Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.     

QT-RR relationship in healthy subjects exhibits substantial intersubject variability and high intrasubject stability

Recently, it was demonstrated that the QT-RR relationship pattern varies significantly among healthy individuals. We compared the intra- and interindividual variations of the QT-RR relationship. Twenty-four-hour 12-lead digital electrocardiograms (ECGs; SEER MC, GE Marquette; 10-s ECG recorded every 30 s) were obtained at baseline and after 24 h, 1 wk, and 1 mo in 75 healthy subjects (42 women, 33 men, age 27.9 +/- 9.6 vs. 26.8 +/- 7.5 yr, P = not significant). QT interval was measured automatically in each ECG by six different algorithms, and the mean of the six measurements was analyzed. In each recording of each individual, QT-RR relationship was assessed by 10 different regression models including linear (QT = beta + alpha x RR) and parabolic (QT = beta x RR(alpha)) models. Standard deviations (SDs) of regression parameters alpha and beta of consecutive recordings of each individual were compared with SD of the individual means. Intrasubject stability and interindividual variability were further tested by ANOVA. With all models, intraindividual SDs of the regression parameters were highly significantly smaller than SD of individual means (P < 10(-5)-10(-9)). The intrasubject stability was further confirmed by ANOVA (P < 10(-19)-10(-30)). The QT-RR relationship exhibits substantial intersubject variability as well as a high intrasubject stability. This has practical implications for a precise estimation of the heart rate-corrected QT interval in which optimized subject-specific rate correction formulas should be used.     

Compliance calibration for fracture testing of equine cortical bone

An experimental compliance calibration method for measuring crack length in fracture toughness tests of cortical bone was developed. Calibration tests were conducted on twenty compact type fracture specimens machined from the mid-diaphysis of five pairs of equine third metacarpal bones. Specimens were oriented for crack propagation in a direction transverse to the longitudinal axis of the bone. Specimen compliance was determined from the load vs. crack opening displacement record over a range of crack lengths from 0.48 to 0.75 times the specimen width. The results demonstrate that the compliance calibration method developed for isotropic materials can be used to determine crack length in bone, which is transversely isotropic. However, specimens from lateral and dorsal regions exhibited significantly different compliance calibrations even after differences in elastic modulus were taken into account in the normalized compliance.     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Integrated control of lung fluid balance

This review summarizes the highlights of the EB2004 symposium that dealt with the integrated aspects of the lung fluid balance. It is apparent that maintenance of lung fluid balance requires the proper functioning of vascular endothelial and alveolar epithelial barriers. Under physiological conditions, the transcytotic pathway requiring repeated fission-fusion events of the caveolar membrane with other caveolae solely transports albumin. Caveolin-1, which forms caveolae, and albumin-binding proteins play a central role in signaling the transcytosis of albumin. Signals responsible for increasing endothelial permeability in lung microvessels in response to inflammatory mediators were also described. These studies in gene knockout mouse models revealed the importance of Ca(2+) signaling via store-operated transient receptor channel 4 and the activation of endothelial myosin light chain kinase isoform in mediating the increase in microvessel permeability. Increases in the cytosolic Ca(2+) in situ in microvessel endothelia can occur by mitochondria-dependent as well as mitochondria-independent pathways (such as the endoplasmic reticulum). Both these pathways, by triggering endothelial cell activation, may result in lung microvascular injury. The resolution of alveolar edema, requiring clearance of fluid from the air space, is another area of intense investigation in animal models. Although beta-adrenergic agonists can activate alveolar fluid clearance, signaling pathways regulating these events in intact alveoli remain to be established. Development of mouse models in which the function of regulatory proteins (identified in cell culture studies) can be systematically analyzed will provide a better and more integrated picture of lung fluid balance. In vivo veritas!     

Limbetazulone: a new 'decahydro-8-oxanaphtho[2,1-f]azulen-7-one' diterpenoid from Ballota limbeta, and occurrence of two conformational isomers in the crystal

A new diterpenoid, limbetazulone (= (3S,4S,4aR,12bS)-1,2,3,4,4a,5,6,11,12,12b-decahydro-3-hydroxy-4-(hydroxymethyl)naphtho[1',2': 5,6]cyclohepta[1,2-b]furan-7(7H)-one; 1), with the very rare 'naphtho[2,1-f]azulene-7-one' skeleton, was isolated from the aerial parts of the Asian medicinal plant Ballota limbeta. Its structure was established by extensive spectroscopic investigations, especially 1D and 2D NMR. X-Ray diffraction studies showed the presence of two conformational isomers (1a and 1b) in the crystal.     

VE-cadherin-induced Cdc42 signaling regulates formation of membrane protrusions in endothelial cells

The cytoplasmic domain of cadherins and the associated catenins link the cytoskeleton with signal transduction pathways. To study the signaling function of non-junctional VE-cadherin, which can form during the loss VE-cadherin homotypic adhesion, wild type VE-cadherin or VE-cadherin cytoplasmic domain (DeltaEXD) was expressed in sub-confluent endothelial cells. We observed that Cdc42 was activated in transfected cells and that these cells also developed Cdc42-dependent >70-microm-long plasma membrane protrusions. The formation of these structures required actin polymerization, and they developed specifically in endothelial cells as compared with epithelial cells. Expression of the VE-cadherin cytoplasmic domain lacking the beta-catenin binding site also induced Cdc42 activation; thus, its activation cannot be ascribed to beta-catenin binding. However, these cells were not able to form the protrusions. These results suggest that the cytoplasmic domain of non-junctional VE-cadherin can serve as a scaffold involved in Cdc42 activation at the endothelial plasma membrane. beta-Catenin and the associated alpha-catenin may serve as support sites for actin polymerization, leading to formation of long plasma membrane protrusions. Thus, non-junctional VE-cadherin actively participates in inside-out signaling at the plasma membrane, leading to the development of endothelial membrane protrusions.     

Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.     

NeSL-1, an ancient lineage of site-specific non-LTR retrotransposons from Caenorhabditis elegans

Phylogenetic analyses of non-LTR retrotransposons suggest that all elements can be divided into 11 lineages. The 3 oldest lineages show target site specificity for unique locations in the genome and encode an endonuclease with an active site similar to certain restriction enzymes. The more "modern" non-LTR lineages possess an apurinic endonuclease-like domain and generally lack site specificity. The genome sequence of Caenorhabditis elegans reveals the presence of a non-LTR retrotransposon that resembles the older elements, in that it contains a single open reading frame with a carboxyl-terminal restriction-like endonuclease domain. Located near the N-terminal end of the ORF is a cysteine protease domain not found in any other non-LTR element. The N2 strain of C. elegans appears to contain only one full-length and several 5' truncated copies of this element. The elements specifically insert in the Spliced leader-1 genes; hence the element has been named NeSL-1 (Nematode Spliced Leader-1). Phylogenetic analysis confirms that NeSL-1 branches very early in the non-LTR lineage and that it represents a 12th lineage of non-LTR elements. The target specificity of NeSL-1 for the spliced leader exons and the similarity of its structure to that of R2 elements leads to a simple model for its expression and retrotransposition.     

Kinetics of recombinant adeno-associated virus-mediated gene transfer

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to beta-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 microgram of hFIX/10(6) cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.