Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-alpha-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-alpha for 24 hr ICAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-alpha treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 microM). The level of HO activity in endothelial cells exposed to heme or TNF-alpha was increased from 24.7 +/- 5.7 pmol bilirubin/mg protein/min in control to 70.0 +/- 9.5 and 36.7 +/- 3.1 pmol bilirubin/mg protein/min in heme- and TNF-alpha-stimulated cells, respectively. These results suggest that upregulation of ICAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.     

Polymorphisms of the human beta-defensin-1 gene

Erythropoietin (EPO) plasma levels were monitored during the perioperative period in 61 consecutive patients (22 males - 39 females), aged 62.5 +/- 9.5 years, scheduled for hip arthroplasty. All patients underwent intraoperative blood salvage (IOBS) and were subdivided into three different groups according to their hemoglobin levels (Hb) 24 hours postoperatively (group A: Hb < 8 g/dl; group B: Hb between 8-9 g/dl; group C: HB > or = 9 g/dl). Seventy-two hours after surgery EPO levels were significantly different in group A (135 +/- 68) compared to group C (54.3 +/- 32), with a positive correlation (p < 0.01) between Hb and EPO levels. On the basis of these results we suggest that a programmed autologous red blood cell collection aimed at obtaining the lowest hemoglobin values during the first 24 hours after surgery, may be of clinical utility in preventing homologous blood needs.     

Linkage-maps and their relations to linkage cluster procedures

Methods of numerical taxonomy should provide a good representation of the relations of the objects and the structure of the groups. Besides dendograms, shade diagrams and minimum spanning trees, linkage-maps are suggested as a good additive tool to accomplish this representation. In this paper linkage-maps and similar technics are described, their relations to some generally known linkage cluster procedures are shown and their advantages are discussed. Their application is demonstrated using a small microbiological data set.     

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region

Monoclonal antibodies were generated against serotonin (5-HT) and the C-terminal portion of the neuronal form of nitric oxide synthase (nNOS), the enzyme producing nitric oxide in neurons. These antibodies were used to compare the distribution of 5-HT- and nNOS-containing neurons in the raphe nuclei of four animal species (rat, mouse, guinea pig, and cat). It was found that the rat was the only species in which the raphe nuclei contain a substantial number of nNOS-immunoreactive (IR) cell bodies. In this species and as observed by other authors, all mesencephalic raphe nuclei contained nNOS-IR cells, the largest group being located in the nucleus raphe dorsalis. The coexistence of nNOS and 5-HT immunoreactivities in these nuclei was visualized by double labeling. In the medulla, the nuclei raphe magnus and obscurus displayed a rather low number of nNOS-IR neurons. In the other species, nNOS-IR cell bodies were found in very low numbers, whatever raphe nucleus was considered. The rostral pole of the nucleus raphe dorsalis and the nuclei raphe magnus and obscurus contained a few nNOS-IR neurons which did not show any coincidence with the 5-HT neurons. In addition, nNOS-IR axons were rare. It is concluded that in the mouse, guinea pig, and cat the involvement of nitric oxide in functions subserved by 5-HT within the raphe nuclei might be minimal.     

Polymorphisms in the HFE gene

BACKGROUND: An important function of the gallbladder is to acidify and concentrate bile. Acidification helps protect against the precipitation of calcium salts, which promote gallstone formation. Altered acidification may result in pigment gallstones. We investigated gallbladder composition in a model of TPN-associated pigment gallstones to test whether changes in acidification may be important in this gallstone model. MATERIALS AND METHODS: Ten miniswine were intravenously fed for 21 to 27 days (mean 23 days). Ten fed pig chow with intravenous infusion of saline served as controls. Gallbladder and hepatic bile electrolytes, lipids, pH, and pCO2 were measured. RESULTS: All animals remained healthy and gained weight. Hepatic bile electrolytes and pH were similar among all animals. Pigs on TPN had a higher gallbladder pH and the [H+] was half the value of controls [8.1 +/- 1.6 x 10(-8) meq/liter (control) versus 3.9 +/- 0.7 x 10(-8) meq/liter (TPN)]. Gallbladder bile pCO2, sodium (Na), and potassium were higher in controls. Biliary lipids [bile salts (BS), phospholipids, and cholesterol] with TPN were decreased in both hepatic and gallbladder bile. CONCLUSIONS: Unlike short-term TPN where gallbladder pH and [BS] are similar, with long-term TPN pH is higher with lower [H+], [Na], and [BS]. Despite a presumed longer residence time in the gallbladder, intravenous feeding without oral intake results in gallbladder bile that is less concentrated and acidified. Enteral stimulation may be an important stimulus for gallbladder acidification and periods without feeding may promote gallstone formation by increasing the pH of gallbladder bile.     

