Growth hormone receptor and insulin-like growth factor-I immunoreactivity in osteoclast-like cells during tooth eruption in the toothless (Osteopetrotic) rat following treatment with colony-stimulating factor-1

In the toothless (tl/tl) osteopetrotic rat, teeth form but fail to erupt. Treatment of tl/tl rats with colony-stimulating factor-1 (CSF-1) activates bone resorption by osteoclasts, permits tooth eruption, and up-regulates the immunoreactivity of bone marrow mononuclear cells to growth hormone receptor (GHr) and insulin-like growth factor (IGF)-I. This study examined the distribution of tartrate-resistant acid phosphatase (TRAP) and immunoreactivity for GHr and IGF-I in osteoclast-like cells located on the alveolar bone margin, adjacent to the lower first molar crown, in 14-day-old normal and tl/tl rats, following treatment with CSF-1. Osteoclast-like cells demonstrated a positive reaction for TRAP, GHr, and IGF-I in all groups. However, in tl/tl tissue, osteoclast-like cells were generally negative for GHr. There was no significant difference in the total number of TRAP, GHr, and IGF-I-positive osteoclast-like cells on the adjacent bone margin in normal, normal treated with CSF-1, and tl/tl rats. CSF-1 treatment of the tl/tl rat significantly increased the total number of osteoclast-like cells, which were positive for TRAP (p < 0.001), GHr (p < 0.05) and IGF-I (P < 0.01).

Nerve growth factor overexpression and autocrine loop in breast cancer cells

We show here that nerve growth factor (NGF), the canonical neurotrophic factor, is synthesized and released by breast cancer cells. High levels of NGF transcript and protein were detected in breast cancer cells by reverse transcription-PCR, Western blotting, ELISA assay and immunohistochemistry. Conversely, NGF production could not be detected in normal breast epithelial cells at either the transcriptional or protein level. Confocal analysis indicated the presence of NGF within classical secretion vesicles. Breast cancer cell-produced NGF was biologically active, as demonstrated by its ability to induce the neuronal differentiation of embryonic neural precursor cells. Importantly, the constitutive growth of breast cancer cells was strongly inhibited by either NGF-neutralizing antibodies or K-252a, a pharmacological inhibitor of NGF receptor TrkA, indicating the existence of an NGF autocrine loop. Together, our data demonstrate the physiological relevance of NGF in breast cancer and its potential interest as a marker and therapeutic target.

Genetic variation in foliar carbon isotope composition in relation to tree growth and foliar nitrogen concentration in clones of the F-1 hybrid between slash pine and Caribbean pine

The objectives of this study were: (1) to quantify the genetic variation in foliar carbon isotope composition (delta(13)C) of 122 clones of ca. 4-year-old F-1 hybrids between slash pine (Pinus elliottii Engelm var. elliottii) and Caribbean pine (Pinus caribaea var. hondurensis Barr.,et Golf.) grown at two field experimental sites with different water and nitrogen availability in southeast Queensland, Australia, in relation to tree growth and foliar nitrogen concentration (N-mass); and (2) to assess the potential of using delta(13)C measurements, in the foliage materials collected from the clone hedges at nursery and the 4-year-old tree canopies in the field, as an indirect index of tree water use efficiency for selecting elite F-1 hybrid pine clones with improved tree growth. There were significant differences in foliar delta(13)C between the nursery hedges and the 4-year-old tree canopies in the field, between the summer and winter seasons, between the two experimental sites, and between the upper outer and lower outer canopy positions sampled. This indicates that delta(13)C measurements in the foliage materials are significantly influenced by the sampling techniques and environmental conditions. Significant differences in foliar delta(13)C, at the upper outer canopy in both field experiments in summer and winter, were detected between the clones, and between the female parents of the clones. Clone means of tree height at age ca. 3 years were positively related to those of the upper outer canopy delta(13)C at both experimental sites in winter, but only for the wetter site in summer. There were positive, linear relationships between clone means of canopy delta(13)C and those of canopy N-mass, indicating that canopy photosynthetic capacity might be an important factor regulating the clonal variation in canopy delta(13)C. Significant correlations were found between clone means of canopy delta(13)C at both experimental sites in summer and winter, and between those at the upper outer and lower outer canopy positions. Mean clone delta(13)C for the nursery hedges was only positively related to mean clone stem diameter at 1.3 m height at age 3 years on the wetter site. The clone by site interaction for foliar delta(13)C at the upper outer canopy was significant only in summer. Overall, the relatively high genetic variance components for foliar delta(13)C and significant, positive correlations between clone means of foliar delta(13)C and tree growth have highlighted the potential of using foliar delta(13)C measurements for assisting in selection of the elite F-1 hybrid pine clones with improved tree growth. (C) 2002 Elsevier Science B.V. All rights reserved.

