Transcriptional profiling of human osteoblast differentiation.

Osteoblast differentiation is a key aspect of bone formation and remodeling. To further our understanding of the differentiation process, we have developed a collection of conditionally immortalized adult human osteoblast cell lines representing discrete stages of differentiation. To evaluate changes in gene expression associated with differentiation, polyA((+)) RNA from pre-osteoblasts, early and late osteoblasts, and pre-osteocytes was subjected to gene chip analysis using the Affymetrix Hu6800 chip in conjunction with an Affymetrix custom chip enriched in bone and cartilage cDNAs. Overall, the expression of 47 genes was found to change threefold or more on both chips between the pre-osteoblastic and pre-osteocytic stages of differentiation. Many of the observed differences, including down-regulation of collagen type I and collagen-processing enzymes, reflect expected patterns and support the relevance of our results. Other changes have not been reported and offer new insight into the osteoblast differentiation process. Thus, we observed regulation of factors controlling cell cycle and proliferation, reflecting decreased proliferation, and increased apoptosis in pre-osteocytic cells. Elements maintaining the cytoskeleton, extracellular matrix, and cell-cell adhesion also changed with differentiation reflecting profound alterations in cell architecture associated with the differentiation process. We also saw dramatic down-regulation of several components of complement and other immune response factors that may be involved in recruitment and differentiation of osteoclasts. The decrease in this group of genes may provide a mechanism for controlling bone remodeling of newly formed bone. Our screen also identified several signaling proteins that may control osteoblast differentiation. These include an orphan nuclear receptor DAX1 and a small ras-related GTPase associated with diabetes, both of which increased with increasing differentiation, as well as a high mobility group-box transcription factor, SOX4, that was down-regulated during differentiation. In summary, our study provides a comprehensive transcriptional profile of human osteoblast differentiation and identifies several genes of potential importance in controlling differentiation of osteoblasts.

Prevalence of abnormal adrenocortical function in human immunodeficiency virus infection by low-dose cosyntropin test.

Recent evidence suggests that 10 microg cosyntropin test has higher sensitivity for detecting hypothalamus-hypophysis-adrenal axis (HHA-A) dysfunction. Our objective was to determine prevalence of glucocorticoid insufficiency with the 10 microg cosyntropin test and the level of the HHA-A defect. One hundred and four HIV-infected patients underwent the 10 microg cosyntropin test. In abnormal and borderline respondents, insulin-induced hypoglycaemia test and human corticotropin releasing hormone test were used to confirm and localize the level of the HHA-A defect. Thirty-two patients with HIV infection and 72 with AIDS were identified. Prevalence of glucocorticoid insufficiency by the 10 microg cosyntropin test was 21.2%. By clinical categories, the frequency in AIDS and HIV infection patients was 26.4% and 9.4%, respectively. Confirmed glucocorticoid insufficiency by insulin-induced hypoglycaemia test was found in 16 out of 19 cases. Twelve cases had primary glucocorticoid insufficiency, 7 had secondary glucocorticoid insufficiency and 3 were false positive. In conclusion, adrenocortical dysfunction occurs in approximately 20% of the cases with HIV disease. Clinical findings commonly occurring in HIV disease as well as adrenocortical insufficiency are not reliable indicators for performing adrenocortical laboratory assessment. Our results suggest screening all AIDS patients with the 10 microg cosyntropin test.

Synthesis and estrogenic activities of novel 7-thiosubstituted estratriene derivatives.

A diastereomerically pure series of 7alpha-thioestratrienes was prepared and evaluated for its affinity for both the human estrogen receptor alpha and the more recently discovered estrogen receptor beta. The functional estrogenic activities of the compounds were measured in a MCF-7 ERE-tk-luciferase assay. The activities and selectivities of the compounds were sensitive to the nature of the thioether side chain.

Immunohistochemical localization of Galphaq, PLCbeta, Galphai1-2, PKA, and the endothelin B and extracellular Ca2+-sensing receptors during early amelogenesis.

