1α,25-Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells

1α,25-Dihydroxyvitamin D₃ increases intracellular calcium in rat osteoblast-like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25-dihydroxyvitamin D₃ (20 nM). This effect was blocked by the inactive epimer 1β,25-dihydroxyvitamin D₃. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25-dihydroxyvitamin D₃, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25-dihydroxyvitamin D₃, 20 nM, had no effect on nuclear calcium but blocked the 1α,25-dihydroxyvitamin D₃ induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25-dihydroxyvitamin D₃. 1α,25-Dihydroxyvitamin D₃ rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

In situ hybridization analysis of polyoma DNA replication in an inducible line of polyoma-transformed cells.

In situ hybridization has been used to study polyoma DNA replication in a clonal derivative of the inducible LPT line of polyoma-transformed cells designated as clone 1A. This study has shown that in clone 1A cultures maintained under normal growth conditions, 4–25 in 10,000 cells are spontaneously induced to synthesize polyoma DNA at an enhanced rate. In cultures exposed to mitomycin C (MMC), the percentage of induced cells remains approximately equal to the spontaneous level for 9 hr, and then increases for at least 24 hr up to 30–57% as more and more cells are asynchronously recruited to replicate the virus DNA.DNA reassociation kinetics and in situ hybridization have been used to determine the amount and distribution of polyoma DNA accumulated within clone 1A cells. These measurements have shown that a single induced cell in an MMCtreated culture produces 24,500 genome-equivalents of the virus DNA; second, that the average yield of virus DNA in a normally growing culture is only 41.7 genome-equivalents per cell; however, a single spontaneously induced cell in this culture produces as much virus DNA as an MMC-induced cell; third, that all the virus DNA molecules are found within the nuclei and many are clustered in aggregates containing up to 2000 genome-equivalents. We discuss the implications of these findings regarding the regulation of polyoma DNA replication in the LPT line.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

Dexamethasone stimulates osteogenic differentiation in vertebral and femoral bone marrow cell cultures: Comparison of IGF-I gene expression

Osteoblast-like cell cultures have been established from the marrow of adult rat vertebrae. We have simultaneously examined the response to dexamethasone (dex) treatment in cultures of young adult female vertebral and femoral marrow cells. Alkaline phosphatase (AP) activity was analyzed as well as the expression of mRNAs for osteocalcin (OC) and insulin-like growth factor I (IGF-I). The vertebral and femoral marrow cells were maintained for 7 days in primary culture with or without 10⁻⁸ M dex and then 6 days in secondary culture without dex or with 10⁻⁸ M or 10⁻⁷ M dex. All cells were examined on day 6 of secondary culture. Vertebral and femoral cultures each expressed the highest AP enzyme levels when grown with dex in primary culture (10⁻⁸ M) and secondary culture (10⁻⁷ M). Under all experimental conditions, vertebral cultures had lower AP enzyme activity than femoral cultures. When dex was omitted from secondary culture, OC gene expression was not detected in either vertebral or femoral passaged cells even if dex was present in primary culture. For dex conditions where OC was expressed, vertebral cultures had higher OC mRNA steady-state levels than femoral cultures. IGF-I gene expression was detected by Northern analysis in both vertebral and femoral secondary cultures. However, vertebral marrow cultures had much higher IGF-I mRNA levels compared to femoral cultures whether or not dex was present in primary culture. These findings demonstrate that dex supports the differentiation of both vertebral and femoral adult marrow osteogenic cells into osteoblasts. Our results support the hypothesis that osteoblastic marrow cultures differ depending upon which location in the skeleton they are from and that there are skeletal site–dependent differences in the insulin-like growth factor system components. J. Cell. Biochem. 71:382–391, 1998. © 1998 Wiley-Liss, Inc.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

Mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models

Antitumor mAb bind to tumors and activate complement, coating tumors with iC3b. Intravenously administered yeast beta-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (iC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of iC3b-coated tumors. Recent data indicated that barley beta-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival. This investigation showed that orally administered yeast beta-1,3;1,6-glucan functioned similarly to barley beta-1,3;1,4-glucan with antitumor mAb. With both oral beta-1,3-glucans, a requirement for iC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3. Barley and yeast beta-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo. Orally administered beta-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow. Within the bone marrow, the macrophages degraded the large beta-1,3-glucans into smaller soluble beta-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes. These granulocytes with CR3-bound beta-1,3-glucan-fluorescein were shown to kill iC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb.     

What is new in the treatment of hypospadias?

Hypospadias is a congenital anomaly characterized by a ventrally placed urethral meatus in a more proximal position on the midline than its normal position in the glanular part of the penis. In 1961, C. E. Horton and C. J. Devine, Jr., developed single-stage modern surgical techniques, namely, local skin flaps and free skin grafts, for urethra reconstruction in hypospadias repair, which may be applied to almost any case with different localizations of the meatus. Later, two new methods, advancement of the urethra and preputial island flap techniques, were added to the surgical algorithm. Because acceptable results were observed, the authors have insisted on using these four techniques for all hypospadias cases since 1972. Complication rates (mainly fistula formation) were quite high (50 percent) in their early series of adults as a result of erection and hematoma formation. The complication rate of their patient population, which is now mainly composed of preschool children, has decreased to 7 to 8 percent, primarily as a result of careful selection of appropriate techniques for each individual case, the development of better surgical materials and equipment, and taking necessary precautions for postoperative care. A brief summary of modern hypospadias repair techniques is presented in four major classes. The results of the authors' 30-year experience and the precautions necessary to avoid postoperative complications are evaluated. The authors conclude that the four modern techniques and their modifications should be performed meticulously for successful hypospadias repair.     

Modulation of monocyte–tumour cell interactions by <Emphasis Type="Italic">Mycobacterium vaccae</Emphasis>

Immunotherapy with Mycobacterium vaccae as an adjuvant to chemotherapy has recently been applied to treatment of patients with cancer. One of the mechanisms of antitumour activity of Mycobacterium bovis bacillus Calmette-Guérin (BCG), the prototype immunomodulator, is associated with activation of monocytes/macrophages. These studies were undertaken to determine how M. vaccae affects monocyte–tumour cell interactions and, in particular, whether it can prevent or reverse deactivation of monocytes that occurrs following their contact with tumour cells during coculture in vitro. Deactivation is characterised by the impaired ability of monocytes to produce tumour necrosis factor (TNF-), interleukin 12 (IL-12), and enhanced IL-10 secretion following their restimulation with tumour cells. To see whether deactivation of monocytes can be either prevented or reversed, three different strains of M. vaccae—B 3805, MB 3683, and SN 920—and BCG were used to stimulate monocytes before or after exposure to tumour cells. Pretreatment of monocytes with M. vaccae MB 3683, SN 920 and BCG before coculture resulted in increased TNF- and decreased IL-10 production. All strains of M. vaccae and BCG used for treatment of deactivated monocytes enhanced depressed TNF- secretion. Strain SN 920 and BCG increased IL-12 release but only BCG treatment inhibited an enhanced IL-10 production by deactivated monocytes. Thus, although some strains of M. vaccae may either prevent or reverse tumour-induced monocyte deactivation, none of them appears to be more effective than BCG.