An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.     

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.     

A geostatistical analysis of small-scale spatial variability in bacterial abundance and community structure in salt marsh creek bank sediments

Small-scale variations in bacterial abundance and community structure were examined in salt marsh sediments from Virginia's eastern shore. Samples were collected at 5 cm intervals (horizontally) along a 50 cm elevation gradient, over a 215 cm horizontal transect. For each sample, bacterial abundance was determined using acridine orange direct counts and community structure was analyzed using randomly amplified polymorphic DNA fingerprinting of whole-community DNA extracts. A geostatistical analysis was used to determine the degree of spatial autocorrelation among the samples, for each variable and each direction (horizontal and vertical). The proportion of variance in bacterial abundance that could be accounted for by the spatial model was quite high (vertical: 60%, horizontal: 73%); significant autocorrelation was found among samples separated by 25 cm in the vertical direction and up to 115 cm horizontally. In contrast, most of the variability in community structure was not accounted for by simply considering the spatial separation of samples (vertical: 11%, horizontal: 22%), and must reflect variability from other parameters (e.g., variation at other spatial scales, experimental error, or environmental heterogeneity). Microbial community patch size based upon overall similarity in community structure varied between 17 cm (vertical) and 35 cm (horizontal). Overall, variability due to horizontal position (distance from the creek bank) was much smaller than that due to vertical position (elevation) for both community properties assayed. This suggests that processes more correlated with elevation (e.g., drainage and redox potential) vary at a smaller scale (therefore producing smaller patch sizes) than processes controlled by distance from the creek bank.     

Catch-up growth and endocrine changes in childhood celiac disease. Endocrine changes during catch-up growth

Childhood celiac disease may lead to a failure of statural growth. After institution of a gluten-free diet most patients exhibit catch-up growth. Catch-up growth is a remarkable phenomenon characterized by a supranormal height velocity. One of the hypothetical mechanisms of catch-up growth is that an increased activity of the somatotrophic axis is involved. In order to provide further insight in the physiology of catch-up growth, auxological and endocrine changes were prospectively studied in 28 children with newly diagnosed celiac disease. The results demonstrate a malnutrition-like state of the somatotrophic axis at the time of diagnosis and a rapid recovery of this axis towards normal functioning after institution of the gluten-free diet. Although several correlations between these endocrine alterations and auxological parameters were detected, it is questionable whether the endocrine changes are the driving force behind catch-up growth.     

Spontaneous nocturnal leptin secretion in children with myelomeningocele and growth hormone deficiency

OBJECTIVE: To examine the spontaneous leptin secretion in patients with myelomeningocele (MMC) and growth hormone deficiency (GHD). METHODS: Serum leptin levels were studied in 10 prepubertal MMC patients with GHD (CA 6.2 +/- 0.5 years), 10 patients with idiopathic GHD (IGHD; CA 7.6 +/- 0.7 years) and 12 children with normal variant short stature (NVSS; CA 7.6 +/- 0.5 years). Mean BMI (kg/m(2)) values of the groups did not differ significantly. Nocturnal leptin levels were analyzed over 10 h (blood samples every 20 min) and measured by specific radioimmunoassay. RESULTS: Mean leptin concentrations did not correlate with BMI in MMC patients. Nocturnal leptin secretion of MMC patients was significantly different to those of children with IGHD and NVSS. Morning leptin levels did not decline as observed in both other groups. CONCLUSION: Since all groups were matched for BMI values, we suggest a hypothalamic dysregulation of leptin secretion in MMC patients.     

T cell substance P receptor governs antigen-elicited IFN-gamma production

Substance P (SP) enhances antigen-dependent T cell IFN-gamma production. It was determined if a T cell neurokinin-1 receptor (NK-1R) was critical for IFN-gamma regulation. T cells from schistosome-infected mice were mixed with splenocytes from uninfected NK-1R knockout (KO) animals. Thus only the schistosome egg antigen-specific T cells expressed NK-1R. The cells were cultured 18 h with or without SP. SP enhanced antigen-induced IFN-gamma production fourfold without affecting IL-4 or IL-5 secretion. NK-1R inhibitor blocked this stimulation. Neither purified T cells nor naive KO splenocytes cultured alone responded to antigen. To further define the importance of T cell NK-1R, we developed a T cell-selective NK-1R KO mouse by reconstituting T cell-deficient Rag mice with NK-1R KO T cells. These mice challanged with schistosomiasis developed abnormal liver granulomas. Granuloma size was smaller in T cell-selective NK-1R KO mice compared with granulomas in Rag reconstituted with normal T cells. Splenocytes and granuloma cells from NK-1R KO mice made less IFN-gamma. The mice also made less IgG2a. Thus T cell NK-1R is important for IFN-gamma regulation.     

Mandibular gland chemistry of four Caribbean species of Camponotus (Hymenoptera: Formicidae)

The volatile components of whole-body extracts of males, females and workers were analyzed in four species of Neotropical ants in the formicine genus, Camponotus. The species, C. kaura, C. sexguttatus, C. ramulorum and C. planatus, represent three different subgenera. Volatile mandibular gland components were found only in male extracts in three of the species. In C. ramulorum, volatile components were found in male and female reproductives and workers. 3,4-Dihydro-3,5-dihydroxy-6-methylpyran-4-one and octanic acid were found in different sets of three of the species. Methyl 6-methyl salicylate was found in two species and the isocoumarin, mellein, was found in a third species. The significance of the mandibular gland secretion for formicid systematics is discussed.     

Leptin and metabolic control of reproduction

Leptin treatment prevents the effects of fasting on reproductive processes in a variety of species. The mechanisms that underlie these effects have not been elucidated. Progress in this area of research might be facilitated by viewing reproductive processes in relation to mechanisms that maintain fuel homeostasis. Reproduction, food intake, and fuel partitioning can be viewed as homeostatic responses controlled by a sensory system that monitors metabolic signals. These signals are generated by changes in intracellular metabolic fuel availability and oxidation rather than by changes in the amount of body fat or by changes in any aspect of body composition. Leptin might be viewed as either a mediator or as a modulator of the intracellular metabolic signal. Consistent with its purported action as a mediator of the metabolic signal, leptin synthesis and secretion are influenced acutely by changes in metabolic fuel availability, and these changes might lead to changes in reproductive function. The effects of leptin treatment on reproduction are blocked by treatments that inhibit intracellular fuel oxidation. Metabolic signals that inhibit reproduction in leptin-treated animals might act via neural pathways that are independent of leptin's action. Alternatively, both leptin and metabolic inhibitors might interact at the level of intracellular fuel oxidation. In keeping with the possibility that leptin modulates the metabolic signal, leptin treatment increases fuel availability, uptake, and oxidation in particular tissues. Leptin might affect reproduction indirectly by altering fuel oxidation or other peripheral processes such as gastric emptying. Reproductive processes are among the most energetically expensive in the female repertoire. Because leptin increases energy expenditure while simultaneously inhibiting energy intake, it may have limited use as a long-term treatment for infertility.