Antibodies specific to Galphaq, PLCbeta, Galphai 1-2, and PKA were immunohistochemically (IHC) localized in the pre-ameloblasts up to initial dentin matrix deposition and continued in the distal ends of the pre-secretory ameloblasts to the beginning of enamel matrix secretion. It was hypothesized that the endothelin B receptor (ETBR) and/or the extracellular Ca2+-sensing receptor (CaR) would localize in the same locations as their known downstream signal transduction pathway (STP) effectors during events related to early amelogenesis. Localization was similar for the 4 signal transduction pathway elements and the CaR. The ETBR was not localized in any of the cells of the enamel organ. These findings indicate that the CaR and its related STPs are expressed in the pre-ameloblasts and pre-secretory ameloblasts in positions where they may be able to detect increases in extracellular Ca2+ concentrations observed in the pre-dentin matrix in a previous study. These observations are consistent with the hypothesis that increased levels of free Ca2+ in the pre-dentin matrix serve as a primary signal for modification of gene expression important to amelogenesis.

Antiviral activity and mechanism of action of calanolide A against the human immunodeficiency virus type-1.

Calanolide A, recently discovered in extracts from the tropical rainforest tree, Calophyllum lanigerum, is a novel inhibitor of the human immunodeficiency virus (HIV) type 1. The compound is essentially inactive against strains of the less common HIV type 2. The present study focused on the further characterization of the selective antiviral activity and mechanism of action of calanolide A. The compound inhibited a wide variety of laboratory strains of HIV type 1, with EC50 values ranging from 0.10 to 0.17 microM. The compound similarly inhibited promonocytotropic and lymphocytotropic isolates from patients in various stages of HIV disease, as well as drug-resistant strains. Viral life-cycle studies indicated that calanolide A acted early in the infection process, similar to the known HIV reverse transcriptase (RT) inhibitor 2', 3'-dideoxycytidine. In enzyme inhibition assays, calanolide A potently and selectively inhibited recombinant HIV type 1 RT but not cellular DNA polymerases or HIV type 2 RT within the concentration range tested. Serial passage of the virus in host cells exposed to increasing concentrations of calanolide A yielded a calanolide A resistant virus strain. RT from the resistant virus was not inhibited by calanolide A but retained sensitivity to other nonnucleoside as well as nucleoside RT inhibitors, including 3'-azido-2',3'-dideoxythymidine triphosphate and nevirapine. The study substantially supports the conclusion that calanolide A represents a novel subclass of nonnucleoside RT inhibitor which merits consideration for anti-HIV drug development.

Immunohistochemical localization of signal transduction pathways during amelogenesis: an initial exploration.

This study was undertaken to map signal transduction pathway (STP) components uniquely associated with the four major receptor groups and their related STPs in association with the events involved in amelogenesis in the rat. Whole-head, freeze-dried sagittal sections were obtained at the level of the maxillary first molars and picked up on transparent adhesive tape. The sections were not decalcified or fixed, providing optimum conditions for immunohistochemical (IHC) localization. Antibodies to pathway components Gs alpha, Gi alpha, Gq alpha, Sos-1, Grb-2, p125Fak, Jak2, and Vav were localized. The respective patterns of localization indicate that the Gq alpha-linked, the receptor tyrosine kinase-initiated, and the integrin receptor-initiated pathways are involved in the proliferating pre-ameloblast cells. In the differentiating and differentiated ameloblasts, the Gs alpha-linked cAMP pathway is involved, apparently reading a factor(s) released by the dentin matrix. The Gq alpha-linked, the receptor tyrosine kinase-initiated, the integrin receptor-initiated, and the cytokine receptor-initiated pathways are also up-regulated in the proximal ends of the ameloblasts. These observations indicate that all four of the major receptor groups are involved in amelogenesis and that the role of classes of ligands not previously implicated in enamel formation must now be considered. It seems that the cells of the enamel organ respond to the appearance and disappearance of autocrine and paracrine growth factors, but they also up-regulate specific STPs to enable them to respond to circulating hormones and growth factors whose concentrations in the extracellular fluids remain relatively constant.

