High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10⁴ and 10⁵. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

High susceptibility of transgenic rats carrying the human c-Ha-ras proto-oncogene to chemically-induced mammary carcinogenesis

A rat line carrying three copies of the human c-Ha-ras proto-oncogenes, including its own promoter region, was established and designated as Hras128. Expression of the transgene was detected in all organs by Northern blot analysis. To examine its influence on susceptibility to mammary carcinogenesis, female rats were treated with N-methyl-N-nitrosourea (MNU) or 7,12-dimethylbenz[a]anthracene (DMBA) at 50 days of age. With MNU, all the transgenic rats rapidly developed multiple mammary carcinomas within as short as 8 weeks (14.1 tumors/rat), in contrast to 0.46 tumors/rat in non-transgenic rats. PCR-RFLP analysis and direct sequencing for the transgene indicated that the large majority of carcinomas (38/44, 86.4%) contained cells with mutations at codon 12 in exon 1. However, comparison of the signal densities of the mutated band to dilution scale bands revealed that the cells with the mutated transgene were not in the majority. By PCR-SSCP analysis for codons 12 and 61 of the rat endogenous c-Ha-ras gene, no mutations were detected. Similarly, with DMBA, almost all (13/14, 92.9%) the transgenic rats developed multiple mammary carcinomas (9.39 tumors/rat) within 16 weeks, and 4 out of 12 (33.3%) non-transgenic rats had only small tumors (0.83 tumors/rat). A lower incidence of mutation of the transgene was found in codon 12 (5/25, 25%) than in MNU-induced tumors, but mutations were detected in codon 61 (7/20, 35%). No mutations were detected in the rat endogenous gene. No mutation was found in the rat endogenous c-Ha-ras gene in non-transgenic rats. As observed in both the MNU- and DMBA-induced tumor cases, the population of cells with the mutated transgene were in the minority. The results thus indicate that rats carrying the transduced human c-Ha-ras proto-oncogene are highly susceptible to MNU- and DMBA-induced mammary carcinogenesis and that this is not primarily due to mutations of the transgene or endogenous c-Ha-ras gene. Furthermore, irrespective of the mechanism of enhanced susceptibility, the Hras128 transgenic rats can be utilized for the screening of mammary carcinogens.     

Functional Role of Metaplastic Paneth Cell Defensins in Helicobacter pylori-Infected Stomach

Background and Aims:  Chronic gastritis is caused by Helicobacter pylori infection, and gastritis is classified as inflammation, atrophy, and intestinal metaplasia. Detailed pathologic studies have shown that H. pylori settles on the surface of gastric mucosa, and that it is eliminated from metaplastic mucosa. However, its mechanism of natural protection is not well known.
Methods:  Antimicrobial human enteric defensin expression was determined in the RNA and protein levels. Recombinant enteric defensins were produced with a bacterial expression system and their anti- H. pylori activities were assessed by bactericidal assay.
Results:  Human enteric defensin (HD)-5 and HD-6 were detected in Paneth cells, which are observed in the gastric metaplastic mucosa as well as small intestinal epithelia. HD-5 protein was coexpressed with trypsin, which is considered to be an activating enzyme of HD-5. Less H. pylori was observed in the intestinal metaplasia with HD-5 expressing Paneth cells. The recombinant defensins showed killing activity against H. pylori at a low concentration in vitro.
Conclusions:  The human defensins that are expressed in the metaplastic Paneth cells eliminate H. pylori . Metaplastic change may be a purposive development of the human stomach.     

Blood-brain barrier transport of a novel micro 1-specific opioid peptide,H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA)

The purpose of this study was to clarify the mechanism of the blood-brain barrier (BBB) transport of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), which is a novel dermorphin analog with high affinity for the micro 1-opioid receptor. The in vivo BBB permeation influx rate of [125I]TAPA after an i.v. bolus injection (7.3 pmol/g body weight) into mice was estimated to be 0.265 +/- 0.025 microL/(min.g of brain). The influx rate of [125I]TAPA was reduced 70% by the coadministration of unlabeled TAPA (33 nmol/g of brain), suggesting the existence of a specific transport system for TAPA at the BBB. In order to elucidate the BBB transport mechanism of TAPA, a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) was used as an in vitro model of the BBB. The acid-resistant binding of [125I]TAPA, which represents the internalization of the peptide into cells, was temperature- and concentration-dependent with a half-saturation constant of 10.0 +/- 1.7 microm. The acid-resistant binding of TAPA was significantly inhibited by 2,4-dinitrophenol, dansylcadaverine (an endocytosis inhibitor) and poly-l-lysine and protamine (polycations). These results suggest that TAPA is transported through the BBB by adsorptive-mediated endocytosis, which is triggered by binding of the peptide to negatively charged sites on the surface of brain capillary endothelial cells. Blood-brain barrier transport via adsorptive-mediated endocytosis plays a key role in the expression of the potent opioid activity of TAPA in the CNS.     

