Developmental and reproductive fitness of Orius laevigatus (Hemiptera: Anthocoridae) reared on factitious and artificial diets.

The developmental and reproductive fitness of the polyphagous predator Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) was compared on two factitious foods and four artificial diets. Adults fed factitious foods (Ephestia kuehniella Zeller eggs and Artemia franciscana Kellogg cysts) performed better than those fed artificial diets. Among the artificial diets, a diet composed of liver and ground beef scored better than meridic diets based on egg yolk. Within the egg yolk-based artificial diets, the developmental fitness varied proportionally with the amount of egg yolk present in the diet. A food switching experiment, in which nymphs and adults of the predator were fed either E. kuehniella eggs or an egg yolk-based artificial diet, showed that the impact of adult food on reproductive capacity was greater than that of nymphal food. An optimal adult food was able to wholly compensate for deficiencies incurred by an inferior artificial diet in the nymphal stage. A strong correlation was found between oocyte counts, lifetime oviposition, and the number of eggs laid after 8 d. A rapid dissection assay may thus be effective to reliably and economically assess the fitness of O. laevigatus as a function of the diet. This method also may prove useful as part of quality assurance procedure for commercially produced predators.

Mechanism and potential of the growth-inhibitory actions of vitamin D and ana-logs.

1alpha,25-Dihydroxyvitamin D(3) [1,25-(OH) (2)D(3)] can exert its biological actions through binding with the nuclear vitamin D receptor (VDR), a ligand-activated transcription factor. Next to control of bone and mineral homeostasis, these actions include an immunomodulatory effect and a potent growth-inhibitory, antiproliferative or prodifferentiating action on a wide variety of cell types. The molecular mechanisms underlying this antiproliferative action form an intriguing research topic and they remain, although thoroughly studied, not completely understood. Important cell cycle regulators are involved such as cyclins, cyclin dependent kinases and their corresponding inhibitors as well as E2F transcription factors and accompanying pocket proteins. Whether 1,25-(OH)(2)D(3) influences the expression of all these proteins directly through the nuclear VDR or rather in an indirect manner is not always clear. The antiproliferative action makes 1,25-(OH) (2)D(3) a possible therapeutic tool to treat hyperproliferative disorders, among which different types of cancer. Clinical application, however, is severely hampered by calcemic effects such as hypercalcemia, hypercalciuria and increased bone resorption. Rational design of chemically modified 1,25-(OH) (2)D(3)-analogs tries to overcome this problem. As such, several thousands of analogs have been synthesized and evaluated, some of which display the desired dissociation between beneficial antiproliferative and unwanted calcemic effects. A number of those analogs are 'superagonistic' and have a several-fold stronger antiproliferative action than the parent compound. This review focuses on recent findings about the complex mechanisms behind the antiproliferative and prodifferentiating effect of 1,25-(OH) (2)D(3). Furthermore, the mode of action and possible clinical application of chemically modified 1,25-(OH) (2)D(3)-analogs will be discussed.

The effect of computer reminders on GPs' prescribing behaviour: a cluster-randomised trial.

