Immune regulation of procalcitonin: a biomarker and mediator of infection.

Procalcitonin (PCT) has recently emerged as a powerful biomarker for an early and accurate diagnosis of bacterial infection. Here we summarize our current understanding of the expression pathways of PCT, its potential cellular sources including immune cells, and factors inducing its secretion. Also addressed is the significance of increased blood PCT concentration, which may allow this molecule not only to act as a clinical biomarker but also as an active participant in the development and progression of infectious processes. Experimental approaches to delineate a better understanding of PCT functions, molecular pathways that modulate its expression and therapeutic opportunities to curtail its biological actions are discussed, as well.

A single receptor identical with that from intestine/T47D cells mediates the action of 1,25-dihydroxyvitamin D-3 in HL-60 cells.

Two anti-vitamin D receptor monoclonal antibodies binding to two different epitopes immunoprecipitate 100% of the HL-60 1,25-dihydroxyvitamin D-3 binding activity, while another monoclonal antibody specific for the porcine receptor precipitates none. Using a rat receptor cDNA probe, a single mRNA species of 4.6 kb was detected by Northern analysis of HL-60 mRNA. Using a cDNA probe from the cloned rat receptor, 10(7) recombinants from a lambda gt11 cDNA library constructed from mRNA isolated from HL-60 cells was screened yielding two positive clones. These clones had sequences identical with the known human receptor sequence from intestinal/T47D sources. Using PCR technology, the entire sequence of the HL-60 1,25-dihydroxyvitamin D-3 receptor was determined. This sequence was found identical with that reported for the human intestinal/T47D cDNA encoding the vitamin D receptor except for a single base. The substitution of this particular base does not alter the amino acid sequence however. Thus, the same receptor likely operates in differentiation and calcium transport functions.

Stabilization of the vitamin D receptor in rat osteosarcoma cells through the action of 1,25-dihydroxyvitamin D3.

A regulatory mechanism for the vitamin D receptor (VDR) in rat osteosarcoma cells (ROS 17/2.8) is stabilization of the receptor through binding of its ligand, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Increased transcription of the gene encoding VDR does not occur upon treatment of these osteoblast-like cells with 1,25-(OH)2D3. When 10 nM 1,25-(OH)2D3 was administered to confluent cultures of ROS 17/2.8 cells, no change in receptor mRNA was detected, as measured by a ribonuclease protection assay. VDR abundance was measured using an immunoradiometric assay at varying time points within a 24-h period after 1,25-(OH)2D3 treatment. Receptor protein levels increased rapidly and continued to rise over 24 h. By 2 h, the level of receptor increased 2.5-fold, achieving a maximum level of 8-fold above the baseline at 18 h. The half-life of the receptor protein is 2 h in the absence of hormone, as determined by blockage of translation in cycloheximide-treated cells. In the presence of hormone, however, receptor levels were unchanged for at least 6 h. The administration of 1,25-(OH)2D3 stabilizes the receptor, thereby resulting in its accumulation in ROS 17/2.8 cells.

Distinct conformations of vitamin D receptor/retinoid X receptor-alpha heterodimers are specified by dinucleotide differences in the vitamin D-responsive elements of the osteocalcin and osteopontin genes.