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Controversies regarding the effects of growth hormone on the heart

Growth hormone (GH) profoundly affects the developing and adult myocardium. Adult patients with GH deficiency (GHD) and GH excess (acromegaly) provide important models in which to understand the effects of GH in adult cardiac physiology. An increasing body of clinical and experimental evidence illustrates the specific physiological abnormalities that are likely associated with the excess cardiovascular mortality observed in both acromegaly and GHD. Because human GH replacement is now available to treat adults with GHD, new questions emerge about the long-term cardiovascular effects of replacement therapy. In multiple trials, GH therapy for congestive heart failure has been proved ineffective in the absence of preexisting GHD. Case reports suggest that, in the setting of GHD, GH therapy can exert a potent beneficial effect on congestive heart failure. Long-term studies addressing cardiovascular morbidity and mortality are needed to assess the role of GH therapy for GHD.

Inhibition of early tumor growth requires Jα18-positive (natural killer T) cells

The role of natural killer T (NKT) cells in the immune response to tumor cells has been largely unexplored. As a model of adoptive tumor immunotherapy, cells from the draining lymph nodes of mice immunized with a tumor-specific or irrelevant antigen were transferred to naive recipients with established tumor. Inhibition of early tumor growth (day 4) required the transfer of both CD8(+) and Jalpha18(+) (NKT) cells from immunized animals without regard to immunogen. In contrast, CD8(+) cells, but not Jalpha18(+) cells, were necessary for the inhibition of late tumor growth (day 8). Thus, the developing tumor changes in sensitivity to NKT-mediated events and the role for NKT cells cannot be replaced by the presence of tumor-specific cells during early tumor growth. This suggests that recruitment/activation of Jalpha18(+) NKT cells is an important consideration during the immune therapy of early stage tumors.

Genetic disruption of the growth hormone receptor does not influence motoneuron survival in the developing mouse

In the rodent central nervous system (CNS) during the five days prior to birth, both growth hormone (GH) and its receptor (GHR) undergo transient increases in expression to levels considerably higher than those found postnatally. This increase in expression coincides with the period of neuronal programmed cell death (PCD) in the developing CNS. To evaluate the involvement of growth hormone in the process of PCD, we have quantified the number of motoneurons in the spinal cord and brain stem of wild type and littermate GHR-deficient mice at the beginning and end of the neuronal PCD period. We found no change in motoneuron survival in either the brachial or lumbar lateral motor columns of the spinal cord or in the trochlear, trigeminal, facial or hypoglossal nuclei in the brain stem. We also found no significant differences in spinal cord volume, muscle fiber diameter, or body weight of GHR-deficient fetal mice when compared to their littermate controls. Therefore, despite considerable in vitro evidence for GH action on neurons and glia, genetic disruption of GHR signalling has no effect on prenatal motoneuron number in the mouse, under normal physiological conditions. This may be a result of compensation by the signalling of other neurotrophic cytokines.

Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3-F442A preadipocytes

Although it is the best characterized in vitro model of GH action, the mechanisms used by GH to induce differentiation of murine 3T3-F442A preadipocytes remain unclear. Here we have examined the role of three transcriptional regulators in adipogenesis. These regulators are either rapidly induced in response to GH [Stra13, signal transducer and activator of transcription (Stat) 3] or of central importance to GH signaling (Stat5). Retroviral transfection of 3T3-F442A preadipocytes was used to increase expression of Stra13, Stat3, and Stat5a. Only Stat5a transfection increased the expression of adipogenic markers peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein (C/EBP)alpha, and adipose protein 2/fatty acid-binding protein in response to GH, as determined by quantitative RT-PCR. Transfection with constitutively active Stat3 and Stat5a revealed that constitutively active Stat5a but not Stat3 was able to replace the GH requirement for adipogenesis. Constitutively active Stat5a but not Stat3 was able to increase the formation of lipid droplets and expression of alpha-glycerol phosphate dehydrogenase toward levels seen in mature adipocytes. Constitutively active Stat5a was also able to increase the expression of transcripts for C/EBPalpha to similar levels as GH, and of C/EBPbeta, peroxisome proliferator-activated receptor gamma, and adipose protein 2/fatty acid-binding protein transcripts to a lesser extent. An in vivo role for GH in murine adipogenesis is supported by significantly decreased epididymal fat depot size in young GH receptor-deleted mice, before manifestation of the lipolytic actions of GH. We conclude that Stat5 is a critical factor in GH-induced, and potentially prolactin-induced, murine adipogenesis.

