Flow cytometric analysis of lipid droplet formation in cells of the human monocytic cell line, U937.

Active uptake of a free fatty acid, oleate, and its incorporation into triacylglycerol and phospholipid in the human monocytic cell line, U937, was identified using 14C-labelled oleate. Excess triacylglycerol accumulates in the form of lipid droplets in the cytoplasm. The extent of lipid droplet formation in each cell can be assessed in the absence of any staining by 90 degrees light scatter intensity, one of the basic parameters of flow cytometry.     

Establishment and characterization of new cell lines from human renal cell carcinoma

We have characterized seven human renal cell carcinoma cell lines established from primary sites of five patients between 1987 and 1989. Two lines, OUR-20P and OUR-20S, were derived from the OUR-20 cells by cloning with a dilution method 3 months after the primary culture. These three cell lines were tumorigenic in athymic nude mice when inoculated subcutaneously. Examined by a dye uptake method, OUR-20 was highly sensitive to interferon-alpha (IFN-alpha); OUR-20P, OUR-20S and OUR-30 showed slight sensitivities, while the other three cell lines were insensitive. All seven cell lines have been maintained for more than 2 years and over 50 passages in vitro. Cytogenetic analyses performed 1.5 to 3 years after the starts of primary cultures indicated that all seven cell lines, which exhibited different morphologies in phase-contrast micrographs, were aneuploid with modal chromosome numbers 41 to 89.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

To lek or not to lek: mating strategies of male fallow deer

We studied the mating system of fallow deer (Dama dama) for6 years in central Italy. Males in this population could defendterritories that were either single, clumped in leks, or satelliteto leks. The most highly successful males in our study werein leks. When we considered all males, there were no significantdifferences in average copulatory success according to territorytype because many lek males did not achieve any copulations,which were seen in only a few lek territories. The variancein copulatory success, however, was much greater for leks thanelsewhere. Single territories were occupied for shorter timesduring the rut than lek territories. Fighting among males wasmore frequent in the lek, even when we excluded highly successfullek males from the analysis. Chases of nonterritorial malesand harem size were correlated with the number of copulationsachieved by individual males, but did not vary according toterritory type. Copulatory success of some individuals increasedwith age, but there were no age differences among males holdingdifferent types of territories. Satellite males switched tolek territoriality in the course of one rut, but switches fromsingle territory to lek territory were rare. We suggest thatmales in single territories are inferior competitors that selecta low-risk, lowbenefit strategy, whereas those in lek territorieswhere no copulations were seen may be attempting to establishthemselves on the lek to increase their copulatory success infuture years.     

Effect of streptokinase on prostacyclin synthesis and phospholipase activity in cultured pulmonary artery endothelial cells

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1-10,000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [3H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacylglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 microM). These results suggest that streptokinase stimulates phospholipase A2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.     

Identification of some abnormal metabolites in psoriatic nail using gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric analysis was used to separate and identify abnormal compounds in the nail of psoriatic patients. The nail was extracted with heated ethanol, and the extract was analyzed with and without trimethylsilylation. Tetradecanoic acid octadecyl ester, hexadecanoic acid octadecyl ester and octadecanoic acid octadecyl ester were first identified in the psoriatic nail, but were not detected in normal nail.     

Fatigue sacral fractures: A case series and literature review

Objectives:Fatigue sacral fractures (FSFs) are rare and often misdiagnosed. This study presents a series of FSFs and a meticulous literature review.Methods:The present is an 11-year (2010-2021) retrospective observational study. The characteristics of all adult patients with FSF, including demographics, fracture type, treatment, history of fatigue fracture and imaging were evaluated.Results:Eight cases (6 females; 75%), suffering from 12 fractures (4 bilateral cases) with mean age=33.4 years were studied. Two patients (25%) had suffered another fatigue fracture in the past. Mean symptoms’ duration prior diagnosis was 8.5 weeks, while mean symptoms’ duration after diagnosis was 10.75. In most cases (7; 87.5%), MRI revealed the fracture. According to the Kaeding-Miller classification; five fractures (42%) were grade III, four (33%) IV and three (25%) II. All patients were treated conservatively, with rest and analgesics, while three received vitamin D and calcium. One patient, due to delayed union, was commenced on teriparatide.Conclusions:FSFs are often misdiagnosed; therefore, they should be included in the differential diagnosis for chronic low back-or-hip pain in athletes. History of other fatigue injuries seems to be a predisposing factor. It is of paramount importance to obtain advanced imaging for identifying a FSF.     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix

Mesenchymal stem cells (MSC) show a very short proliferative life span and readily lose the differentiation potential in culture. However, the growth rate and the proliferative life span of the stem cells markedly increased using tissue culture dishes coated with a basement membrane-like extracellular matrix, which was produced by PYS-2 cells or primary endothelial cells. Furthermore, the stem cells expanded on the extracellular matrix, but not those on plastic tissue culture dishes, retained the osteogenic, chondrogenic, and adipogenic potential throughout many mitotic divisions. The extracellular matrix had greater effects on the proliferation of MSC and the maintenance of the multi-lineage differentiation potential than basic fibroblast growth factor. Mesenchymal stem cells expanded on the extracellular matrix should be useful for regeneration of large tissue defects and repeated cell therapies, which require a large number of stem or progenitor cells.