Assignment of the muscle-eye-brain disease gene to 1p32-p34 by linkage analysis and homozygosity mapping

Muscle-eye-brain disease (MEB) is an autosomal recessive disease of unknown etiology characterized by severe mental retardation, ocular abnormalities, congenital muscular dystrophy, and a polymicrogyria-pachygyria-type neuronal migration disorder of the brain. A similar combination of muscle and brain involvement is also seen in Walker-Warburg syndrome (WWS) and Fukuyama congenital muscular dystrophy (FCMD). Whereas the gene underlying FCMD has been mapped and cloned, the genetic location of the WWS gene is still unknown. Here we report the assignment of the MEB gene to chromosome 1p32-p34 by linkage analysis and homozygosity mapping in eight families with 12 affected individuals. After a genomewide search for linkage in four affected sib pairs had pinpointed the assignment to 1p, the MEB locus was more precisely assigned to a 9-cM interval flanked by markers D1S200 proximally and D1S211 distally. Multipoint linkage analysis gave a maximum LOD score of 6.17 at locus D1S2677. These findings provide a starting point for the positional cloning of the disease gene, which may play an important role in muscle function and brain development. It also provides an opportunity to test other congenital muscular dystrophy phenotypes, in particular WWS, for linkage to the same locus.     

LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production

The role of CD28/B7 and LFA-1/ICAM costimulation in proliferation and Th1/Th2 differentiation of naive CD4+ T cells was addressed using T cells from DO11.10 TCR transgenic mice stimulated by dendritic cells. The blockade of either CD28/B7 or LFA-1/ICAM interactions partially inhibited T cell proliferation. By comparison, blocking CD28/B7 costimulation inhibited IL-4 and IL-5 (Th2 cytokine) production, whereas blocking LFA-1/ICAM-1 or LFA-1/ICAM-2 led to a significant increase (15- to 40-fold) of Th2 cytokines. The combination of anti-ICAM-1 and anti-ICAM-2 mAbs had a synergistic effect with a 100- to 1000-fold increase of Th2 cytokine production. Thus, these two costimulatory pathways have opposing roles in the regulation of Th2 development.     

T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells

Transendothelial migration is a crucial step in the complex process of lymphocyte extravasation during lymphocyte homing, immunosurveillance and inflammation. However, little is known about the precise role of cell adhesion molecules (CAM) involved in this particular event. To define the CAM involved in T cell adhesion versus transendothelial migration, we have previously established an in vitro transendothelial migration system using mouse T cells and mouse endothelioma cells. We demonstrate here that, using ICAM-1-deficient endothelioma cells derived from ICAM-1 mutant mice, transendothelial migration of T cells was inhibited to a much greater extent when compared to migration across wild-type cells treated with a blocking anti-ICAM-1 monoclonal antibody. This unexpected result was confirmed by a rescue experiment using retroviral transfer of wild-type ICAM-1 into ICAM-1-deficient endothelial cells. Additional experiments showed that, in the absence of functional ICAM-1, only ICAM-2 was involved in transendothelial migration, but not PECAM-1, VCAM-1, or E-selectin. Taking this novel approach, we show that ICAM-1 and ICAM-2 are essential for transendothelial migration of T cells.     

A novel microsatellite polymorphism in the human OB gene: a highly polymorphic marker for linkage analysis

The mouse ob gene and its human homologue OB have recently been cloned. The mutations in the ob gene are known to be associated with extreme obesity. The relationship between the human OB gene and disease, however, is largely unknown due to the lack of suitable markers within or adjacent to the OB gene. To obtain informative markers, we searched for simple tandem repeat polymorphisms in the genomic sequence of the human OB gene and identified a novel tetranucleotide repeat in the 3' flanking region. Fifteen alleles were detected in this marker with a heterozygosity of 0.85 and polymorphism information content of 0.83, indicating a highly informative nature of this marker. Two-point linkage mapping in two Centre Etude Polymorphisme Humaine (CEPH) reference families suggested that this marker is located in the interval between D7S514 and D7S530, the same interval where the OB gene is located (recombination fractions with D7S514 and D7S530 were 0.026 and 0.034, respectively). Although allele frequency distributions of this marker did not differ between 84 control subjects and 69 NIDDM patients, there was a tendency to higher body weight in control subjects with class I/class I genotype than in those without this genotype (68.8 +/- 11.1 vs 60.8 +/- 10.3 kg, p = 0.05). The highly polymorphic nature of this marker and its location in the OB gene makes this marker useful for linkage studies of the OB gene with a number of phenotypes, such as obesity, non-insulin-dependent diabetes mellitus, hypertension and the insulin resistance syndrome.     

