Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Syngeneic leukemia models using lentiviral transgenics

Animal models are necessary to study cancer and develop treatments. After decades of intensive research, effective treatments are available for only a few types of leukemia, while others are currently incurable. Our goal was to generate novel leukemia models in immunocompetent mice. We had achieved abilities for overexpression of multiple driving oncogenes simultaneously in normal primary cells, which can be transplanted and followed in vivo. Our experiments demonstrated the induction of primary malignant growth. Leukemia lines that model various types of leukemia, such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), were passaged robustly in congenic wild-type immunocompetent mice. These novel leukemia lines, which may complement previous models, offer the flexibility to generate tailored models of defined oncogenes of interest. The characterization of our leukemia models in immunocompetent animals can uncover the mechanisms of malignancy progression and offer a unique opportunity to stringently test anti-cancer chemotherapies.Subject terms: Cancer models, Haematopoietic stem cells, Leukaemia     

Prophylactic intervention in radiation-leukemia-virus-induced murine lymphoma by the biological response modifier polysaccharide K

Polysaccharide K (PSK) is a biological response modifier used for adjuvant immunotherapy of malignant diseases. We studied the potential applicability of PSK for preventing tumor progression using an experimental model of murine lymphoma. Mice inoculated with the radiation leukemia virus (RadLV) develop thymic lymphomas after a latency of 3–6 months. However, 2 weeks after virus inoculation, prelymphoma cells can already be detected in the thymus. We found that PSK treatment induced hyperresponsiveness to concanavalin A and heightened production of interleukin-2 (IL-2) and IL-4 in spleen cells of both control and prelymphoma mice. The response was transient and was accompanied with a dominant usage of T cells expressing Vβ8, but other T cell subsets were also stimulated by PSK. T lymphoma cells expressing Vβ8.2 underwent apoptosis when incubated with PSK. Treatment of RadLV-inoculated mice with PSK delayed the onset of overt lymphoma (and mortality) but could not protect the mice from the disease. Combined treatment with PSK and a RadLV-specific immunotoxin prevented synergistically the progression of the prelymphoma cells to frank lymphoma. The results suggest that PSK contains a superantigen-like component that selectively activates Vβ8⁺ T cells. Its administration prelymphoma mice interfered with the process of lymphoma progression.     

Diagnosing Growth Hormone Deficiency: Can a Combined Arginine and Clonidine Stimulation Test Replace 2 Separate Tests?

ObjectiveGiven the large number of false-positive growth hormone deficiency (GHD) diagnoses from a single growth hormone (GH) stimulation test in children, 2 different pharmacologic tests, performed on separate days or sequentially, are required. This study aimed to assess the reliability and safety of a combined arginine-clonidine stimulation test (CACST).MethodsThis was a retrospective, single-center, observational study. During 2017-2019, 515 children aged >8 years underwent GH stimulation tests (CACST: n = 362 or clonidine stimulation test [CST]: n = 153). The main outcome measures used to compare the tests were GH response (sufficiency/deficiency) and amplitude and timing of peak GH and safety parameters.ResultsPopulation characteristics were as follows: median age of 12.2 years (interquartile range [IQR]: 10.7, 13.4), 331 boys (64%), and 282 prepubertal children (54.8%). The GHD rate was comparable with 12.7% for CACST and 14.4% for CST followed by a confirmatory test (glucagon or arginine) (P = .609). Peak GH was higher and occurred later in response to CACST compared with CST (14.6 ng/mL [IQR: 10.6, 19.4] vs 11.4 ng/mL [IQR: 7.0, 15.8], respectively, P < .001; 90 minutes [IQR: 60, 90] vs 60 minutes [IQR: 60, 90], respectively, P < .001). No serious adverse events occurred following CACST.ConclusionOur findings demonstrate the reliability and safety of CACST in detecting GHD in late childhood and adolescence, suggesting that it may replace separate or sequential GH stimulation tests. By diminishing the need for the second GH stimulation test, CACST saves time, is more cost-effective, and reduces discomfort for children, caregivers, and medical staff.     