OBJECTIVE: It is difficult to control drug-prescribing behaviour in general practice, despite the development and distribution of guidelines. The purpose of this study was to assess the effect on drug-prescribing behaviour of implementing prescribing guidelines by means of a reactive computer reminder system (CRS). DESIGN: Cluster-randomised controlled trial with an incomplete block design in the south of the Netherlands: 25 GPs (7 GP practices) received reminders about antibiotics and asthma/COPD prescriptions, 28 GPs (7 GP practices) received reminders about cholesterol prescriptions. Prescription guidelines were integrated into the computerised GP information system. MEASUREMENTS: Both performance indicators and prescription volumes were calculated as the main outcome measures. Next to individual volume measure, sum scores were constructed on the volume measures per drug group (antibiotics, asthma/COPD and cholesterol). RESULTS: Variation between GPs turned out to be larger and more skewed than expected. No differences between groups were found for indicators and volumes related to recommendations advocating certain drugs. Although there was a tendency towards clinically relevant results for prescription volumes that were supposed to drop, the difference in sum score between the groups was not significant. For antibiotic prescriptions that were supposed to drop, the sum score for the intervention group was 28.2 (95% CI: 20.8-44.5) prescriptions per 1000 patients per GP, while this was 39.7 (95% CI: 29.7-64.1) for the control group (p 0.2). For prescriptions asthma/COPD that were supposed to drop, the sum score for the intervention group was 1.1 (95% CI: 0.6-2.6) prescriptions per 1000 patients per GP, while this was 2.2 (95% CI: 1.4-4.3) for the control group (p 0.1). On three specific recommendations (on quinolones for cystitis, corticosteroids for CPOD, and antibiotics for acute sore throat) significant differences were found. CONCLUSIONS: This study turned out to be underpowered due to high inter doctor variation in prescribing behaviour. Nevertheless, computerised reminders sometimes have a favourable effect on restricting certain drugs that are not or no longer indicated in general practice.

Three-Layer Model for the design of a Protocol Support System.

OBJECTIVE: The aim of the PropeR project is to investigate the impact of Active Computerized Protocol Support (ACPS) on daily care processes in different settings (home care and hospital care). ACPS consists of an active Protocol Support System (PSS) that is linked to an Electronic Patient Record system. The aim of this paper is to describe how we have taken the organizational and social aspects into account in the hospital setting and the consequences of this approach for the design of the PSS. METHODS: Socio-technical approaches have been applied. Observations and interviews with various health care providers were performed at the hematology and oncology department of the University Hospital Maastricht. Ten extensive sessions with a specialist physician and research nurse took place to further elaborate a study protocol and to discuss how it is integrated in daily practice. The knowledge editor component of Gaston was used to build a computer interpretable version of the selected protocol. RESULTS AND CONCLUSIONS: To support the representation of a study protocol integrated in routine clinical care, a Three-Layer Model was developed. This model distinguishes the protocol description, local adaptations to the protocol and communication as three separate layers. These layers have been incorporated into the knowledge acquisition tool Gaston. The Three-Layer Model makes easy updating possible, and also supports transferability of computerized (study) protocols to other organizations.

Nymphal development and feeding preference of Podisus maculiventris (Heteroptera: Pentatomidae) on eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) parasitised or not by Trichogramma brassicae (Hymenoptera: Trichogrammatidae)

Avaliou-se a predação de ninfas de Podisus maculiventris sobre ovos de Ephestia kuehniella parasitados (em fase de pupa) ou não por Trichogramma brassicae. Esses ovos foram colados em cartelas retangulares e oferecidos às ninfas de P. maculiventris como alimento. Esse Pentatomidae só atingiu a fase adulta quando se alimentou de ovos não parasitados desse Pyralidae, indicando que os mesmos podem ser usados como hospedeiro alternativo para criação desse predador. Ninfas de P. maculiventris que receberam ovos parasitados morreram antes do quarto estádio. Em teste de escolha, ninfas desse predador mostraram preferência por ovos não parasitados em vez de por aqueles parasitados que continham em seu interior uma pupa de T. brassicae. Esses resultados mostram que é possível associar o uso de P. maculiventris com liberações de T. brassicae em programas de controle de lepidópteros pragas.