Annual and seasonal changes in foraging site and diving behavior in Adélie penguins

Foraging sites, diet, and diving behavior of chick-rearing Adélie penguins, Pygoscelis adeliae, in fast sea-ice areas were investigated during two consecutive seasons with contrasting sea-ice conditions. During 1995/1996, fast sea ice covered the foraging range of penguins during the whole breeding season. In contrast, during 1996/1997, sea ice covered the area in December 1996, but gradually thinned and finally broke up, so that open sea appeared along the coast during February 1997. Foraging sites were concentrated in a small area in 1995/1996 and spread over a wider area in 1996/1997 as more small open-water areas were available. In both seasons, parents traveled to more distant foraging sites as the season progressed and, consequently, the foraging-trip duration increased. In both years, Euphausia superba and Pagothenia borchgrevinki dominated the diet in the early part of the season, while later in the season penguins fed mainly on E. superba in 1995/1996 and Pagothenia borchgrevinki and E. crystallorophias in 1996/1997. In 1995/1996, penguins tended to dive deeper—albeit for a relatively shorter duration—when feeding mainly on krill compared to when feeding on fish. In 1996/1997, there was no difference in the dive depth and duration between krill- and fish-eating trips. Our results suggest that prey distribution changes annually and seasonally, probably according to sea-ice conditions, and that consequently penguins modify their foraging sites, diving patterns, and diet according to these changes.     

Importance of permafrost as a source of water for plants in east Siberian taiga

Stable oxygen isotope ratios of plant water (sap water) were observed at Spasskaya Pad experimental forest near Yakutsk, Russia in 1997–1999. The ¹⁸O of sap water in larch trees (Larix gmelinii) decreased soon after leaf unfolding every year, indicating that snowmelt water was used in the beginning of summer. During mid to late summer, a clear difference in the water source used by plants was observed between wet summers and severe drought summers. The ¹⁸O values of water in larch trees were high (–17.8 to –16.1) in August 1999 (wet summer), but low (–20.4 to –19.7) in August 1998 (drought summer). These results indicated that plants used rainwater during a wet summer, but meltwater from permafrost was used by plants during a drought summer. One important role of permafrost is to provide a direct source of water for plants in a severe drought summer; another role is to keep surplus water in the soil until the next summer. If this permafrost system is disturbed by future global warming, unique monotypic stands of deciduous larch trees in east Siberia might be seriously damaged in a severe drought summer.     

ESR study on synthetic glyceroglycolipid liposomal membranes

We previously reported that glyceroglycolipid liposomes without cholesterol activated mouse peritoneal macrophages in vivo and in vitro, whereas glyceroglycolipid liposomes containing equimolar cholesterol did not. In order to characterize the properties of the glyceroglycolipid membranes, ESR spectroscopic studies were carried out with an acyl spin-labeled galactosyl ceramide (SL-GC) or a headgroup spin-labeled phospholipid (SL-6-DPPA) in 1,2-dipalmitoyl[beta-cellobiosyl-(1'---3)]glycerol (Cel-DAG) liposomal membranes. The ESR spectrum of the SL-GC in the Cel-DAG liposomes at 37 degrees C was a single broad line, indicating that the SL-GC molecules were excluded almost completely from Cel-DAG domains and formed clusters in the membranes. The spectrum of SL-6-DPPA in the Cel-DAG liposomes at 37 degrees C showed broad resonance lines with the central peak being the highest, while that at 60 degrees gave narrow lines with the low-field peak being the highest. This observation and rotational correlation time analysis showed that the molecular motions of spin-label moiety of the SL-6-DPPA were extremely restricted at 37 degrees C but not above Tc. These results suggest that below Tc the Cel-DAG molecules are packed tightly and restricted in motion in the membrane. Incorporation of cholesterol into the Cel-DAG liposomal membranes gave (1) the spectra of the SL-GC triplet, and (2) the spectra of the SL-6-DPPA narrow resonance with the low-field peak being the highest. These results suggest that cholesterol disturbs the rigid-packed structure of the Cel-DAG membrane and increases the molecular motions of the Cel-DAG. The DSC analysis of Cel-DAG with and without cholesterol agreed well to the results of the ESR technique. Thus we assume that peritoneal macrophages recognize the rigid-packed carbohydrate residues which are restricted in motion on the Cel-DAG membranes.     