Epitope map for a growth hormone receptor agonist monoclonal antibody, MAb 263

Monoclonal antibody (MAb) 263 is a widely used monoclonal antibody that recognizes the extracellular domain (ECD) of the GH receptor. It has been shown to act as a GH agonist both in vitro and in vivo, and we report here that it must be divalent to exert its effect on the full-length receptor. To understand the mechanism of its agonist action, we have determined the precise epitope for this antibody using a novel random PCR mutagenesis approach together with expression screening in yeast. A library of 5200 clones of rabbit GH receptor ECD mutants were screened both with MAb 263 and with an anticarboxy-tag antibody to verify complete ECD expression. Sequencing for clones that expressed complete ECD but were not MAb 263 positive identified 20 epitope residues distributed in a discontinuous manner throughout the ECD. The major part of the epitope, as revealed after mapping onto the crystal structure model of the ECD molecule, was located on the side and upper portion of domain 1, particularly within the D - E strand disulfide loop 79 - 96. Molecular dynamics docking of an antibody of the same isotype as MAb 263 was used to dock the bivalent antibody to the 1528-Angstrom(2) epitope and to visualize the likely consequences of MAb binding. The minimized model enables the antibody to grasp two receptors in a pincer-like movement from opposite sides, facilitating alignment of the receptor dimerization domains in a manner similar to, but not identical with, GH.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Differences in lung function, growth parameters and frequency of hospital admissions between adolescents with cystic fibrosis-related diabetes and controls

As survival of patients with CF increases,glucose intolerance and cystic fibrosisrelated diabetes (CFRD),ar e increasingly recognised common complications. CFRD may be preceded by a pre-diabetic state. Using markers identified as being associated with CFRD may improve targeted screening. Aim: To identify features consistently predicting CFRD in paediatric patients. Patients diagnosed with CFRD between January 1997–January 2002 were compared with age and sex matched controls. Clinical,micr obiological, and hospitalisation data was collected at time of CFRD diagnosis,and at six monthly intervals for 3 yr prior to diagnosis. Eight patients with CFRD were identified,mean age 13.7 yr (S.D. 3.49) at time of diagnosis. Control patients underwent OGTT to ensure normal glucose tolerance. Patients with CFRD had a lower FEV1 up to 12 months prior to diagnosis however, this was only significant at diagnosis. There was no difference in weight and height z scores between the 2 groups; however,the decrease in weight and height z scores in the CFRD group over 3 yr prior to diagnosis was significant. Mean number of days in hospital and admissions per patient significantly increased in the CFRD group,6 months prior to diagnosis. No other significant differences were observed between the 2 groups. Conclusions: This study has shown a difference in lung function,gr owth parameters and frequency of hospital admissions between patients with CFRD and controls. These differences may be utilised as tools for targeted screening in the paediatricyadolescent population. Further larger scale studies are required to improve guidelines for targeted screening in this population.

Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt(-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates. (C) Wiley Periodicals, Inc.

Production of proteinases by psychrotrophic bacteria in raw milk stored at low temperature

Raw milk samples from two different sources were stored at 2degreesC, 4degreesC and 7degreesC for 10 days and the growth of psychrotrophic bacteria, production of proteinase and proteolysis in the milks were measured during storage. Peptide analyses by the fluorescamine method and RP-HPLC were used in determination of proteolysis and proteinase activity. The average times taken for the psychrotroph counts to reach 10(7) cfu/mL at 2degreesC, 4degreesC and 7degreesC were approximately 9, 7 and 4 days, although there was considerable variation in growth rates in the different milks. There was little correlation between psychrotroph counts and either proteolysis or proteinase activity levels. At 2degreesC, no milk stored showed significant proteolysis by the fluorescamine method after 10 days' storage, but significant proteinase activity could be measured in some of these milks at 8 and 10 days. RP-HPLC analysis was a more sensitive means of detecting peptides than the fluorescamine method.