Macromolecular accessibility of fluorescent taxoids bound at a paclitaxel binding site in the microtubule surface

The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax (7-O-{N-[6-(fluorescein-4'-carboxamido)-n-hexanoyl]-l-alanyl}paclitaxel) and 4-4-20 IgG (a monoclonal antibody against fluorescein) has been observed by means of sedimentation and electron microscopy methods. The kinetics of binding of the antibody to microtubule-bound Hexaflutax has been measured. The quenching of the observed fluorescence is fast (k+ 2.26 +/- 0.25 x 10(6) m(-1) s(-1) at 37 degrees C), indicating that the fluorescein groups of Hexaflutax are exposed to the outer solvent. The velocity of the reaction is linearly dependent on the antibody concentration, indicating that a bimolecular reaction is being observed. Another fluorescent taxoid (Flutax-2) bound to microtubules has also been shown to be rapidly accessible to polyclonal antibodies directed against fluorescein. A reduced rate of Hexaflutax quenching by the antibody is observed in microtubule-associated proteins containing microtubules or in native cellular cytoskeletons. It can be concluded that the fluorescent taxoids bind to an outer site on the microtubules that is shared with paclitaxel. Paclitaxel would be internalized in a further step of binding to reach the known luminal site, this step being blocked in the case of the fluorescent taxoids. Because the fluorescent ligands are able to induce microtubule assembly, binding to the outer site should be enough to induce assembly by a preferential binding mechanism.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Trypanosoma cruzi (Kinetoplastida Trypanosomatidae): ecology of the transmission cycle in the wild environment of the Andean valley of Cochabamba, Bolivia

An active Trypanosoma cruzi transmission cycle maintained by wild rodents in the Andean valleys of Cochabamba Bolivia is described. Wild and domestic Triatoma infestans with 60% infection with T. cruzi were found and was evidenced in 47.5% (rodents) and 26.7% (marsupial) by parasitological and/or serologycal methods. Phyllotis ocilae and the marsupial species Thylamys elegans, are the most important reservoirs followed by Bolomys lactens and Akodon boliviensis. In spite of both genotypes (TCI and TCII) being prevalent in Bolivia, in our study area only T. cruzi I is being transmitted. Our data suggest that wild T. infestans and wild small mammals play an important role in the maintenance of the transmission cycle of T. cruzi. Furthermore, the finding of high prevalence of T. cruzi infection in wild T. infestans point to the risk of the dispersion of Chagas' disease.     

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rond?nia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

Lipid-induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: determination and biological specificity

While antimicrobial and cytolytic peptides exert their effects on cells largely by interacting with the lipid bilayers of their membranes, the influence of the cell membrane lipid composition on the specificity of these peptides towards a given organism is not yet understood. The lack of experimental model systems that mimic the complexity of natural cell membranes has hampered efforts to establish a direct correlation between the induced conformation of these peptides upon binding to cell membranes and their biological specificities. Nevertheless, studies using model membranes reconstituted from lipids and a few membrane-associated proteins, combined with spectroscopic techniques (i.e. circular dichroism, fluorescence spectroscopy, Fourier transform infra red spectroscopy, etc.), have provided information on specific structure-function relationships of peptide-membrane interactions at the molecular level. Reversed phase-high performance chromatography (RP-HPLC) and surface plasmon resonance (SPR) are emerging techniques for the study of the dynamics of the interactions between cytolytic and antimicrobial peptides and lipid surfaces. Thus, the immobilization of lipid moieties onto RP-HPLC sorbent now allows the investigation of peptide conformational transition upon interaction with membrane surfaces, while SPR allows the observation of the time course of peptide binding to membrane surfaces. Such studies have clearly demonstrated the complexity of peptide-membrane interactions in terms of the mutual changes in peptide binding, conformation, orientation, and lipid organization, and have, to a certain extent, allowed correlations to be drawn between peptide conformational properties and lytic activity.     

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.     

Adenylate Uptake by Proplastids from Cultured Cells of Tobacco {Nicotiana tabacum L. cv. BY2) Indicates That an Adenylate Translocator is Present in All Types of Plastid

The presence of an adenylate translocator in the envelope membranesof proplastids isolated from the cultured cells of tobacco (Nicotianatabacum L. cv. BY2) was examined by means of transport experimentsusing the silicone oil filtering centrifugation technique. Itwas observed that proplastids can import [³H]ATP, [³H]ADP, [³H]AMPand less specifically ADP-[¹⁴C]Glc which can eventually be usedfor starch biosynthesis. The effects of specific inhibitorsof the mitochondrial adenylate translocator, i.e. atractyloside,bongkrekic acid and carboxyatractyloside were tested. Similarto the case of amyloplasts isolated from the cultured cellsof sycamore and chloroplasts isolated from spinach leaves, onlyATP and ADP-Glc uptake were shown to be partially inhibitedby carboxyatractyloside. On the other hand, neither atractylosidenor bongkrekic acid exerted a significant inhibitory effecton adenylate uptake. (Received August 8, 1992; Accepted November 26, 1992)     

Phosphate-buffered saline-based nucleofection of primary endothelial cells

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.