Nymphal development and feeding preference of Podisus maculiventris (Heteroptera: Pentatomidae) on eggs of Ephestia kuehniella (Lepidoptera: Pyralidae) parasitised or not by Trichogramma brassicae (Hymenoptera: Trichogrammatidae)

Predation by Podisus maculiventris nymphs, a predatory pentatomid, was evaluated with eggs of the flour moth Ephestia kuehniella (Pyralidae), parasitised or not by Trichogramma brassicae (pupae stage). Eggs of this pyralid were glued on rectangular cardboard and presented to nymphs of P. maculiventris as food. The pentatomid successfully reached adult stage when feeding on unparasitised eggs, indicating that flour moth eggs can be used as a factitious food for rearing this predator. Pentatomid nymphs that received only parasitised eggs died before reaching fourth instar. In choice tests, P. maculiventris showed a preference for preying on unparasitised eggs of E. kuehniella rather than those containing pupae of T. brassicae. These results show that it is possible to combine the use of P. maculiventris with releases of T. brassicae in control programs of lepidopteran pests.

Avaliou-se a predação de ninfas de Podisus maculiventris sobre ovos de Ephestia kuehniella parasitados (em fase de pupa) ou não por Trichogramma brassicae. Esses ovos foram colados em cartelas retangulares e oferecidos às ninfas de P. maculiventris como alimento. Esse Pentatomidae só atingiu a fase adulta quando se alimentou de ovos não parasitados desse Pyralidae, indicando que os mesmos podem ser usados como hospedeiro alternativo para criação desse predador. Ninfas de P. maculiventris que receberam ovos parasitados morreram antes do quarto estádio. Em teste de escolha, ninfas desse predador mostraram preferência por ovos não parasitados em vez de por aqueles parasitados que continham em seu interior uma pupa de T. brassicae. Esses resultados mostram que é possível associar o uso de P. maculiventris com liberações de T. brassicae em programas de controle de lepidópteros pragas,

The PropeR way to support medical doctors in daily practice. Developing the protocol based DSS.

This paper describes the first phase of the development of a Protocol based Decision Support System (PDSS) that will be linked to an Electronic Patient Record system (EPR system). The protocol system will be pro-active: the physician will be automatically prompted from the EPR of a particular patient if the protocol that applies for that patient defines it necessary. The PropeR project studies the impact of a PDSS that is linked to an EPR on daily care processes. There are two areas of research: hospital and home care. This paper describes the application in the hospital. The protocol that is being computerized is a treatment protocol for Acute Myelogenous Leukaemia (AML) that also studies treatment alternatives (conventional versus experimental treatment). This paper based AML protocol has been translated into a formal representation. The KA-tool Gaston is used to make this representation. Twenty-eight subprotocols have been organized in a hierarchical structure with three levels. One of the aims of the project is to make a representation of the AML protocol that can be used in other organizations as well. The main problem we encountered is that the representation not only contains the content of the protocol, but also aspects of application of that protocol in daily care of the hospital and aspects of support. The solution to this problem is the creation of two layers of representation: the first layer is an exact copy of the protocol and thus sharable and the second layer focuses on the support of the protocol in the daily working processes and is mainly domain specific: for the University Hospital Maastricht. At the moment, this division into two layers is being discussed.

Interaction of two novel 14-epivitamin D3 analogs with vitamin D3 receptor-retinoid X receptor heterodimers on vitamin D3 responsive elements.

This study provides a detailed and exact evaluation of the interactions between vitamin D3 receptor (VDR), retinoid X receptor (RXR), and vitamin D3 responsive elements (VDREs) mediated by two novel 14-epianalogs of 1,25-dihydroxyvitamin D [1,25(OH)2D3], 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527). Both analogs were more potent (14- and 75-fold, respectively) than 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation. However, DNA-independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA-dependent assays such as gel shift experiments revealed that those analogs were slightly more potent (3-7 times) than 1,25(OH)2D3 in enhancing binding of VDR-RXR heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE. The functional consequences of the ligand-VDR-RXR-VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14-epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)2D3 in COS-1 and MCF-7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell-specific process because maximal transcriptional induction and the half-maximal transactivation concentration for each reporter construct were different in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF-7 cell proliferation. These data clearly indicate that superagonistic activity starts beyond the binding of the ligand-heterodimer (VDR-RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.

Development of analogues of 1alpha,25-dihydroxyvitamin D3 with biased side chain orientation: methylated des-C,D-homo analogues.

The discovery that 1alpha,25-dihydroxyvitamin D3 is effective in the inhibition of cellular proliferation and in the induction of cellular differentiation has led to a search for analogues in which these activities and the classical calcemic activity of this hormone are separated. In this context, the synthesis and biological evaluation are reported of the three stereoisomeric CD-ring modified structural analogues in order to enforce a particular and different orientation of the 25-hydroxylated side chain. Comparison of the results of the biological evaluation and conformational analysis of the side chain suggests one defined and "active" geometry.

An unusual metastatic motilin-secreting neuroendocrine tumour with a 20-year survival. Pathological, biochemical and motility features.

Motilin-secreting neuroendocrine tumours have been rarely described. Immunohistochemical, biochemical and motility investigations were performed in a 62-year-old man with liver and bone metastases of a motilin-secreting neuroendocrine tumour originating from a rectal polyp removed 14 years previously. Symptoms related to liver metastases were reduced by a right hepatectomy whereas plasma motilin levels were decreased. The patient also underwent two operations for spinal cord decompression and survived 6 more years under medical treatment, mainly octreotide. Immunohistochemistry revealed predominant expression of motilin-containing cells, with rare cells expressing somatostatin and pancreatic polypeptide, and staining for only one panendocrine marker, neurone-specific enolase. A liver tumour extract contained 17.9 microg motilin per gram of tissue, which permitted to isolate and characterize human motilin, which was identical to porcine motilin. Plasma column gel chromatography revealed a main peak corresponding apparently to porcine motilin. The patient had no symptoms of disturbed motility. Gastric emptying and gastroduodenojejunal motility were found within normal limits. The absence of alterations of gut motility was perhaps related to sustained autonomous motilin production. The long evolution of this type of tumour suggests that plasma motilin determination should be added to the investigations for neuroendocrine tumours.

Two novel 14-Epi-analogues of 1,25-dihydroxyvitamin D3 inhibit the growth of human breast cancer cells in vitro and in vivo.

The biological activity of two novel 14-epi-analogues of 1,25(OH)2D3, 19-nor-14-epi-23-yne-1,25(OH)2D3 (TX 522) and 19-nor-14,20-bisepi-23-yne-1,25(OH)2D3 (TX 527), is described. Both analogues were at least 10 times more potent than 1,25(OH)2D3 in inhibiting in vitro cell proliferation and had much lower in vivo calcemic effects than 1,25(OH)2D3. Treatment with 1,25(OH)2D3, TX 522, or TX 527 in vitro was accompanied by an accumulation of cells in the G1 phase of the cell cycle. Protein levels of cyclin C and cyclin D1 in in vitro cultures of MCF-7 cells were down-regulated to 50 and 30%, respectively, of control levels at 72 and 120 h after stimulation. Protein levels of p21 and p27 at 72 h were significantly enhanced by 1,25(OH)2D3 and TX 522 but surprisingly not by TX 527. The inability of TX 527 to up-regulate p21 seemed to be cell type specific because p21 was induced in other cell types. Diminished phosphorylation of the retinoblastoma protein after treatment with 1,25(OH)2D3, TX 522, or TX 527 may ultimately contribute to the growth inhibition caused by these compounds. According to the data presented, the induction of apoptosis seemed not to be a major mechanism responsible for the growth-inhibitory effect of 1,25(OH)2D3 and analogues. Both 14-epianalogues significantly retarded tumor progression (40% reduced compared with control mice) in an in vivo model of MCF-7 breast cancer cells established in nude mice. In conclusion, these novel analogues have the eligible profile to be tested as therapeutic agents for the treatment of hyperproliferative diseases such as breast cancer.

Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1alpha,25-dihydroxyvitamin D analogues, diastereomeric at C17 and C20.