The 1 alpha,25-dihydroxyvitamin D3 (VD3)-dependent stimulation of osteocalcin (OC) and osteopontin (OP) gene transcription in bone tissue is mediated by interactions of trans-activating factors with distinct VD3-responsive elements (VDREs). Sequence variation between the OC- and OP-VDRE steroid hormone half-elements provides the potential for recognition by distinct hormone receptor homo- and heterodimers. However, the exact composition of endogenous VD3- induced complexes recognizing the OC- and OP-VDREs in osteoblasts has not been definitively established. To determine the identity of these complexes, we performed gel shift immunoassays with nuclear proteins from ROS 17/ 2.8 osteoblastic cells using a panel of monoclonal antibodies. We show that VD3- inducible complexes interacting with the OC- and OP-VDREs represent two distinct heterodimeric complexes, each composed of the vitamin D receptor (VDR) and the retinoid X receptor-alpha (RXR). The OC- and OP-VDR/RXR alpha heterodimers are immunoreactive with RXR antibodies and several antibodies directed against the ligand-binding domain of the VDR. However, while the OC-VDRE complex is also efficiently recognized by specific monoclonal antibodies contacting epitopes in or near the VDR DNA-binding domain (DBD) (between amino acids 57-164), the OP-VDRE complex is not efficiently recognized by these antibodies. By systematically introducing a series of point-mutations in the OC-VDRE, we find that two internal nucleotides of the proximal OC-VDRE half-site (nucleotide -449 and -448; 5'-AGGACA) determine differences in VDR immunoreactivity. These results are consistent with the well established polarity of RXR heterodimer binding to bipartite hormone response elements, with the VDR recognizing the 3'-half-element. Furthermore, our data suggest that the DBD of the VDR adopts different protein conformations when contacting distinct VDREs. Distinctions between the OC- and OP-VDR/RXR alpha complexes may reflect specialized requirements for VD3 regulation of OC and OP gene expression in response to physiological cues mediating osteoblast differentiation.

An enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein.

In this paper, we detail an enzyme-linked immunoassay for the 1,25-dihydroxyvitamin D3 receptor protein. The receptor protein of cell and tissue homogenates is bound between two monoclonal antibodies specific for different epitopes on the receptor protein. The first antibody is bound to the well of an ELISA plate and the second is biotinylated. The receptor-antibody complex is detected with avidin-alkaline phosphatase and rho-nitrophenyl phosphate. The amount of receptor in each sample is determined by comparison with a standard curve made from purified receptor protein. This assay is highly sensitive, measuring as little as 2 fmol of receptor, and has an intra-assay coefficient of variation of 6.6% and an interassay coefficient of variation of 13.8%. The assay can be used to measure the receptor from mammalian and avian species and is independent of the presence of hormone. By eliminating the need for a radio-iodinated monoclonal antibody and incorporating the ease of a plate assay, we have a significantly improved method for measuring the vitamin D receptor protein. This paper also presents Western analysis of the antibodies used to demonstrate that they do not recognize other steroid hormone receptors.

Association between intestinal vitamin D receptor, calcium absorption, and serum 1,25 dihydroxyvitamin D in normal young and elderly women.

The exact mechanism for the decrease in intestinal calcium absorption with age is not yet understood. A decrease with age in serum 1,25-dihydroxyvitamin D (1,25(OH)2D) or a decrease in the intestinal vitamin D receptor (VDR) protein concentration are possible causes. The objective of this study was to examine the effect of age on these factors. Fifty-nine young women age 25-35 years were compared with 41 elderly women age 65-83 years who underwent measurements of VDR, calcium absorption using a 20 mg and 100 mg calcium carrier, and calciotropic hormones. Calcium absorption by both tests was lower in the elderly women compared with the young women (p < 0.05). Serum 1,25(OH)2D and duodenal VDR protein concentration were not significantly different between the two age groups. Serum 1,25(OH)2D correlated with the 20 mg calcium absorption test in both young (r = 0.35, p < 0.007) and elderly women (r = 0.58, p < 0.0001) and with the 100 mg calcium absorption in the elderly (r = 0.32; p < 0.05). VDR did not correlate with calcium absorption in young women or elderly women, nor did VDR correlate with serum 1,25(OH)2D and serum 25-hydroxyvitamin D. In summary, the decrease in calcium absorption cannot be explained by a decrease in intestinal VDR. The correlation between serum 1,25(OH)2D and both calcium absorption tests only accounts for 12-30% of the variance in the age-related change in the calcium absorption tests. Other factors, not yet understood, are responsible for the decline in calcium absorption with age.

Evidence for involvement of 17beta-estradiol in intestinal calcium absorption independent of 1,25-dihydroxyvitamin D3 level in the Rat.

The sex steroid 17beta-estradiol (17beta-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 17beta-E2 in 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 17beta-E2 pellet 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 17beta-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17beta-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17beta-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17beta-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)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (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 17beta-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. 17beta-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17beta-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17beta-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17beta-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption. (J Bone Miner Res 1999;14:57-64)

New 1alpha,25-dihydroxy-19-norvitamin D3 compounds of high biological activity: synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and 2-methylene analogues.

