Modulation of nuclear pore topology by transport modifiers

The nuclear pore complex (NPC) represents the only pathway for macromolecular communication between the nuclear and cytoplasmic compartments of the cell. Nucleocytoplasmic transport requires the interaction of transport receptors with phenylalanine-glycine (FG)-repeats that line the transport pathway through the NPC. Here we examine the effects of transport receptors and amphipathic alcohols on NPC topology using scanning force microscopy. We show that transport receptors that irreversibly bind FG-repeats increase NPC vertical aspect, whereas transport receptors that weakly interact with FG-repeats increase NPC diameter. Interestingly, small polar alcohols likewise increase NPC diameter. These opposing effects agree with the inhibition or enhancement of nuclear transport, respectively, previously ascribed to these agents.     

Expression of galP and glk in a Escherichia coli PTS mutant restores glucose transport and increases glycolytic flux to fermentation products

In Escherichia coli, the uptake and phosphorylation of glucose is carried out mainly by the phosphotransferase system (PTS). Despite the efficiency of glucose transport by PTS, the required consumption of 1 mol of phosphoenolpyruvate (PEP) for each mol of internalized glucose represents a drawback for some biotechnological applications where PEP is a precursor of the desired product. For this reason, there is considerable interest in the generation of strains that can transport glucose efficiently by a non-PTS mechanism. The purpose of this work was to study the effect of different gene expression levels, of galactose permease (GalP) and glucokinase (Glk), on glucose internalization and phosphorylation in a E. coli PTS(-) strain. The W3110 PTS(-), designated VH32, showed limited growth on glucose with a specific growth rate (mu) of 0.03 h(-1). A low copy plasmid family was constructed containing E. coli galP and glk genes, individually or combined, under the control of a trc-derived promoter set. This plasmid family was used to transform the VH32 strain, each plasmid having different levels of expression of galP and glk. Experiments in minimal medium with glucose showed that expression of only galP under the control of a wild-type trc promoter resulted in a mu of 0.55 h(-1), corresponding to 89% of the mu measured for W3110 (0.62 h(-1)). In contrast, no increase in specific growth rate (mu) was observed in VH32 with a plasmid expressing only glk from the same promoter. Strains transformed with part of the plasmid family, containing both galP and glk genes, showed a mu value similar to that of W3110. Fermentor experiments with the VH32 strain harboring plasmids pv1Glk1GalP, pv4Glk5GalP, and pv5Glk5GalP showed that specific acetate productivity was twofold higher than in W3110. Introduction of plasmid pLOI1594, coding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis, to strain VH32 carrying one of the plasmids with galP and glk caused a twofold increase in ethanol productivity over strain W3110, also containing pLOI1594.     

The role of acute wall recoil and late restenosis: results of the OCBAS trial (Optimal Coronary Balloon Angioplasty with Provisional Stenting versus Primary Stent)

Early deterioration of minimal luminal diameter immediately after PTCA, has been associated with an increase of late restenosis. Lesions with no early loss after PTCA have a low restenosis rate. Coronary stents reduce restenosis in lesions exhibiting early wall recoil. The purpose of the OCBAS study was to compare two strategies during coronary interventions; provision vs. elective stenting. 116 patients with good PTCA results were randomized to stent (n = 57) or to optimal PTCA (n = 59). After randomization in PTCA group, 13.5% of the patients crossed over to stent due to early loss (provisional stenting). Baseline demographic and angiographic characteristics were similar in both groups of patients. At 7.6 months, 96.6% of the entire population had a follow-up angiographic study; 98.2% in the stent and 94.9% in the PTCA group. Immediate and follow-up angiographic data showed that acute gain was significantly higher in the stent than in the PTCA group (1.95 vs. 1.5 mm; P < 0.03). However, late loss was significantly higher in the stent than the PTCA groups (0.63 +/- 0.59 vs. 0.26 +/- 0.44, respectively; P = 0.01). Hence, net gain with both techniques was similar (1.32 +/- 0.3 vs. 1.24 +/- 0.29 mm for the stent and PTCA groups respectively; P = NS). Angiographic restenosis rate at follow-up (19.2% in stent vs. 16.4% in PTCA; P = NS) and TVR (17.5 in stent vs. 13.5% in PTCA; P = NS) were also similar. Furthermore, event-free survival was 80.8% in the stent versus 83.1% in the PTCA group (P = NS). Overall costs (hospital and follow-up) were US$591,740 in the stent versus US$398,480 in the PTCA group (P < 0.02). The strategy of the PTCA with delay angiogram and provisional stent if early loss occurs, had similar restenosis rate and TVR than universal use of bare stents.     

Effects of betamethasone,sulindac and quinacrine drugs on the inflammatory neoangiogenesis response induced by polyurethane sponge implanted in mouse

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.     

Stenurus globicephalae Baylis et Daubney, 1925 (Nematoda: Pseudaliidae) from a false killer whale,Pseudorca crassidens (Cetacea: Delphinidae), stranded on the coast of Uruguay

Stenurus globicephalae Baylis et Daubney, 1925 (Nematoda: Pseudaliidae) was found in the cranial air sinuses of a false killer whale, Pseudorca crassidens (Owen), stranded on the coast of Uruguay in 1999. Although this species has been reported once in P. crassidens from the North Atlantic, this is the first record for South America. A total of 920 specimens were obtained, of which 663 were females (body length: 4.34 +/- 0.45 cm) and 257 were males (2.99 +/- 0.18 cm). Morphometric details are presented for S. globicephalae in this host, which do not show significant differences from those parasitizing Globicephala melas (Traill), but are distinct from those parasitizing Peponocephala electra (Gray). The host's skull revealed loss of osseous mass with the disappearance of the left zygomatic arch, and the left jaw had three osseous fenestrations in the region related to the organ of acoustic reception. These lesions support the hypothesis that this infection, known as stenurosis, was related to the stranding.     

Polymorphism in allergens

Allergens are antigens that elicit an IgE-mediated immune response; they originate from diverse sources such as pollens, mites, molds, mammal exudates, insects and food. Allergenic molecules can contain several antigenic determinants, termed epitopes. Allergenic proteins have been discovered with polymorphisms, i.e., a mixture of similar molecules with minor variations in their amino acid sequences. These are called isoallergens or allergenic variants depending on the degree of similarity. Polymorphism may be defined by the presence of several alleles of the same gene or as families of related genes. Polymorphisms can have an important effect on the epitopes recognized by T lymphocytes, monoclonal antibodies and IgE of allergic patients. Individual polymorphisms can affect the basal level of allergenicity as well as the cross-reactivity with other allergens. The use of isoforms with low or total absence of IgE binding capacity but with high capacity to stimulate T cell response has been suggested as an alternative to the conventional immunotherapy for allergic diseases. Standardization of allergenic compounds can be affected by the differing proportions of isoforms in allergenic sources from different regions.     

Route of jejunal mucosa absorption of trypsin demonstrated by immunofluorescence

Previous studies have demonstrated the absorption of porcine trypsin in isolated jejunal loops from male Wistar rats by open-loop perfusion. The possible routes of absorption were examined in the study reported here. Trypsin (0.5 mg/ml) was dissolved in tyrode solution and perfused at a rate of 0.5 ml/min, at 37 degrees C, for 40 min. Using immunoperoxidase and immunofluorescence techniques, strong reactivity towards anti-TLCK-trypsin antibody was demonstrated through out the enterocyte cytosol. The present data indicate that trypsin was absorbed by enterocytes, probably through a transcellular route.     

Characterization of Cah,a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.     

Protein origami: therapeutic rescue of misfolded gene products

Receptor mutations that elicit loss of function are sometimes equated with defects that ablate receptor-ligand binding or receptor-effector interactions. Similarly, mutationally defective enzymes and ion channels are often viewed as compromised in substrate or ion recognition, respectively. Recent observations, however, suggest that an alternate mechanism may be surprisingly common, namely, that mutations in structural genes may not interfere with the inherent functionality of the affected protein, but nevertheless cause disease by preventing the cell's trafficking machinery from placing the affected protein at the appropriate subcellular compartment (e.g., at the cell membrane). Accordingly, therapies may be devised to ensure the placement of receptors (or other proteins) at locations where they can support cell function.     

Understanding p27(kip1) deregulation in cancer: down-regulation or mislocalization

There is considerable evidence that the inactivation of the cyclin-dependent kinase inhibitor p27(kip1) is a fundamental step for the development of human malignancies. In particular, reduced expression of p27(kip1), due to increased protein degradation, correlates with poor prognosis of patients affected by various types of cancer. The purpose of this mini-review is to present an overview of the current understanding of the alteration of p27(kip1) function in human cancer and to describe the different mechanisms that contributes to it. Particular emphasis is placed on the novel finding of p27(kip1) mislocalization in tumor cells and on the biochemical pathways responsible for p27(kip1) cytosolic accumulation. Finally, we review the possible clinical implications of these observations with respect to prognosis and novel anticancer therapies.