X-ray studies of water structure in 2 Zn insulin crystal

The water structure of rhombohedral 2 Zn insulin crystal which contains about 280 water molecules and 0.55-0.60 mol citrate molecules per dimer has been studied by X-ray crystallographic refinement with 1.1 A resolution data. Atomic parameters of 83 fully occupied and 258 partially occupied water molecules and 0.3 mol of citrate were obtained. Full matrix least-squares method with isotropic temperature factor was used for the refinement of partially occupied water molecules. The water molecules in this crystal exist in one of the three states: fully occupied water, partially occupied water and water continuum, and a schematic model of water structure in protein crystal was proposed. The flexibility of water molecules is described.     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.     

Arabidopsis GCP3-interacting protein 1/MOZART 1 is an integral component of the γ-tubulin-containing microtubule nucleating complex

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain γ-tubulin and a family of homologous γ-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of γ-tubulin 1 (MOZART1), a recently identified component of the γ-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis γ-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the γ-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis γ-tubulin complexes.     

Normalizing the bone marrow microenvironment with p38 inhibitor reduces multiple myeloma cell proliferation and adhesion and suppresses osteoclast formation

The multiple myeloma (MM) bone marrow (BM) microenvironment plays a critical role in supporting tumor growth and survival as well as in promoting formation of osteolytic lesions. Recent results suggest that the p38 mitogen-activated protein kinase (MAPK) is an important factor in maintaining this activated environment. In this report, we demonstrate that the p38alpha MAPK inhibitor, SCIO-469, suppresses secretion of the tumor-supportive factors IL-6 and VEGF from BM stromal cells (BMSCs) as well as cocultures of BMSCs with MM cells, resulting in reduction in MM cell proliferation. Additionally, we show that SCIO-469 prevents TNFalpha-induced adhesion of MM cells to BMSCs through an ICAM-1- and VCAM-1-independent mechanism. Microarray analysis revealed a novel set of TNFalpha-induced chemokines in BMSCs that is strongly inhibited by SCIO-469. Furthermore, reintroduction of chemokines CXCL10 and CCL8 to BMSCs overcomes the inhibitory effect of SCIO-469 on TNFalpha-induced MM adhesion. Lastly, we show that SCIO-469 inhibits secretion and expression of the osteoclast-activating factors IL-11, RANKL, and MIP-1alpha as well as prevents human osteoclast formation in vitro. Collectively, these results suggest that SCIO-469 treatment can suppress factors in the bone marrow microenvironment to inhibit MM cell proliferation and adhesion and also to alleviate osteolytic activation in MM.     

Sex differences in the excretion of fecal glucocorticoid metabolites in the Syrian hamster

We verified the relevance of measuring fecal glucocorticoid metabolites (FGM) to assess the stress response of the Syrian hamster. Male and female hamsters (n = 10 each) were submitted to an adrenocorticotropic hormone (ACTH) challenge test, whereas animals in the control group received 0.5 mL of sterile isotonic saline solution. All feces voided by each animal were collected at 4 h intervals from 24 h before (baseline) until 48 h after injections. FGM were quantified using an 11-oxoetiocholanolone enzyme immunoassay (EIA). Basal concentrations of FGM were almost four times higher in males than in females. Following ACTH administration, FGM levels started rising from 8 h onwards, reaching peak concentrations 20 or 28 h post injection in males and females, respectively. Despite the much higher absolute concentrations present in males, the relative increase (500%) in response to the ACTH stimulation was similar in both sexes. Sex differences in FGM levels are in accordance with results reported by others regarding the hamster adrenal physiology. The comparison of the adrenocortical response of males and females to an ACTH challenge provided new information about the amplitude and the timing of such a response and the excretion of glucocorticoids in both sexes. We demonstrated for the first time in the Syrian hamster that adrenocortical activity can be monitored in fecal samples in a noninvasive way. Our study provides a humane, practical, and noninvasive alternative to blood removal and therefore a powerful tool for stress-related studies in a species frequently used as an animal model in medical research.     

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.     

The expression of TGF-β3 for epithelial-mesenchyme transdifferentiated MEE in palatogenesis

The fate of the palatal medial edge epithelial (MEE) cells undergoes programming cell death, migration, and epithelial-mesenchymal transdifferentiation (EMT) coincident with the process of palatal fusion and disappearance of MEE. Mesenchymal cells in the palate have both cranial neural crest (CNC) and non-CNC origins. The objectives of this study were to identify the populations of palatal mesenchymal cells using β-galactosidase (β-gal) and DiI cell lineage markers, and to determine whether MEE-derived cells continued to express transforming growth factor-β3 (TGF-β3) and transforming growth factor-β type III receptor (TβR-III), which were specific for MEE. A model has been developed using Wnt1 tissue specific expression of Cre-recombinase to activate β-gal solely in the CNC. The expressions of TGF-β3 and TβR-III in MEE were temporally correlated with critical events in palatogenesis. Three cell populations could be distinguished in the palatal mesenchymal CNC-derived, non-CNC derived and MEE-derived. After fusion, β-gal (−) and DiI (+) mesenchymal cells continued to express TGF-β3, however TβR-III was expressed only in the epithelial MEE, as well as keratin expression. In addition, we performed laser capture microdissection to identify mRNA expression of isolated DiI (+) MEE cells. Both epithelial and transdifferentiated MEE have expressed TGF-β3, however, TβR-III was only expressed in epithelium. Extracellular matrix, especially MMP13 has been expressed coincident with fused stage which can be strongly associated with TGF-β3. These results demonstrate that combining a heritable marker and a cell lineage dye can distinguish different populations of mesenchymal cells in the developing palate. Furthermore, TGF-β3 and MMP13 could be strongly associated with EMT in palatogenesis.     

In vitro male germ cell cultures of zebrafish

Transgenic modification of sperm before fertilization has the advantages of a much shorter timeline for the production of transgenic animals. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation of spermatogonia to functional sperm can occur in vitro, allows us to introduce foreign DNA into the cultured sperm and to produce transgenics from the sperm. This chapter describes methods for the co-culture of male germ cells and a Sertoli cell feeder layer and the introduction of foreign DNA with retroviruses. This male germ cell culture system should prove useful not only in producing genetically modified sperm, but also in analyzing the regulatory function of Sertoli cells for spermatogenesis in vertebrates.     

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.