New highly active isomers of the natural hormone 1alpha, 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 1alpha,25-(OH)2D3 was performed, resulting in 1alpha,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 1alpha-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 1alpha-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. 2alpha-Methyl-substituted vitamins proved to be much more active in vivo than the corresponding epimers with 2beta-configuration. All of the 2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2alpha-substituted in the 20S-series being especially potent. The present studies imply that the axial orientation of the 1alpha-hydroxy group is necessary for biological activity of vitamin D compounds.

Synthesis and biological activity of 1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3.

1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 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. 1 alpha, 25-Dihydroxy-18-norvitamin D3 was found more potent than 1 alpha, 25-dihydroxyvitamin D3 in binding to the porcine intestinal vitamin D receptor (5-10x), in differentiation of HL-60 cells (5-10x), and in inhibition of HL-60 proliferation. 1 alpha, 25-Dihydroxy-18, 19-dinorvitamin D3 appeared equally active as 1 alpha, 25-dihydroxyvitamin D3 in these activities. In vivo, 1 alpha, 25-dihydroxy-18-norvitamin D3 was only slightly less active than 1 alpha, 25-dihydroxyvitamin D3 in intestinal calcium transport and bone calcium mobilization, while 1 alpha, 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 1 alpha, 25-dihydroxyvitamin D3.

Contribution of several metabolites of the vitamin D analog 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-(OH)(2) vitamin D(3) (KH 1060) to the overall biological activity of KH1060 by a shared mechanism of action.

The synthetic 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) analog 20-epi-22-oxa-24a,26a,27a-tri-homo-1,25-(OH)(2)vitamin D(3) (KH1060) is considerably more potent than its cognate hormone. The mechanism of action of KH1060 includes interaction with the vitamin D receptor (VDR). We previously showed that KH1060 increases VDR stability in ROS 17/2.8 osteoblastic cells by inducing a specific conformational change in the VDR. KH1060 is metabolized, both in vivo and in vitro, into several stable products. In the present study, we investigated whether these metabolites might contribute to the increased biological activity of KH1060. We found that the potencies of two of these metabolites, 24a-OH-KH1060 and 26-OH-KH1060, were similar to that of 1,25-(OH)(2)D(3) in inducing osteocalcin production by the osteoblast cell line ROS 17/2.8. This report further showed that these metabolites had the same effects as KH1060 on VDR: they increased VDR stability in ROS 17/2.8 cells, while limited proteolytic analysis revealed that they caused a conformational change in the VDR, resulting in an increased resistance against proteolytic cleavage. Furthermore, as shown in gel mobility shift assays, both compounds clearly induced VDR binding to vitamin D response elements. Together, these results show that the potent in vitro activity of KH1060 is not only directed by the effects on the VDR conformation/stabilization of the analog itself, but also by certain of its long-lived metabolites, and emphasizes the importance of detailed knowledge of the metabolism of synthetic hormonal analogs.

Vitamin D-dependency rickets type II: truncated vitamin D receptor in three kindreds.

Fibroblasts from three patients with vitamin D-dependency rickets type II were used to study mutations in the 1,25-dihydroxyvitamin D3 receptor responsible for this hereditary disease. Normal human fibroblasts contain 43 +/- 13 fmol receptor/mg protein as determined by immunoradiometric assay and 22 +/- 3 fmol/mg by ligand binding assay. The fibroblasts from the rachitic patients contained no receptor detectable by either method. The 1,25-(OH)2D receptor cDNA for cells from each kindred was produced from total RNA using reverse transcription and polymerase chain reaction amplification. When these cDNAs were sequenced, it was found that each cell line contained a nucleotide substitution resulting in a stop codon in the coding sequence. The predicted resultant receptor protein is 69 amino acids long in one family, and 148 and 291 amino acids long in two other families. These truncated proteins have little or no 1,25-dihydroxyvitamin D3-binding domain accounting for 1,25-dihydroxyvitamin D resistance.

Protein crystal growth in microgravity-temperature induced large scale crystallization of insulin.