Characterization of the human class Mu glutathione S-transferase gene cluster and the GSTM1 deletion

A partial physical map has been constructed of the human class Mu glutathione S-transferase genes on chromosome 1p13.3. The glutathione S-transferase genes in this cluster are spaced about 20 kilobase pairs (kb) apart, and arranged as 5'-GSTM4-GSTM2-GSTM1-GSTM5-3'. This map has been used to localize the end points of the polymorphic GSTM1 deletion. The left repeated region is 5 kb downstream from the 3'-end of the GSTM2 gene and 5 kb upstream from the beginning of the GSTM1 gene; the right repeated region is 5 kb downstream from the 3'-end of the GSTM1 and 10 kb upstream from the 5'-end of the GSTM5 gene. The GSTM1-0 deletion produces a novel 7.4-kb HindIII fragment with the loss of 10.3- and 11.4-kb HindIII fragments. The same novel fragment was seen in 13 unrelated individuals (20 null alleles), suggesting that most GSTM1-0 deletions involve recombinations between the same two regions. We have cloned and sequenced the deletion junction that is produced at the GSTM1-null locus; the 5'- and 3'-flanking regions are more than 99% identical to each other and to the deletion junction sequence over 2.3 kb. Because of the high sequence identity between the left repeat, right repeat, and deletion junction regions, the crossing over cannot be localized within the 2.3-kb region. The 2.3-kb repeated region contains a reverse class IV Alu repetitive element near one end of the repeat.     

Evolutionary analysis of the hisCGABdFDEHI gene cluster from the archaeon Sulfolobus solfataricus P2

While sequencing the genome of the archaeon Sulfolobus solfataricus P2, we found an 8,313-bp sequence containing a cluster of nine histidine biosynthesis genes in an order different from that of any known his operon. Results of phylogenetic analysis of the coding regions in the putative operon give conflicting evolutionary histories for individual his genes.     

Cloning, sequencing and analysis of cDNA encoding bovine intercellular adhesion molecule-1 (ICAM-1)

Intercellular adhesion molecule-1 (ICAM-1) is an inducible glycoprotein that interacts with the leukocyte beta 2-integrins, LFA-1 and Mac-1. We have isolated and analyzed a cDNA clone coding for the putative bovine ICAM-1 gene and compared it with known comparative sequences from other species as well as bovine ICAM-3. The 3398-bp bovine ICAM-1 cDNA sequence codes for 535 amino acids and shows 57% homology with human ICAM-1 and 47% homology with bovine ICAM-3 at the amino acid levels. The predicted number and positions of cysteine residues in bovine ICAM-1 are all conserved among species including bovine ICAM-3. It has two arginine-glycine-aspartate (RGD) sites in the extracellular region and a serine residue in the cytoplasmic tail. Northern blot results show that the bovine ICAM-1 gene is expressed in stimulated leukocytes whereas bovine ICAM-3 is expressed predominantly in resting neutrophils.     

Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene

Pseudomonas putida ML2 contains a large catabolic plasmid, pHMT112, carrying genes that encode the dioxygenase and dehydrogenase involved in the catabolism of benzene via the ortho or beta-ketoadipate pathway. pHMT112 was derived from a larger and less stable plasmid in P. putida ML2 following growth on succinate as carbon and energy source but was, however, stably maintained in P. putida even in the absence of selection for growth on benzene. Cleavage sites for the restriction endonucleases DraI, XbaI, and BamHI were mapped on the plasmid. A region of the plasmid, downstream of the benzene dioxygenase genes (bedC1C2BA), was found to encode the cis-benzene dihydrodiol dehydrogenase gene (bedD). Recombinant Escherichia coli containing bedC1C2BAD genes was found to express benzene dioxygenase and dehydrogenase activity, indicated by the production of catechol when incubated in the presence of benzene. Hybridization using benzene dioxygenase genes as probes was used to construct a restriction map of the 35.5-kb XhoI-DraI fragment on which the bed operon was located.