The synthesis of four CD-ring-modified 19-nor-1alpha, 25-dihydroxyvitamin D(3) derivatives lacking C15, referred to as 6C analogues, and diastereomeric at C17 and C20 is described. The synthesis involves an Ireland-Claisen rearrangement of a 3-methyl-substituted ester of (1R)-3-methyl-2-cyclohexen-1-ol as the key step, followed by elaboration of the side chain, transformation into a C8 cyclohexanone derivative, and final Wittig-Horner coupling with the 19-nor A-ring phosphine oxide. Despite possessing a more flexible side chain than the parent hormone, biological evaluation showed an unexpected superagonistic antiproliferative and prodifferentiating activity (10-50 times higher as compared to that of 1alpha,25(OH)(2)D(3)) for the diastereomer with the "natural" configuration at C17 and C20. The other diastereomers exhibit a 25-90% decrease in activity. All four analogues show a decreased binding affinity (45% or less), and their calcemic activity is 4-400 times less than that of 1alpha,25(OH)(2)D(3). The conformational behavior of their side chain was studied using molecular mechanics calculations, and the result is presented as volume maps. A relative activity volume was determined by subtraction of the volume map of the least active analogue from the volume map of the most active one. This shows three regions corresponding to preferred orientations in space of the side chain of the active analogue. One of these regions was found to overlap with the region that is preferentially occupied by the most active of the four diastereomeric 22-methyl-substituted 1alpha,25(OH)(2)D(3) analogues.

The biological activity of nonsteroidal vitamin D hormone analogs lacking both the C- and D-rings.

1alpha,25-dihydroxyvitamin D is a key calcium-regulating hormone but also displays potent differentiating and antiproliferative activities on many cell types. The structural requirements of this secosteroid hormone have been extensively studied for the A-ring and side chain, whereas relatively little is known about the requirements of the natural CD-ring structure for the vitamin D-like biological activity. We have embarked on a vast program in which derivatives were synthesized and evaluated characterized by profound structural changes in the central C/D-region. This first series of nonsteroidal analogs consists of (1R,3S)-5-((Z,2E)-4-((1S,3S)-3-(4-hydroxy-4-methylpentyl)-1,2,2-++ +trimethylcyclopentyl)-2-butenylidene)-4-methylenecyclohexan e-1,3-diol (KS 176) and derivatives thereof. These analogs are characterized by the absence of normal C- and D-rings and by the presence of an unnatural five-membered ring which we call the E-ring. KS 176 with the otherwise natural side chain structure of 1alpha,25(OH)2D3 has between 10 and 30% of the biological activity of 1alpha,25(OH)2D3 when tested in vitro (prodifferentiating effects on HL-60 and MG-63; antiproliferating activity on MCF-7 and keratinocytes) but has minimal in vivo calcemic effects. Introduction of several side chain modifications created analogs with increased intrinsic noncalcemic biological properties, whereas their calcemic potency remains very low. These data demonstrate that the full CD-rings are not mandatory for the biological activity of 1alpha,25(OH)2D3 since they can be replaced by a new ring structure which generates an appropriate spacing of the A-seco B-rings in relation to the side chain. The biological activity of these nonsteroidal analogs probably involves a classical genomic activation since they are also active in transfection assays using an osteocalcin vitamin D responsive element coupled to a human growth hormone reporter gene.

Isolation and sequencing of the cDNA encoding the motilin precursor from sheep intestine.

