Expression, purification, and functional characterization of the carboxyl-terminal domain fragment of bacteriophage 434 repressor

The repressor protein of bacteriophage 434 binds to DNA as a dimer of identical subunits. Its strong dimerization is mediated by the carboxyl-terminal domain. Cooperative interactions between the C-terminal domains of two repressor dimers bound at adjacent sites can stabilize protein-DNA complexes formed with low-affinity binding sites. We have constructed a plasmid, pCT1, which directs the overproduction of the carboxyl-terminal domain of 434 repressor. The protein encoded by this plasmid is called CT-1. Cells transformed with pCT1 are unable to be lysogenized by wild-type 434 phage, whereas control cells are lysogenized at an efficiency of 1 to 5%. The CT-1-mediated interference with lysogen formation presumably results from formation of heteromeric complexes between the phage-encoded repressor and the plasmid-encoded carboxyl-terminal domain fragment. These heteromers are unable to bind DNA and thereby inhibit the repressor's activity in promoting lysogen formation. Two lines of evidence support this conclusion. First, DNase I footprinting experiments show that at a 2:1 ratio of CT-1 to intact 434 repressor, purified CT-1 protein prevents the formation of complexes between 434 repressor and its OR1 binding site. Second, cross-linking experiments reveal that only a specific heterodimeric complex forms between CT-1 and intact 434 repressor. This latter observation indicates that CT-1 interferes with 434 repressor-operator complex formation by preventing dimerization and not by altering the conformation of the DNA-bound repressor dimer. Our other evidence is also consistent with this suggestion. We have used deletion analysis in an attempt to define the region which mediates the 434 repressor-CT-1 interaction. CT-1 proteins which have more than the last 14 amino acids removed are unable to interfere with 434 repressor action in vivo.     

Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.     

Evidence for a luteinizing hormone surge center in the hypothalamus of the pig

Lipoprotein-X (Lp-X) is an abnormal low-density lipoprotein frequently found in liver disease. It is regarded as the most sensitive and specific biochemical parameter for the diagnosis of intra- and extrahepatic cholestasis. Moreover, Lp-X is supposed to contribute to the development of hypercholesterolemia in cholestatic liver disease, because it fails to inhibit de novo cholesterol synthesis. This investigation will focus on the relationship between the presence of Lp-X and serum lipid concentrations in cirrhosis. The significance of Lp-X in the diagnosis of cholestasis, compared with alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), and bilirubin levels, will be assessed as well. The present cross-sectional study includes 212 patients with histopathologically proven cirrhosis. The detection of Lp-X and the quantification of -, beta-, and pre-beta-cholesterol was based on agar gel electrophoresis and polyanion precipitation. For the characterization of liver function, the concentrations of albumin and bilirubin, the activities of liver enzymes, and coagulation times were assessed. In a subgroup of 40 individuals, liver biopsies were re-evaluated to confirm or exclude intrahepatic cholestasis. As a result, there was no association between the appearance of Lp-X and total cholesterol concentrations. While all patients with Lp-X showed intrahepatic cholestasis (predictive value of the positive test = 1), only 16 of 28 patients with cholestasis formed Lp-X (sensitivity = 0.57). The activities of AP and of GGT, as well as the concentrations of bilirubin, were strongly elevated in most patients, with and without cholestasis. The predictive values of AP, GGT, and bilirubin were 0.77, 0.69, and 0.74 for the positive test and 0.5, 0, and 0.6 for the negative test, respectively. We conclude that Lp-X is not related to hypercholesterolemia in cirrhosis. The positive, but not the negative, Lp-X test has high predictive value for the diagnosis of cholestasis in cirrhosis. The biochemical parameters traditionally used for the assessment of extrahepatic cholestasis, AP, GGT, and bilirubin, do not support the diagnosis of intrahepatic cholestasis caused by cirrhosis.     

Multiple adhesive phenotypes linked to rosetting binding of erythrocytes in Plasmodium falciparum malaria

The cerebral form of severe malaria is associated with excessive intravascular sequestration of Plasmodium falciparum-infected erythrocytes (PRBC). Retention and accumulation of PRBC may lead to occlusion of brain microvessels and direct the triggering of acute pathologic changes. Here we report that by selection, cloning, and subcloning, we have identified rare P. falciparum parasites expressing a pan-adhesive phenotype linked to erythrocyte rosetting, a previously identified correlate of cerebral malaria. Rosetting PRBC not only bound uninfected erythrocytes but also formed autoagglutinates, adhered to endothelial cells, and bound to CD36, immunoglobulins, and the blood group A antigen. The linkage of rosetting, autoagglutination, and cytoadherence involved the coexpression on a single PRBC of ligands with multiple specificities and the binding to two or more receptors on erythrocytes and to at least two other cell adhesion molecules, including a new endothelial cell receptor for P. falciparum-infected erythrocytes. Limited proteolysis that differentially cleaved the rosetting ligand PfEMP1 from the PRBC surface abrogated all the binding phenotypes of these parasites, implicating the variant antigen PfEMP1 as a carrier of multiple ligand specificities. The results encourage the further study of pan-adhesion as a potentially important parasite phenotype in the pathogenesis of severe P. falciparum malaria.     

The use of fluorescent probes for the detection of under-film corrosion

An inexpensive and practical method for detecting under-film aluminium corrosion using a hand-held UV inspection lamp is described. A novel fluorescent probe for under-film iron corrosion is demonstrated. Studies of light transmission and ion transport through three types of paint film show polyester to be the best barrier to ions and epoxy to be the most transparent in the near UV.     

Antimicrobial properties of human lysozyme transgenic mouse milk

To examine whether malignant mesothelioma due to asbestos has genetic alterations in the Ha- and Ki-ras oncogenes or in the p53 suppressor gene, we analyzed the point mutations of these genes in paraffin-embedded autopsy samples of the primary tumors of malignant mesothelioma in seven asbestos patients who died from malignant mesothelioma. The genetic analysis was conducted by the polymerase chain reaction-single strand comformation polymorphysms (PCR-SSCP) method in all patients, and through the sequencing of deoxyribonucleic acid (DNA) bases in one patient. No genetic alterations were found in exons 1 or 2 of Ha- and Ki-ras oncogenes, or in exons 5 to 9 of the p53 gene, in any of the patients. Further studies on a larger number of patients are required to reach a definite conclusion concerning the genetic effects of asbestos on malignant mesothelioma.     

Brief report: two case studies using virtual reality as a learning tool for autistic children

The objective of this study was to evaluate the osteogenic capacities of a vascularized periosteal flap reinforced by a bone substitute, coral, to validate the possibility of creating prefabricated bone flaps with shapes adapted to a recipient zone, while limiting the donor site sequelae. 24 periosteal flaps, with preserved vascular pedicles, were raised from the medial femoral epiphyses of 12 ewes. In the same animal, these flaps were reinforced with a cylinder of coral and a cylinder of autologous graft. After implantation for 2 or 8 weeks depending on the animal, the flaps were submitted to histopathological and histomorphometric examination. The results of this examination demonstrated a similar course of the flaps regardless of the type of reinforcement, both in terms of implant resorption and the quantity of newly formed bone.