chlorophyll a'.

Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a'. To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-13(2)(R)-methoxy-pheophorbide a and zinc-13(2)(S)-methoxy-pheophorbide a, zinc-13(2)(R)-methoxy-pyropheophorbide a and zinc-chlorine6-13(1), 15(2)-dimethylester]. We conclude that compounds which have the 13(2)-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 13(2)-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occurring chlorophyll a' must be formed by epimerization after esterification.

Bone loss in experimental diabetes. Comparison with the model of inflammation mediated osteopenia.

Osteopenia occurs in diabetes mellitus. Since bone status is altered in Inflammation Mediated Osteopenia (IMO), it was appropriate to study its course in diabetic animals in order to investigate the mechanism of diabetic osteopenia. To this end, we compared the bone loss in streptozotocin (STZ) diabetes with that of IMO. Female rats were studied in total of 8 groups: Control, IMO, Diabetes, IMO with Diabetes (IMO-DIA) on the 3rd and similar groups on the 6th week after induction of IMO with 8 s.c. injections of talcum suspension. Femoral mineral content as reflected by ash weight per femoral volume after 3 weeks was lower in IMO compared to control rats (p < 0.05) while after 6 weeks this difference was not significant. The femur ash weight per volume of diabetic rats was lower than the one of intact rats with and without IMO both after 3 and 6 weeks. Diabetic rats with and without IMO exhibited no difference in this respect. Spleen weight as a measure of the extent of inflammation per body weight was increased only in the IMO group. The diabetic rats with and without IMO did not differ significantly with regard to spleen weight. Similar changes were observed 6 weeks after the induction of IMO. However the difference between IMO and diabetes rats was of borderline significance and no difference existed between the IMO and IMO-diabetic group. The serum calcium levels of intact, IMO and diabetic rats showed no change during both experimental periods. Those of diabetic rats with IMO were higher than those of the diabetic and IMO animals after 6 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on regulation of insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 production in human bone cells.

Previous studies have shown that the actions of IGF-II in bone are determined not only by its concentration, but also by the concentration of IGFBP-4 as well as other IGFBPs. In this study, we sought to determine by Western ligand blotting the effects of growth hormone, IGF-I and IGF-II on the production of IGFBP-3 and IGFBP-4 in TE89 human osteosarcoma cells and in untransformed normal human bone cells derived from rib. Human growth hormone at 10 micrograms/l decreased the amount of IGFBP-4 but had no effect on the IGFBP-3 level in the conditioned medium of low density cultures of TE89 cells and human bone cells derived from rib. Human growth hormone had no effect on IGFBP-3 or IGFBP-4 levels in the conditioned medium of high density human bone cell cultures. IGF-I and IGF-II, which increased human bone cell proliferation, decreased the level of IGFBP-4 (30% of control at 100 micrograms/l IGF-I and IGF-II) but increased the level of IGFBP-3 (3-10 fold at 100 micrograms/l IGF-I and IGF-II) after 48 h of treatment in the conditioned medium of both low and high density TE89 cell cultures. Similar changes in IGFBP-3 and IGFBP-4 levels were also seen in the conditioned medium of human bone cells derived from rib after treatment with IGF-I and IGF-II. Studies to determine the underlying molecular mechanisms by which IGF-II decreased the amount of IGFBP-4 in the conditioned medium revealed that IGF-II decreased the IGFBP-4 mRNA abundance and increased the IGFBP-3 mRNA abundance in human bone cells. Based on the above findings, we conclude that the production of both IGFBP-3 and IGFBP-4 is regulated in bone cells and that local and systemic agents may modulate the responsiveness of bone cells to IGFs by regulated secretion of IGFBP-3 and IGFBP-4.

Influence of parathyroidectomy, 1,25-dihydroxyvitamin D3 and high dietary calcium intake on demineralized bone matrix powder-induced bone formation in the rat.

Demineralized bone matrix induces ectopic endochondral bone formation. We used this model to study the effect of parathyroidectomy (PTX), 1,25-dihydroxyvitamin D3 treatment, and calcium enriched diet on bone formation in the rat. Hypocalcemia and hyperphosphatemia in PTX rats were corrected by 1,25-dihydroxyvitamin D3 treatment (2 x 12.5 ng/day) or by calcium enriched diet (3% calcium). Serum 1,25-dihydroxyvitamin D3 concentration was decreased in PTX rats and in intact rats with high dietary calcium intake. Calcium content of ectopic new bones (42 days after bone matrix implantation) was reduced in PTX rats compared with intact control rats. This could be prevented by 1,25-dihydroxyvitamin D3 treatment. In contrast, calcium enriched diet led to diminished mineralization of ectopic bones both in intact and PTX rats. We conclude that the effect of parathyroidectomy on bone formation may be mediated by 1,25-dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 directly stimulates bone formation in this model and this effect is not simply the result of increasing serum calcium concentration.

