An adaptor strategy to subclone entire cDNA libraries as single insert recombinants

The current methods for subcloning entire cDNA libraries usually result in a significant portion of recombinants containing multiple inserts, since in most instances the inserts derived from the library to be subcloned are released as a bulk of self-ligatable DNA fragments. By use of an adaptor strategy, a method is presented to confer noncompatible ends to primarily self-ligatable inserts, resulting in efficient subcloning of entire libraries as single insert recombinants.     

Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.     

Neuromuscular, anaerobic, and aerobic performance characteristics of elite power athletes

Various aspects of neuromuscular, anaerobic, and aerobic performance capacity were investigated in four powerlifters, seven bodybuilders, and three wrestlers with a history of specific training for several years. The data (means +/- SD) showed that the three subject groups possessed similar values for maximal isometric force per unit bodyweight (50.7 +/- 9.6, 49.3 +/- 4.1, and 49.3 +/- 10.9 N/kg, respectively). However, significant (P less than 0.05) differences were observed in the times for isometric force production, so that e.g., times to produce a 30% force level were shorter for the wrestlers and bodybuilders (28.3 +/- 3.1 and 26.4 +/- 6.6 ms) than that (53.3 +/- 23.7 ms) for the powerlifters. Utilization of elastic energy by the wrestlers was significantly (P less than 0.05) better than that of the other two subject groups, as judged from differences between the counter-movement and squat jumps at 0, 40, and 100 kg's loads. No differences were observed between the groups in anaerobic power in a 1-min maximal test, but the values for VO2 max were higher (P less than 0.05) among the wrestlers and bodybuilders (57.8 +/- 6.6 and 50.8 +/- 6.8 ml X kg-1 X min-1) as compared to the powerlifters (41.9 +/- 7.2 ml X kg-1 X min-1). Within the limitations of the subject sample, no differences of a statistical significancy were observed between the groups in fibre distribution, fibre areas, or the area ratio of fast (FT) and slow (ST) twitch fibres in vastus lateralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mesocyclops longisetus effects on survivorship of Aedes aegypti immature stages in car tyres

Abstract .The effect of the introduction of the entomophagous copepod Mesocyclops longisetus (Acuacultura F.C.B. strain) on the survival of Aedes aegypti immature stages in car tyres was evaluated under semi-natural conditions in the municipality of Merida, Yucatan, Mexico. Life tables were constructed for the immature stages of the mosquito in the presence and absence of M. longisetus , and the survival data were compared using log–linear models. The data set was adjusted using the GLIM statistical package and the quality of adjustment was evaluated with a chi-squared test . Survivorship curves were constructed for each treatment.
In the absence of M. longisetus , the survivorship of Ae. aegypti immature stages averaged 9%. The highest mortality rate was observed during the fourth larval instar (54%) and the resulting survival pattern corresponded to a type II survivorship curve. The mortality rate of Ae. aegypti first-instar larvae (fifty per tyre) increased more than 200-fold in the presence of M. longisetus (twenty per tyre) and the highest mortality was during the first two larval instars, where it reached 98.9%, with a resulting survivorship of 0.2%. Overall mortality was sixfold greater in the presence of the copepod than in its absence. The survival pattern of immature stages of Ae. aegypti in the presence of the copepod corresponded to a type III survivorship curve. As M. longisetus was so effective against Ae. aegypti immature stages in tyres under semi-natural conditions, its long-term effectiveness should be evaluated under socially and ecologically realistic field conditions in Mexico.     

U-Shape Suppressive Effect of Phenol Red on the Epileptiform Burst Activity via Activation of Estrogen Receptors in Primary Hippocampal Culture

Phenol red is widely used in cell culture as a pH indicator. Recently, it also has been reported to have estrogen-like bioactivity and be capable of promoting cell proliferation in different cell lines. However, the effect of phenol red on primary neuronal culture has never been investigated. By using patch clamp technique, we demonstrated that hippocampal pyramidal neurons cultured in neurobasal medium containing no phenol red had large depolarization-associated epileptiform bursting activities, which were rarely seen in neurons cultured in phenol red-containing medium. Further experiment data indicate that the suppressive effect of the phenol red on the abnormal epileptiform burst neuronal activities was U-shape dose related, with the most effective concentration at 28 µM. In addition, this concentration related inhibitory effect of phenol red on the epileptiform neuronal discharges was mimicked by 17-β-estradiol, an estrogen receptor agonist, and inhibited by ICI-182,780, an estrogen receptor antagonist. Our results suggest that estrogen receptor activation by phenol red in the culture medium prevents formation of abnormal, epileptiform burst activity. These studies highlight the importance of phenol red as estrogen receptor stimulator and cautions of careful use of phenol red in cell culture media.     

Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

Clinical significance of platelet alloantibodies detected in immunofluorescence test (neonatal alloimmune thrombocytopenia and post-transfusion purpura)

Three cases of neonatal alloimmune thrombocytopenia and one patient with post-transfusion purpura could be diagnosed only by introducing the platelet immunofluorescence test. Thrombocytopenia was caused by anti-PlA1 platelet alloantibodies detected neither in the agglutination nor by the complement fixation test.     

Fenestrated capillaries in the ventral sebaceous gland of the Djungarian hamster, Phodopus sungorus

The ventral sebaceous gland of the Djungarian hamster is a macroscopically visible organ situated in the midventral area of the abdominal wall. It consists of densely packed acini arranged in lobules with common excretory ducts. The rich vascular network of the gland is characterized by fenestrated capillaries. Fenestrated endothelium has not yet been reported as a characteristic and regular finding within sebaceous glands. Results are discussed with regard to proliferation rate of sebocytes and the demand of fluid and nutrient supply.     

Resolution of the conformational distribution and dynamics of a flexible molecule using frequency-domain fluorometry

We report the first resolution of both the conformational distribution and end-to-end diffusion coefficient of a flexible molecule. This molecular information was recovered using only the donor intensity decay in a single solvent at a single viscosity, as observed by the technique of frequency-domain fluorometry. This technique can be extended to measurements of structural fluctuations of biological macromolecules.     

Stability of the Transthyretin Molecule as a Key Factor in the Interaction with A-Beta Peptide - Relevance in Alzheimer's Disease

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.