One of the major stumbling blocks that prevents rapid structure determination using x-ray crystallography is macromolecular crystal growth. There are many examples where crystallization takes longer than structure determination. In some cases, it is impossible to grow useful crystals on earth. Recent experiments conducted in conjunction with NASA on various Space Shuttle missions have demonstrated that protein crystals often grow larger and display better internal molecular order than their earth-grown counterparts. This paper reports results from three Shuttle flights using the Protein Crystallization Facility (PCF). The PCF hardware produced large, high-quality insulin crystals by using a temperature change as the sole means to affect protein solubility and thus, crystallization. The facility consists of cylinders/containers with volumes of 500, 200, 100, and 50 ml. Data from the three Shuttle flights demonstrated that larger, higher resolution crystals (as evidenced by x-ray diffraction data) were obtained from the microgravity experiments when compared to earth-grown crystals.

Assessment of speech production with dentures by electromagnetic articulography.

This report investigates the use of electromagnetic articulography (EMA) to compare basic speech patterns between a patient with traditional dentures to those of a normally dentate person. The goal is to assess the efficacy of traditional dentures in order to generate clinical data and works towards the improvement of denture design. Kinematic and acoustic data were acquired for these two subjects using a variety of repetitive vowel-consonant-vowel tasks. Spatiotemporal parameters indicating dynamic properties of the tongue blade and jaw movements, and timing coordination of the movements between them and with the output acoustic signal, were measured and compared within and between the participants. The results show significant differences in both spatial and temporal patterns and variation between individual tasks within each subject's data, as well as a difference in the two subjects' performance of the same task (cross-subject) for select calculated kinematic and latency parameters. It is concluded that there is more variation in spatiotemporal parameters in speech patterns for patients with dentures than without; in particular, latencies of the tongue blade and jaw movements and acoustic landmarks of the consonants, show strategies of movements timing coordination, typical of the speaker with denture.

The C-terminal sequences of the heavy chains of human immunoglobulin G myeloma proteins of differing isotopes and allotypes.

The sequences of the C-terminal octadecapeptides obtained by cyanogen bromide cleavage of the gamma-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 gamma(2b), gamma(2c) and gamma(2d) 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.

Enzymic degradation of the Fc fragment of rabbit immunoglobulin IgG.

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.

Allotype-related sequence variation of the heavy chain of rabbit immunoglobulin G.

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 gamma-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.

Third component of human complement: purification from plasma and physicochemical characterization.

The third component of complement has been purified from fresh human plasma employing an initial fractionation with poly(ethylene glycol) followed by sequential depletion of plasminogen by affinity adsorbents, chromatography on diethylaminoethylcellulose, gel filtration on agarose, and batch adsorption/desorption on hydroxylapatite. Final recoveries of C3 were 33% of the initial protein, as quantitated by radial immunodiffusion, and 31% of the initial hemolytic activity. Apparent homogeneity is indicated by immunological criteria and by polyacrylamide gel electrophoresis. A partial specific volume of 0.736 +/- 0.003 mlgm-1 was determined for C3 by the mechanical oscillator technique. "Low speed" sedimentation equilibrium yielded an apparent weight average molecular weight for the protein of 187 650 +/- 5650. Based upon this molecular weight, a molar extinction coefficient of 1.82 X 10(5) 1. mole-1 cm-1 at 280 nm was calculated from boundary-spreading experiments in the ultracentrifuge and as assumed refractive index increment. Amino acid analyses revealed no unusual or distinctive characteristics. Automated Edman degradation revealed a double N-terminal sequence, Ser-Val,Pro-Glx,Met-Lee,Tyr-Thr,Ser-Glx,Ile-Lys,Gly-Arg,Thr-Met,Pro-Asx, in agreement with the two chain structure observed on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and revealing both chains are available to degradation. Serine is postulated as the initiating sequence in both chains based upon high recoveries of dinitrophenylserine upon hydrolysis of dinitrophenylated C3, and our inability to identify any other dinitrophenyl or phenylthiohydantoin derivatives in this position. Alanine is the ultimate carboxy-terminal amino acid of at least one of the chains, as indicated by the action of carboxypeptidases on C3 in the presence of sodium dodecyl sulfate.