Construction of linear plasmid vectors for cloning in Escherichia coli cells]

Prophage of the temperate coliphage N15 is a linear plasmid with covalently closed ends. The central part of plasmid N15 genome responsible for vegetative phage growth was replaced by DNA fragments containing genes for selective markers which have unique restriction sites. As a result a family of linear plasmid vectors was constructed. Their size is about 20 kb and their capacity is comparable with that of cosmid vectors.     

Transmission light microscopy of structurally colored semithin cartilage sections

 Conversion of osmified tracheal cartilage constituents into an array of laminar interference gratings has been attained by three tandem reactions. Oxidation of semithin, LR white-embedded cartilage sections by acetic anhydride in dimethyl sulfoxide is the first step in the conversion process. Subsequent addition reactions of oxidized cartilage pyranoses and furanoses with thiocarbohydrazide constitutes the second step. Reduction of silver proteinate by thiocarbohydrazones and the concomitant coating of cartilage constituents with silver gratings completes the conversion of cartilage sections into a system of layered interference filters. In transmitted light, all components of converted cartilage display vivid structural colors, which allow detailed microscopic analysis of structurally colored cellular and extracellular cartilage constituents. Accepted: 10 June 1997     

A modified periodic acid-thiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by transmission electron microscopy

Oxidation of araldite-embedded liver sections by 1% w/v aqueous H5IO6 for 15 min and a 5-min reaction of carbonyls with 1% w/v thiocarbohydrazide in 10% v/v acetic acid was employed for subsequent staining of glycogen with silver-proteinate (S-P). The network of branching intracellular glycogen aggregates was revealed by 15-min staining with S-P, whereas 24 hr incubation in S-P was necessary to enhance the contrast of glycogen inclusions. We conclude that the proposed modification of glycogen staining readily affords the means for its localization at a desired level of contrast and resolution.     

The in situ determination of melting-solidification points of lipid inclusions in fixed cultured cells

Melting-solidification points of microscopic lipid inclusions in fetal human kidney cultures were determined visually by gradual heating of formol-fixed cell monolayers while immersed in dilute aqueous solutions of Nile blue A. Intracellular lipid inclusions stained red when their melting point was reached and reverted to blue color at the solidification temperature. The melting-solidification points of intracellular lipid inclusions in kidney cells were determined to fall within 19-25, 40-50, and 60-65 degrees C range.     

Cytotoxic effect of dimethylsulfoxide on the ultrastructure of cultured Rhesus kidney cells

Cultured Rhesus kidney cells were incubated in the 7.5 and 15% solutions at 4 and 25 ° C and for periods ranging from 10 to 60 min.The ultrastructural alterations, while varying from cell to cell, were evident even following the 10-min incubation interval. The most frequent and least severe structural changes included lipid accumulation, increased number of lysosomes, dilation and degranulation of rough surfaced endoplasmic reticulum, as well as the swelling of mitochondria and damage to mitochondrial membranes.The more severe instances of cellular lesions were represented by the extensive damage to cell membranes, karyorrhexis, total obliteration of the internal structure of mitochondria, and areas of complete loss of hyaloplasm.The extent and frequency of cellular lesions appeared to be determined primarily by the concentration of DMSO with the temperature and duration of incubation being important ancillary determinants.     

Correction with thymic and bone marrow peptides of radiation injuries of immuno- and hemopoiesis

Exposure of an organism to ionizing radiation causes a drastic decrease of immunity and nonspecific resistance as well an impairment of haemopoiesis. Peptide preparations from the thymus and bone marrow normalize the immunological reactivity and haemopoiesis of irradiated animals. Their mixture favors the recovery of haemopoiesis and immunogenesis after irradiation.     

Changes in sperm stores, ejaculate size, fertilization success, and sexual motivation over repeated matings in the common toad, Bufo bufo (Anura: Bufonidae)

Fertilization is of central importance in the determination of reproductive success for both males and females. In species where males have the chance to mate repeatedly within a short period of time, sperm stocks may become depleted and males may have to carefully economize on their sperm reserves. Also, intensive intrasexual competition for females and repeated matings may lead to exhaustion on the behavioural level. To determine whether the reproductive potential of males is limited and if such a limitation is due to behavioural exhaustion or sperm depletion, we experimentally investigated changes in sperm stores, sperm expenditure, fertilization success, and sexual motivation over three repeated matings in the common toad, Bufo bufo , where the breeding season is short and sequential polygyny occurs. At the end of the breeding season, the number of sperm stored in the testes of males mated repeatedly was close to 50% lower than in testes of unmated males. Ejaculate size, which was estimated by applying a novel method allowing direct quantification, decreased by 88% from first to third matings. We also observed a drop in fertilization success from the first two to third matings by 65%, which was largest in males that had started the reproductive season in bad body condition. Some of these males also showed a decreased interest in females in the third mating round. Our results suggest that sperm depletion and loss of sexual motivation may together set a limit to the reproductive potential of common toad males. The present study draws attention to a limitation in reproductive potential, which may occur more often than currently anticipated and has the potential to strongly influence several aspects of reproductive behaviour.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 361–371.     

Newly developed polymorphic microsatellite markers in Job's tears (Coix lacryma‐jobi L.)

A microsatellite‐enriched library of Job's tears (Coix lacryma‐jobi L. var. Ma‐yuen Stapf) was constructed using a modified biotin–streptavidin capture method. After screening, 17 polymorphic microsatellites were used for diversity analysis among 30 Job's tears accessions. The number of alleles ranged from one to five alleles per locus with an average of 2.8 alleles. Expected heterozygosity (H_E) and polymorphism information content (PIC) ranged from 0 to 0.676 and from 0 to 0.666, respectively. The newly developed microsatellite markers are expected to be very valuable tools for evaluation of genetic diversity among large germplasm collection of Job's tears present in our Korean Gene Bank.