Problems associated with estimation of net calcium uptake during enamel formation using 45Ca.

45Ca uptake in mineralizing tissues may occur by net Ca uptake or by isotopic exchange. It is rarely possible to differentiate between these effects, making interpretation of the findings difficult. Unfortunately, this problem is not often considered, and 45Ca uptake is usually regarded as representative of only net calcium uptake. The study reported here was undertaken to estimate the extent to which 45Ca uptake in mineralizing enamel is due to net Ca deposition or to isotopic exchange, and to consider the implications. The enamel surfaces of the lower incisors of adult rats were notched at the gingival line, and the eruption distance over 16 hours was measured. This distance was used to establish the position of a 0.3-mm-wide increment of enamel at the beginning and end of the 16-hour period, during which it passed through the early-maturation stage of enamel formation. The rate of Ca uptake was determined by chemical assay. Other rats were injected with 45Ca, mean plasma specific activity values for the experimental period determined, and the rate of Ca uptake through the same area of enamel formation was estimated. The estimates were from two- to nearly ten-fold greater than those established by chemical assay, indicating that from 50 to 90% of the 45Ca uptake occurred by isotopic exchange. 45Ca uptake may indicate more about the labile state of Ca in mineralizing enamel than about the rate of mineral deposition.

Stages of emotion: an adult adoptee's postreunion perspective.

Increasing numbers of adoptees are searching for and finding their biological parents. This article is an attempt to alert counselors to the particular emotional state of adoptees postreunion. The theory presented is drawn from the author's personal experience and integrated with her perspective as a professional counselor. From these two vantage points, the author has concluded that there are four postreunion emotional stages: paralysis, eruption, loss and grief, and empowerment. Three factors have a direct bearing on the four stages: definition of reunion success, when reunion occurs, and the "female factor."

HIV-inhibitory natural products. 11. Comparative studies of sulfated sterols from marine invertebrates.

A total of 22 sulfated sterols isolated from marine sponges, ophiuroids (brittle stars), and asteroids (sea stars) were comparatively evaluated for their antiviral activity against HIV-1 and HIV-2. In general, sterols with sulfate groups at position 2, 3, or 6 were the most active, with EC50 values of 3-13 microM against HIV-1 (RF) and 2-8 microM against HIV-2 (CBL20). Those compounds which were sulfated on the sterol D ring were completely inactive against both HIV-1 and HIV-2. Overall, sulfated sterols active against HIV-1 were also active against HIV-2.

Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.

A semiautomated multiparameter approach for anti-HIV drug screening.

We are implementing a series of complementary assays for initial follow-up confirmation and prioritization of new active anti-HIV compounds identified by the U.S. National Cancer Institute's large-scale in vitro primary anti-HIV screen. Two different kinds of cellular viability assays, in addition to specific assays for total cellular DNA content, supernatant reverse transcriptase activity, p24 core antigen production and the synthesis of infectious HIV virions are all performed from a single well of a 96-well microtiter plate containing human host cells infected with HIV. Antiviral activities of several known prototype HIV inhibitors including 3'-azido,3'-deoxythymidine, 2',3'-dideoxycytidine, dextran sulfate and phorbol myristate acetate were compared in these multiparameter assays as a means of validation. Procedures to automate the method optimally, as well as to maximize the safety of the technicians working with HIV and HIV-infected cells have been emphasized. The resulting semiautomated, highly reproducible battery of assays yields a maximum amount of antiviral and cytotoxicity information from a minimum amount of sample. This is especially crucial when analyzing new synthetic compounds and natural product extracts or fractions where the available amounts of sample may be very limited.

Application of microwave irradiation to immunohistochemistry: preservation of antigens of the extracellular matrix.