Phosphoinositide breakdown in isolated rat parotid membranes. Stimulation by cholinergic and alpha-adrenergic agonists

Parotid gland membranes labelled with [3H]inositol were challenged with the cholinergic agonist, carbamylcholine, or with epinephrine in the presence of propranolol. Both agonists caused a significant breakdown of labelled phosphoinositides (17.5%) in membranes suspended in Krebs-Ringer bicarbonate buffer. This effect was abolished by the respective antagonists, atropine or phentolamine. The carbamylcholine-induced breakdown of labelled phosphoinositides did not require cytosol. The addition of cytosol alone, or the exposure of membranes to a medium of low ionic strength caused a significant breakdown of phosphoinositides (10-40%). No further breakdown due to the addition of carbamylcholine was observed under these conditions. It is suggested that neurotransmitter-induced breakdown of phosphoinositides is effected by membrane-associated enzyme(s) and can be observed only in a medium of high ionic strength.     

Long-term heat adaptation results in an enhanced efficiency of muscarinically-induced water secretion in rat submaxillary glands

1. Carbamylcholine-induced 86Rb+ and 36Cl- efflux, as markers of calcium mobilization and water secretion, respectively, were studied during 30 days of heat acclimation (at 34 degrees C) in rat submaxillary gland slices using perifusion techniques. 2. The fractional rate of 36Cl- efflux was markedly elevated with acclimation, reaching its maximal level on day 30, while that of 86Rb+, after an initial rise, returned to non-acclimated control levels. The total carbamylcholine-induced efflux of both ions markedly increased throughout the 30 days' acclimation. 3. The rapid increase in ion fluxes was accompanied by a transient increase in Na+ concentrations in the gland and a decrease in the saliva. 4. The data suggest that the acclimation-induced increase in secretory capacity is bi-phasic: initially, a rapid transient rise in ion fluxes accompanies a transient rise in muscarinic receptor density (Kloog et al., 1985). 5. Long term acclimation is characterized by increased efficiency of the cellular secretory mechanism(s), as demonstrated by the chronically increased efflux of ions.     

Mechanism of membrane electrical response to thyrotropin-releasing hormone in Xenopus oocytes injected with GH3 pituitary cell messenger ribonucleic acid

TRH evoked a complex electrical membrane response in Xenopus laevis oocytes injected with either total cytosolic or poly(A)(+)-enriched RNA from GH3 pituitary cells but not in uninjected oocytes. A typical response consisted of a transient, rapid depolarizing current followed by a prolonged depolarizing current with superimposed current fluctuations. The reversal potentials of the rapid and the slow components of the response were -23.0 and -22.6 mV, respectively, and were markedly affected by CI- concentration indicating that the TRH response was mainly an increase in Cl- conductance. The response to TRH was dose dependent and was inhibited by the TRH antagonist, chlordiazepoxide. TRH caused rapid hydrolysis of labeled phosphatidylinositol 4,5-bisphosphate and a marked, prolonged increase in 45Ca2+ efflux from injected oocytes. The depolarizing response to TRH was not diminished in oocytes incubated in a Ca2(+)-free medium, but was inhibited by microinjection of EGTA. These data suggest that TRH evokes an electrophysiological response in oocytes injected with RNA from GH3 cells via activation of the same biochemical pathway that mediates its actions in GH3 cells. This pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate, forming inositol trisphosphate that causes mobilization of cellular Ca2+. We suggest that oocytes injected with GH3 cell RNA, because of their large size and easy access to their intracellular milieu, will be a useful intact cell model in which to define the molecular details of signal transduction by TRH.     

Processing of snake venom -amino acid oxidase during intracellular transport

The isoelectrofocusing patterns of -amino acid oxidase (LAO) from venom gland homogenates and of the secreted venom of Vipera palaestinae have been compared. The LAO isozyme profile of actively synthesizing gland spans over a wider range of pIs (4.8–6.0) and includes more variants as compared with the profile of the secreted venom. A basic shift of the isoelectrofocusing pattern of LAO obtained by treatment of the gland homogenate or the venom with neuraminidase indicates that sialic acid residues are responsible for the changes in the electronegativity of the isozymes. Analyses of subcellular fractions show that the microsomal fraction of the venom gland homogenate exhibits the highest multiplicity of molecular forms of LAO, whereas the fraction including the secretory granules has an isozyme profile similar to the venom. Double labelling experiments show that the newly synthesized LAO include isozymes which span over a wide range of pIs, whereas later on labelling of the more acidic isozymes is prominent. The results obtained may suggest that the sialic acid residues which are attached to LAO during its transport serve as “markers” for secretion.