Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha.

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.     

Clostridium thermocellum: Adhesion and sporulation while adhered to cellulose and hemicellulose

Summary During growth in the presence of fibers composed of cellulose or hemicellulose, various strains of the thermophilic soil bacterium Clostridium thermocellum and several newly isolated thermophilic anaerobic soil bacteria adhered to the fibers. Attachment occurred via a fibrous ruthenium red-staining material. C. thermocellum sporulated while attached to the fibers when the pH dropped below 6.4. It is postulated that the attachment is involved in cellulose breakdown and that C. thermocellum gaines an advantage by remaining attached to its insoluble substrates when the environment is not suitable for rapid growth. The tendency to adhere to cellulose fibers was used in the purification of thermophilic cellulolytic anaerobes.     

Wall and membrane biogenesis in the unusual labyrinthulid-like organismSorodiplophrys stercorea

Summary Electron microscopic investigations show the scale subunits of the walls ofSorodiplophrys stercorea to be produced by the Golgi apparatus. Their chief monosaccharide subunit is identified tentatively as arabinose. Membrane flow involving the nuclear envelope, endoplasmic reticulum, Golgi apparatus, Golgi vesicles, plasmalemma, and possibly prominent refractive bodies composed of membranes is suggested. Cytochemical data supporting ultrastructural evidence concerning the sites of scale and membrane biogenesis are presented.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Differential indicators of loneliness among elderly. The importance of type of partner relationship, partner history, health, socioeconomic status and social relations

Using data from the 1992 NESTOR-survey 'Living arrangements and social networks of older adults' (N = 4494), the aim of the present study is to identify specific categories of older adults who are most vulnerable to loneliness. By looking at different types of partner relationships (first, second, and third marriages; consensual unions; partners who are not household members) and at partner histories (never married, ever divorced, ever widowed, remarried), this study elaborates on previous research which has tended to look only at the presence versus the absence of partner relationships. Findings indicate that different types of partner relationships provide differential protection against loneliness. There appears to be a 'shadow of the past' of a previous divorce or widowhood in second and third partnerships, which accounts for generally higher levels of loneliness. Single men tend to be more lonely than single women. Moreover, there are no differences in loneliness between men who have always been single and those previously married. Among single women, differences in partner history are relevant: never married single women tend to be least vulnerable to loneliness. The differences in loneliness between older adults with different types of partner relationships and partner histories are only partially attributable to network and social participation differences. The latter independently contribute to the explanation of loneliness. The role of non-social determinants (health and socioeconomic position) is also examined. The results underscore the socially isolating effects of sensory impairments. Older adults with functional limitations, and those with visual or auditory problems tend to be more lonely, findings which are only partially attributable to differences in the number and quality of social relationships. Socioeconomic circumstances primarily have an indirect influence on loneliness. Those with higher levels of educational attainment and higher incomes tend to have more extensive social networks and are therefore less prone to loneliness.     

Floating the raft hypothesis for immune receptors: access to rafts controls receptor signaling and trafficking

The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.     

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro

The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.