Microwave irradiation as a means of fixation was evaluated for the preservation of extracellular matrix antigens such as collagen III, IV, fibronectin and laminin in both lung and liver specimens. Small tissue samples were placed in normal saline or periodate-lysine-paraformaldehyde (PLP) and irradiated for 30 sec to bring them to a temperature of 50 C. The tissue was then processed rapidly in a tissue processor adjusted to a 2 hr cycle and embedded in paraffin. Sections were immunostained. For comparison, routine cryostat sections as well as sections of formalin fixed tissue were used. Microwave irradiation in saline gave excellent morphological detail, comparable to that in formalin fixed tissue. All four antigens evaluated were well preserved without the necessity of prior pepsin digestion. Microwave fixation is promising for preservation of antigenicity and morphological detail, and considerably reduces the time required for processing.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on IgA serum and bile levels in rats.

Serum IgA is actively transported from blood to bile against a concentration gradient in the liver by the binding of dimeric IgA to secretory component, endocytosis and transport to the bile canaliculus by vesicles. As 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to elicit hepatotoxicity, the effects of TCDD on rat serum and bile IgA levels were investigated. Rats were orally administered 50 micrograms TCDD/kg body weight in 95% corn oil: 5% acetone. At days 5, 10, 15, 20 and 30 after treatment, rats were anesthetized and a cannula inserted into the bile duct for collection of bile. In addition, blood was drawn, and, after euthanasia, the liver and thymus weights were recorded. Enzyme-linked immunosorbent assay (ELISA) techniques were employed to determine IgA in serum and bile and IgG levels in serum. Rocket immunoelectrophoresis was carried out to support ELISA results. It was found that serum IgA increased with time while serum IgG remained unchanged. In addition, while serum IgA levels were increasing, there was a concomitant decrease in biliary IgA. Thymus and liver weight changes were also observed. The data indicate that TCDD affects hepatic clearance of serum dimeric IgA and suggests that liver damage may be reflected by increased serum levels of IgA.

Artifacts from pulsatile flow in MR imaging.

Previous investigators have examined the effect of blood flow on the apparent blood vessel signal intensity. These studies reported flow brightening and darkening effects within vessels. In this paper we have investigated another type of flow artifact, which originates from the pulsatile nature of blood flow. These flow artifacts have characteristic bright and dark "ghosting" patterns which appear close to small vessels, usually arteries, which are bright in slow flow. Similar to the amplitude-of-motion artifacts caused by patient motion (e.g., breathing and cardiac motion) the ghosting artifacts due to pulsatile flow are best characterized as frequency modulated spectral sidebands. The pulsatile artifacts can have both dark and bright structures and usually appear close to the "moving" vessel that generates the artifact. In this paper we present a study of the chief features of these pulsatile flow artifacts, and we develop a theoretical description of their origins in terms of "accidental" velocity-encodings that occur strongly in most magnetic resonance imaging sequences.

Verification and evaluation of internal flow and motion. True magnetic resonance imaging by the phase gradient modulation method.

We report qualitative and quantitative evaluation and verification studies of the bipolar phase gradient modulation method for true MR imaging of internal flow and motion velocities. Velocity encoding modulations provide speed-of-motion and direction-sensitive images using special phase-sensitive reconstructions. True motion MR imaging does not depend upon subject parameters, T1 or T2, nor upon selective active-volume time-of-flight calculations, nor is it limited strictly to fluid-flow velocities. Conventional MR sequences often induce strong accidental phase gradient modulations that can cause severe artifacts in conventional MR scans and limit the useful sensitivities of true motion MR. Multiple steps of velocity encoding allow resolution of separate elements of the velocity spectrum, and enable suppression of all such phase-artifact difficulties. Some view-to-view phase inconsistencies are intrinsic to the subject being scanned, e.g., strong motion variations during the heart cycle; limitations due to such effects require external modifications in the scanning, such as cardiac gating. Since conventional density information remains in the data, independent of velocity encoding modulations, we suggest a multiple encoding sequence and saving the MR raw data. These evaluations and verifications demonstrate exciting potential in clinical application for the phase gradient modulation method of true flow and motion MR imaging.