The nucleotide sequence of sheep prepromotilin has been determined from cDNA clones. The nucleotide sequence revealed an open reading frame of 345 nucleotides encoding 115 amino acids. The amino acid sequence deduced from the nucleotide sequence consists of a 25 amino acid signal peptide, followed by the 22 amino acid motilin sequence, an endoproteinase cleavage site (Lys23-Lys24) and a 66 amino acid motilin associated peptide (MAP). Compared with human and pig motilin we observed two substitutions at positions 10 (Leu-->Val) and 19 (Asn-->Tyr). The second one may explain the poor cross-reactivity of ovine motilin with C-terminally directed antibodies against porcine motilin. The sheep motilin precursor exhibits the same structure as the motilin precursors from rabbit, pig and man. However, while there is considerable identity in the amino acid sequences as well as in the nucleotide sequences of the signal peptide and motilin, the MAP strongly differs between the species. This may be a result of 'mosaic evolution' at the molecular level.

Biological evaluation of epoxy analogs of 1 alpha,25-dihydroxyvitamin D3.

The biological activity of 16-epoxy side-chain analogs of 1 alpha,25-dihydroxyvitamin D3, (1 alpha,25(OH)2D3) was evaluated in vitro and in vivo. Compared to 1 alpha,25(0H)2D3, all analogs had lower affinities for the pig duodenal vitamin D receptor and also for the human serum vitamin D binding protein. The in vitro effects on cell proliferation or differentiation of human promyeloid leukemia (induction of superoxide production in HL-60 cells), human osteosarcoma MG-63 cells (osteocalcin secretion), or human breast cancer cells (incorporation of thymidine in MCF-7 cells), was markedly inhibited by several epoxy analogs, compared to 1 alpha,25(OH)2D3, but the rank order of their activity widely varied among different cancer cells. The most potent analogs (24S,25S-24-hydroxy-25,26-epoxy-22-ene-1 alpha-OHD3, 25,26-epoxy-23-yne-1 alpha-OHD3, and 25,26-epoxy-23-yne-20-epi-1 alpha-OHD3 or compounds, 16, 5, and 7, respectively) were equipotent (16 and 5) or 30-fold (compound 7 on MG-63 cells) to 40-fold (compound 7 on MCF-7 cells) more active than 1 alpha,25-(OH)2D3. These analogs were nevertheless poorly antirachitic (< 3%) when tested in vitamin D-deficient chicks (using serum and bone calcium, serum osteocalcin and duodenal calbindin D-28K, as end points). Compound 7 was also 100-fold more active than 1 alpha,25-(OH)2D3 in inhibition of proliferation of human foreskin keratinocytes. Some epoxy analogs of 1 alpha,25-(OH)2D3 thus display interesting dissociations between their receptor affinity and their potency to induce cell differentiation, whereas their effect on cell proliferation/differentiation exceed their calcemic effects more than 100- to 1000-fold.

Purification and amino acid sequence of human motilin isolated from a motilin containing liver metastasis.

The acid extract of a liver metastasis from a patient with elevated plasma motilin levels contained large quantities of motilin (3.37 micrograms/ml). The extract was concentrated on a C18-column and motilin was isolated by gel chromatography (Sephadex G-50) followed by cation ion exchange chromatography (HR5/5 Mono-S) and three successive steps of reverse phase chromatography (Nucleosil 300-5 C18). The pure peptide was sequenced and the identity of porcine and human motilin was confirmed. This is the first report of a tumor containing large amounts of motilin.

The biological activity of 23-oxa-, 23-oxa-24-oxo-, and 23-thia-dihydroxyvitamin D3.

Three analogs of 1 alpha,25-(OH)2D3 with an oxygen or another heteroatom at position 23 were synthesized in search of separating the cell-differentiating from the calcemic effects of the vitamin D hormone. Their ability to induce superoxide production in human myeloid leukemia cells (HL-60) was 1 alpha,25-(OH)2D3 > 23-oxa-24-oxo-1 alpha,25-(OH)2D3 > 23-thia-1 alpha,25-(OH)2D3 > 23-oxa-1 alpha, 25-(OH)2D3. 23-oxa-24-oxo-1 alpha, 25(OH)2D3 was slightly more potent than 1 alpha,25-(OH)2D3 in inhibiting cell proliferation in MCF-7 cells and 23-thia- and 23-oxa-1 alpha,25(OH)2D3 were less potent. Their in vitro potency to produce osteocalcin in MG-63 cells was 1 alpha,25-(OH)2D3 > 23-oxa-24-oxo-1 alpha,25-(OH)2D3 > 23-thia-1 alpha,25-(OH)2D3 = 23-oxa-1 alpha,25-(OH)2D3. All three analogs had reduced receptor and DBP affinity compared to 1 alpha,25-(OH)2D3. When these analogs were injected in rachitic chicks, only little calcemic effects were observed. The introduction of a heteroatom in carbon 23 of 1 alpha,25-(OH)2D3 thus creates analogs with dissociated action on cell differentiation and calcium homeostasis.