[PTH-related protein (PTHrP) in serum of patients with tumor hypercalcemia]. / PTH-related protein (PTHrP) im Serum von Patienten mit Tumorhyperkalzämie.

Parathyroid hormone-related protein (PTHrP) is a recently described hormone, that was isolated from malignant tumors. It shows many properties of parathyroid hormone (PTH) and is related to the pathogenesis of humoral hypercalcemia of malignancy. Therefore, we analyzed PTHrP in the sera of 30 patients with hypercalcemia of malignancy and compared the values with those obtained in patients with primary hyperparathyroidism, Paget's disease of bone, and normal subjects. PTHrP was quantitated with radioimmunoassay (RIA) using aminoterminal antibodies without and with chromatographical sample purification applying SEP-PAK C18 cartridges. Measurements of PTHrP without sample purification yielded high values in all patient groups. There was no differentiation between patient groups. However, quantitation of PTHrP after SEP-PAK C18 purification of the samples resulted in values above the normal range only in tumour patients. In 30 normal subjects PTHrP levels were 110 +/- 75 pg-eq/ml. Eight out of 30 patients with malignant tumours displayed PTHrP-concentrations above 335 pg-eq/ml. PTHrP levels in patients with primary hyperparathyroidism or Paget's disease of bone were within the normal range. PTHrP concentrations were not affected from renal function. We conclude, that determination of PTHrP after sample purification may contribute to the differential diagnosis of malignant disease.

Osteolytic activity of Walker carcinosarcoma 256 is due to parathyroid hormone-related protein (PTHrP).

The hypercalcemic Walker carcinosarcoma 256 of the rat is an animal model for humoral hypercalcemia of malignancy. Previous in vivo studies suggested the production of a parathyroid hormone-related protein (PTHrP) by the Walker tumor. Therefore, we have measured immunoreactive PTHrP in serum-free conditioned medium from cells derived from this tumor using an antibody raised against human PTHrP(1-34). Walker tumor cell conditioned medium (WCM) displaced 125I-hPTHrP(1-34) from the antibody in a dose dependent manner, whereas control medium contained no immunoreactive PTHrP. In contrast, we detected no secretion of immunoreactive rat parathyroid hormone (rat PTH) by the Walker tumor cells using a midregional radioimmunoassay for rat PTH. WCM stimulated adenylate cyclase in osteoblast like cells, the dose-response curve paralleling that of hPTHrP(1-34). This effect could be inhibited by the PTH antagonist (8Nle, 18Nle, 34Tyr)bPTH(3-34) and by the addition of anti-hPTHrP(1-34) antibody. Bone resorbing activity of WCM in organ culture (calvaria of fetal rats) was not inhibited by indomethacin and glucocorticoids, suggesting a prostaglandin independent mechanism of osteoclast activation in this model.

Bone mass reduction after estrogen deprivation by long-acting gonadotropin-releasing hormone agonists and its relation to pretreatment serum concentrations of 1,25-dihydroxyvitamin D3.

Estrogen deficiency results in bone mass reduction of largely varying extent in postmenopausal females, indicating that additional mechanisms influence the response of bone. They are by no ways identified in either the animal experiment or under clinical conditions. In search for factors, conditioning the response of bone to estrogen deficiency, we have conducted a study in females under treatment with the GnRH agonist decapeptyl (D-Trp6-LHRH). This drug blocks ovarian function and was administered for treatment of endometriosis or uterine leiomyoma. We determined spinal (dual photon absorptiometry) and forearm (single photon absorptiometry) bone mineral density before and 3 and 6 months after the onset of therapy and measured biochemical parameters of bone metabolism. Our results showed an increase in bone turnover after initiation of estrogen deficiency, as indicated by the elevation of alkaline phosphatase and osteocalcin. This resulted in a secondary decrease in serum intact PTH and 1,25-dihydroxy-vitamin D3. Furthermore, we found a positive correlation between pretreatment values of serum 1,25-dihydroxyvitamin D3 as well as its decrease and the reduction in bone mass during GnRH agonist treatment. This demonstrates that the patients' metabolic conditions predict their response to estrogen deficiency.

