Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

Differential human impact and vegetation history in two adjacent Pyrenean valleys in the Ariège basin,southern France,from 3000 B.P. to the present

Detailed palynological studies in two adjoining French Pyrenean valleys, complemented by the study of archives, demonstrate that under similar climatic conditions, the forest history of each valley from the Bronze Age to present time was essentially determined by socio-economical constraints, possibly modified by natural characteristics such as topography. The studies show why the expansion of Fagus (beech) at c. 4000 B.P. was asynchronous on the northern slope of the Pyrenees and emphasize the effects of the human impact on the recent lowering of the tree-line.A contribution to the 8th IPC, Aix-en-Provence, 1992     

A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites

Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for β-glucosidase (GBA) and the β-polypeptide 1 of the Na⁺, K⁺-transporting ATPase (ATP1B1). Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097). The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88 CM. This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene. In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4pl2-pl3. The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E. coli pilus antigens. One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization.     

Malaria-induced reduction of fecundity during the first gonotrophic cycle of Anopheles Stephensi mosquitoes

Abstract. Anopheles stephensi mosquitoes which had fed upon mice infected with Plasmodium yoelii nigeriensis malaria parasites produced significantly fewer eggs than mosquitoes fed on an uninfected mouse. Fecundity reduction was more pronounced when the bloodmeal contained malaria gametocytes and the mosquitoes developed oocysts. Egg production and haematin excretion were correlated for uninfected bloodfed mosquitoes; the presence of P.y. nigeriensis in the blood affected this relationship. Reduced fecundity was associated with a significant reduction of bloodmeal size (measured by haematin excretion) in mosquitoes which ingested gametocytaemic blood. The bloodmeal size in mosquitoes fed on parasitaemic blood without gametocytes was not significantly reduced. The use of haematin assays for determination of bloodmeal size in mosquitoes is discussed.     

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, −10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.     

An actuated dissipative spring-mass walking model: Predicting human-like ground reaction forces and the effects of model parameters

Simple models are widely used to understand the mechanics of human walking. The optimization-based minimal biped model and spring-loaded-inverted-pendulum (SLIP) model are two popular models that can achieve human-like walking patterns. However, ground reaction forces (GRF) from these two models still deviate from experimental data. In this paper, we proposed an actuated dissipative spring-mass model by integrating these two models to realize more human-like GRF patterns. We first explored the function of stiffness, damping, and weights of both energy cost and force cost in the objective function and found that these parameters have distinctly different influences on the optimized gait and GRF profiles. The stiffness and objective weight affect the number and size of peaks in the vertical GRF and stance time. The damping changes the relative size of the peaks but has little influence on stance time. Based on these observations, these parameters were manually tuned at three different speeds to approach experimentally measured vertical GRF and the highest correlation coefficient can reach 0.983. These results indicate that the stiffness, damping, and proper objective functions are all important factors in achieving human-like motion for this simple walking model. These findings can facilitate the understanding of human walking dynamics and may be applied in future biped models.     

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

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Insights into the relationship between the proteasome and autophagy in human and yeast cells

In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions.