Enhanced Engraftment of HTLV-I-Infected Human T Cells in Severe Combined Immunodeficiency Mice by Anti-asialo GM-1 Antibody Treatment

The effects of anti-asialo GM-1 antibody (AAGM) treatment on the engraftment of human T-cell leukemia virus type I (HTLV-I)-infected human T cells in severe combined immunodeficiency (SCID) mice were studied. The frequency of tumor formation in an HTLV-I-transformed human T-cell line, MT-2 cells, at the site of inoculation was significantly higher in AAGM-treated than untreated mice (P<0.05): 16/18 (89%) and 16/26 (62%), respectively. The promotive effect of AAGM treatment on tumor development was marked in the early stage (less than 3 weeks), suggesting that the immediate reaction of natural killers to the inoculated cells may be important for the prevention of tumor development. The surface phenotypes and clonality of the tumor cells were the same as the MT-2 cells inoculated. Inoculation of peripheral blood mononuclear cells (PBMC) from one of the 4 adult T-cell leukemia/lymphoma (ATL) patients resulted in the development of tumors in AAGM-treated SCID mice. However, the surface phenotypes of the cells from these tumors were a mixture of B cells and T cells, suggesting that these tumors consisted of Epstein-Barr virus-transformed B cells and HTLV-I-transformed T cells. In addition, HTLV-I was detected by polymerase chain reaction in various organs of the mice inoculated with PBMC from the ATL patient and the asymptomatic carrier examined. These results suggest that elimination of natural killer function by AAGM treatment is important, although such treatment is not always necessary for the engraftment of HTLV-I-infected cells in SCID mice.     

Analysis of the recJ gene and protein from Deinococcus radiodurans

The mechanism by which double-strand DNA breaks are repaired in the radiation-resistant bacterium Deinococcus radiodurans is not well understood. This organism lacks the RecBCD helicase/nuclease, which processes broken DNA ends in other bacteria. The RecF pathway is an alternative pathway for recombination and DNA repair in E. coli, when RecBCD is absent due to mutation, and D. radiodurans may rely on enzymes of this pathway for double-strand break repair. The RecJ exonuclease is thought to process broken DNA ends for the RecF pathway. We attempted to delete the recJ gene from D. radiodurans, using homologous recombination to replace the gene with a streptomycin-resistance cassette. We were unable to obtain a complete deletion mutant, in which the gene is deleted from all of the chromosome copies in this polyploid organism. Quantitative real-time PCR shows that the heterozygous mutants have a recJ gene copy that is ca. 10–30% that of the wild-type. Mutants with reduced recJ gene copy grow slowly and are more sensitive than wild-type to UV irradiation, gamma irradiation, and hydrogen peroxide. The mutants are as resistant as wild-type to methyl-methanesulfonate. The D. radiodurans RecJ protein was expressed in E. coli and purified under denaturing conditions. The re-folded protein has nuclease activity on single-stranded DNA with specificity similar to that of E. coli RecJ exonuclease.     

Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes

We have used combinations of subcellular fractionation, specific cytochemical tracers, and quantitative immunoadsorption to determine when, where, and in which intracellular structure internalized asialoglycoproteins (ASGPs) are segregated from their receptor. All membrane vesicles containing the receptor (R+ vesicles) were quantitatively immunoadsorbed from crude microsomes with Staphylococcus aureus cells and affinity-purified anti-ASGP receptor. Using this assay, we varied the time and temperature of exposure of perfused livers to 125I-asialoorosomucoid (125I-ASOR) and followed the movement of ligand from R+ to R- vesicles. After 2.5 min at 37 degrees C, 98% of the internalized ligand could be immunoadsorbed and thus was in R+ vesicles. Over the next 12 min of continuous 37 degrees C perfusion with 125I-ASOR, an increasing fraction of the ligand was not immunoadsorbed and therefore was present in R- vesicles. A maximum of 30% of the ligand could be found in R- vesicles (14-44 min). When livers were maintained at 16 degrees C, ligand was internalized but remained in R+ vesicles. Furthermore, ligand accumulating in R- vesicles at 37 degrees C remained there when livers were cooled to 16 degrees C. R- endosomes could be separated from R+ endosomes by flotation on sucrose density gradients and visualized by the presence of sequestered ASOR-horseradish peroxidase (ASOR-HRP). These structures resembled those labeled by ASOR-HRP in situ: R+ vesicles were relatively dense (1.12 g/cc), frequently tubular or spherical and small (100-nm diam), corresponding to the peripheral and internal tubular endosomes; R- structures were of lower density (1.09 g/cc), large (400-nm diam), and resembled internal multivesicular endosomes (MVEs). Endocytosed ASOR-HRP was found in both the peripheral and internal tubular endosomes in situ under conditions where 95% of the ligand was present in R+ vesicles by immunoadsorption, whereas MVEs containing ASOR-HRP were predominant in situ when ligand was found in R- vesicles and were often in continuity with the tubular internal endosomes. All of these results suggest that complete segregation of ligand and receptor occurs after arrival in the Golgi-lysosome region of the hepatocyte and that MVEs are R- and represent the final prelysosomal compartment.     

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.     

BMI and Neuronal Integrity in Healthy,Cognitively Normal Elderly: A Proton Magnetic Resonance Spectroscopy Study

Recent studies associated excess body weight with brain structural alterations, poorer cognitive function, and lower prefrontal glucose metabolism. We found that higher BMI was related to lower concentrations of N‐acetyl‐aspartate (NAA, a marker of neuronal integrity) in a healthy middle‐aged cohort, especially in frontal lobe. Here, we evaluated whether NAA was also associated with BMI in a healthy elderly cohort. We used 4 Tesla proton magnetic resonance spectroscopy (¹H MRS) data from 23 healthy, cognitively normal elderly participants (69.4 ± 6.9 years; 12 females) and measured concentrations of NAA, glutamate (Glu, involved in cellular metabolism), choline‐containing compounds (Cho, involved in membrane metabolism), and creatine (Cr, involved in high‐energy metabolism) in anterior (ACC) and posterior cingulate cortices (PCC). After adjustment for age, greater BMI was related to lower NAA/Cr and NAA/Cho ratios (β < ?0.56, P < 0.008) and lower Glu/Cr and Glu/Cho ratios (β < ?0.46, P < 0.02) in ACC. These associations were not significant in PCC (β > ?0.36, P > 0.09). The existence of an association between NAA and BMI in ACC but not in PCC is consistent with our previous study in healthy middle‐aged individuals and with reports of lower frontal glucose metabolism in young healthy individuals with elevated BMI. Taken together, these results provide evidence that elevated BMI is associated with neuronal abnormalities mostly in frontal brain regions that subserve higher cognitive functions and impulse control. Future studies need to evaluate whether these metabolite abnormalities are involved in the development and maintenance of weight problems.     

Priming and memory of stress responses in organisms lacking a nervous system

Experience and memory of environmental stimuli that indicate future stress can prepare (prime) organismic stress responses even in species lacking a nervous system. The process through which such organisms prepare their phenotype for an improved response to future stress has been termed ‘priming’. However, other terms are also used for this phenomenon, especially when considering priming in different types of organisms and when referring to different stressors. Here we propose a conceptual framework for priming of stress responses in bacteria, fungi and plants which allows comparison of priming with other terms, e.g. adaptation, acclimation, induction, acquired resistance and cross protection. We address spatial and temporal aspects of priming and highlight current knowledge about the mechanisms necessary for information storage which range from epigenetic marks to the accumulation of (dormant) signalling molecules. Furthermore, we outline possible patterns of primed stress responses. Finally, we link the ability of organisms to become primed for stress responses (their ‘primability’) with evolutionary ecology aspects and discuss which properties of an organism and its environment may favour the evolution of priming of stress responses.     

Effect of peak pressure and pressure gradient on subsurface shear stresses in the neuropathic foot

The pressure distribution on the plantar surface of the foot may provide insights into the stresses within the subsurface tissues of patients with diabetes mellitus and peripheral neuropathy (PN) who are at risk for skin breakdown. The purposes of this study were to (1) estimate the stress distribution in the subsurface soft tissue from a measured surface pressure distribution and determine any differences between values in the forefoot and rearfoot, and (2) determine the relationship between maximum shear stress (MSS) (magnitude and depth) and characteristics of the pressure distribution. The measured in-shoe pressure distributions during walking characterized by the peak plantar pressure and maximum pressure gradient on the plantar surface of the feet for 20 subjects with diabetes, PN and history of a mid foot or forefoot plantar ulcer were analyzed. The effects of peak pressure and maximum pressure gradient at the peak pressure location on the stress components in the subsurface soft tissue were studied using a potential function method to estimate the subsurface tissue stress. The calculated MSSs are larger in magnitude and located closer to the surface in the forefoot, where most skin breakdown occurs, compared to the rearfoot. In addition, the MSS (magnitude and depth) is highly correlated with the pressure gradient (r=-0.77 & 0.61) and the peak pressure (r=-0.61 & 0.91). The peak pressure and the maximum pressure gradient obtained from the surface pressure distribution appear to be important variables to identify where MSSs are located in the subsurface tissues on the plantar foot that may lead to skin break down.     

STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.