Antiestrogen and antiandrogen administration reduce bone mass in the rat.

In mature female and male rats sex hormone deficiency was produced by surgical castration and by antiestrogen or antiandrogen administration. For the latter purpose we used the nonsteroidal antiestrogens tamoxifen, keoxifene (LY156758) and tetramethylhexestrol, and the steroidal antiandrogen cyproterone acetate. Dosages of 0.4 mg tamoxifen/rat/day and isomolar dosages of keoxifene and tetramethylhexestrol led to a bone mass reduction which was comparable to ovariectomized rats. Cyproterone acetate showed, at 10 mg/rat/day, a similar decrease in bone mass like orchidectomy. The often discussed intrinsic estrogen activity of the antiestrogens was present only in the highest dosage tested of tamoxifen. Keoxifene and tetramethylhexestrol showed no estrogenic effects, but this may be a dosage problem. Cyproterone acetate revealed no androgenic side-effects. These results indicate that antigonadal hormone drugs reduce bone mass to a varying extent.

1,25-Dihydroxyvitamin D3 prevents the decrease of bone mineral appositional rate in rats with inflammation-mediated osteopenia (IMO).

We have studied the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on bone mass and bone mineral appositional rate in intact rats and rats with inflammation-mediated osteopenia (IMO), where osteoblast number and mineral appositional rate are decreased. 1,25(OH)2D3 prevents IMO-specific bone loss when given in a daily dose of 25 ng per rat, but does not when given in higher doses. The hormone was effective, when given over the complete duration of the experiment (21 days), but not when given over shorter time periods (7 and 14 days, respectively). 1,25(OH)2D3 prevents IMO-dependent reduction in mineral appositional rate and leads to an only moderate increase in intact rats. We conclude, that 1,25(OH)2D3 is more effective in stimulating mineral appositional rate in rats with IMO where mineral apposition is impaired.

The hypercalcemic Walker carcinosarcoma 256 of the rat causes an increase in serum 1,25-dihydroxyvitamin D3.

We have studied vitamin D metabolism in rats with the transplantable hypercalcemic Walker carcinosarcoma 256, which is a well characterized animal model for humoral hypercalcemia of malignancy. 25-Hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) concentrations were determined in blood samples obtained from parathyroidectomized (PTX) female rats at different time intervals after intramuscular tumor cell inoculation. We observed a dramatic increase in serum 1,25(OH)2D3 (280 +/- 184 vs. 98 +/- 31 pmol/l) 6 days after tumor cell injection and 4 days after the initial rise of serum calcium, whereas 25(OH)D3 tended to decrease. In a separate control experiment we compared this to the effect of exogenous parathyroid hormone in PTX rats and found similar results. In contrast, rats exhibited no change in vitamin D metabolite blood concentration after inoculation of the normocalcemic Yoshida sarcoma, which obviously does not interfere with vitamin D metabolism. We conclude that the humoral bone-resorbing agent produced by the Walker tumor cells causes elevation of serum 1,25(OH)2D3 concentration by this fulfilling an additional criterion of PTH-like activity.

Incorporation of phytol precursors into chlorophylls of tobacco cell cultures.

The incorporation of [1-(3)H] geranylgeranyl diphosphate (GGPP), [1-(3)H] geranylgeranyl monophosphate (GGMP) and [U-(14)C] phytyl diphosphate (PhPP) into chlorophylls a and b in growing tobacco cell cultures was investigated. The substrates were effectively incorporated into chlorophylls a and b, 3.2% of the total activity of applied GGPP or GGMP and 12.4% of the total activity of applied PhPP being found in chlorophylls a and b after 24 h incubation. The radioactivity was found in phytyl chlorophyllide through-out which means effective hydrogenation of the alcohol moiety in the case of GGPP and GGMP. With increasing substrate concentration, the specific radioactivity of chlorophyll increased up to a saturation level which was reached either at 20-40 µM PhPP or at 60 µM GGPP and GGMP. The specific radioactivity of the chlorophyll formed during the 24-h incubation period was the same as that of the applied substrate at saturating substrate concentration. The specific radioactivity of chlorophyll a was higher than that of chlorophyll b only in the case of PhPP.