A Mutant of Arabidopsis thaliana That Defines a New Locus for Glycine Decarboxylation

A novel photorespiratory mutant of Arabidopsis thaliana, designatedgld2, was isolated based on a growth requirement for abnormallyhigh levels of atmospheric CO₂. Photosynthetic CO₂ fixationwas inhibited in the mutant following illumination in air butnot in atmosphere containing 2% O₂. Photosynthetic assimilationof ¹⁴CO₂ in an atmosphere containing 50% O₂ resulted in accumulationof 48% of the soluble label in glycine in the mutant comparedto 9% in the wild type. The rate of glycine decarboxylationby isolated mitochondria from the mutant was reduced to 6% ofthe wild type rate. In genetic crosses, the mutant complementedtwo previously described photorespiratory mutants of A. thalianathat accumulate glycine during photosynthesis in air due todefects in glycine decarboxylase (glyD, now designated gld1)and serine transhydroxymethylase (stm). Because glycine decarboxylaseis a complex of four enzymes, these results are consistent witha mutation in a glycine decarboxylase subunit other than thataffected in the gld1 mutant. The two gld loci were mapped tochromosomes 2 and 5, respectively. ³Present address: Department of Crop and Soil Sciences, MichiganState University, East Lansing, MI 48824, U.S.A. ⁴Present address: Department of Applied Bioscience, Facultyof Agriculture, Hokkaido University, Kita-Ku, Sapporo, 060 Japan ⁵Present address: Department of Biology, Carnegie Institutionof Washington, 290 Panama Street, Standford, CA 94305, U.S.A.     

Biaxial mechanical properties of the inferior vena cava in C57BL/6 and CB-17 SCID/bg mice

Multiple murine models have proven useful in studying the natural history of neovessel development in the tissue engineering of vascular grafts. Nevertheless, to better understand longitudinal changes in the biomechanics of such neovessels, we must first quantify native tissue structure and properties. In this paper, we present the first biaxial mechanical data for, and nonlinear constitutive modeling of, &QJ;the inferior vena cava from two models used in tissue engineering: wild-type C57BL/6 and immunodeficient CB-17 SCID/bg mice. Results show that inferior vena cava from the latter are significantly stiffer in the circumferential direction, both materially (as assessed by a stored energy function) and structurally (as assessed by the compliance), despite a lower intramural content of fibrillar collagen and similar wall thickness. Quantifying the natural history of neovessel development in different hosts could lead to increased insight into the mechanisms by which cells fashion and maintain extracellular matrix in order to match best the host stiffness while ensuring sufficient vascular integrity.     

Circadian rhythm of intracellular protein synthesis signaling in rat cardiac and skeletal muscles

Intracellular signaling exhibits circadian variation in the suprachiasmatic nucleus and liver. However, it is unclear whether circadian regulation also extends to intracellular signaling pathways in the cardiac and skeletal muscles. Here, we examined circadian variation in the intracellular mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase 1 (p70S6K) and extracellular signal-regulated kinase (ERK) pathways, which regulate protein synthesis in rat cardiac and skeletal muscles. Seven-week-old male Wistar rats were assigned to six groups: Zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, ZT18, and ZT22 (ZT0, lights on; ZT12, lights off). The cardiac, plantaris, and soleus muscles were removed after a 12-h fasting period, and signal transducers involved in protein synthesis (mTOR, p70S6K, and ERK) were analyzed by western blotting. Circadian rhythms of signal transducers were observed in both cardiac (mTOR, p70S6K, and ERK) and plantaris (p70S6K and ERK) muscles (p<0.05), but not in the soleus muscle. In the cardiac muscle, the phosphorylation rate of mTOR was significantly higher at ZT6 (peak) than at ZT18 (bottom), and the phosphorylation rate of p70S6K was significantly higher at ZT2 (peak) than at ZT18 (bottom). In contrast, in the plantaris muscle, the phosphorylation rate of ERK was significantly lower at ZT2 (bottom) than at ZT18 (peak). Our data suggested that protein synthesis via mTOR/p70S6K and ERK signaling molecules exhibits circadian variation in rat cardiac and fast-type plantaris muscles.