Biologic activity of dihydroxylated 19-nor-(pre)vitamin D3.

Vitamin D3 and its hydroxylated metabolites are normally in thermal equilibrium with their previtamin D isomers. To evaluate the biologic activity of 1 alpha, 25-dihydroxyprevitamin D3, we synthesized 19-nor analogs of 1 alpha, 25-dihydroxy(pre)vitamin D3 because the absence of a C19 methylene group prevents the isomerization of these analogs. The affinity of 1 alpha, 25-(OH)2D3-19-nor-D3 for the intestinal vitamin D receptor and plasma vitamin D binding protein was mildly decreased [30 and 20% of the affinity of 1 alpha, 25-(OH)2D3, respectively], but the affinity of 1 alpha, 25-(OH)2-19-nor-previtamin D3 was only 1 and 6% of that of 1 alpha, 25-(OH)2D3 for the receptor and DBP, respectively. The in vitro effects on human promyeloid leukemia (HL-60 cell) differentiation and osteocalcin secretion by human osteosarcoma (MG-63) cells by 1 alpha, 25-(OH)2-19-nor-D3 were nearly identical to those of 1 alpha-25-(OH)2D3, whereas 19-nor-previtamin D3 showed poor activity (2%). The in vivo calcemic effects of both analogs, studied in vitamin D-deficient chicks treated for 10 consecutive days with the analogs, showed no activity of the previtamin D3 analog and reduced calcemic effects (< or = 10%) of 1 alpha, 25-(OH)2-19-nor-D3. We conclude that the previtamin D form of 1 alpha, 25-(OH)2D3 has lost most of its biologic activity in vitro and in vivo.

Structure function analysis of vitamin D analogs with C-ring modifications.

Analogs of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) with substitutions on C-11 were synthesized. Small apolar substitutions (11 alpha-methyl, 11 alpha-fluoromethyl) did not markedly decrease the affinity for the vitamin D receptor, but larger (11 alpha-chloromethyl or 11 alpha- or 11 beta-phenyl) or more polar substitutions (11 alpha-hydroxymethyl, 11 alpha-(2-hydroxyethyl] decreased the affinity to less than 5% of that of 1 alpha,25-OH)2D3. Their affinity for the vitamin D-binding protein, however, increased up to 4-fold. The biological activity of 11 alpha-methyl-1 alpha,25-(OH)2D3 closely resembled that of the natural hormone on normal and leukemic cell proliferation and bone resorption, whereas its in vivo effect on calcium metabolism of the rachitic chick was about 50% of that of 1 alpha,25-(OH)2D3. The 11 beta-methyl analog had a greater than 10-fold lower activity. The differentiating effects of the other C-11 analogs on human promyeloid leukemia cells (HL-60) agreed well with their bone-resorbing activity and receptor affinity, but they demonstrated lower calcemic effects in vivo. Large or polar substitutions on C-11 of 1 alpha,25-(OH)2D3 thus impair the binding of the vitamin D receptor but increase the affinity to vitamin D-binding protein. The effects of many C-11-substituted 1 alpha,25-(OH)2D3 analogs on HL-60 cell differentiation exceeded their activity on calcium metabolism.