Use of modified Fraser's stain in Promoting Activity Test (PAT)

The Promoting Activity Test (PAT) requires a staining procedure that allows rapid, accurate and reliable counting of mitotic figures. We propose use of Fraser's kernechtrot-crystal violet technique, but eliminating the picric-alcoholic differentiation to avoid fading. This modified protocol gives higher mitotic counts in adult mouse adrenal cortex than the hematoxylin-eosin originally used, especially with respect to less conspicuous prophases.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Chemotherapy of Aspergillus fumigatus keratitis: An experimental study

An experimental Keratitis study of Aspergillus fumigatus was performed in 130 rabbits divided into 12 groups of ten animals each. Three antifungal drugs (myconazole, amphotericin B and pimaricin) were tested using two procedures (topical drops and subconjunctival injections) and two different concentrations (500 and 10 000 times the MIC). In each case, the drugs were applied every 3 h starting 14 h after inoculation. Miconazole was useful at 10 mg/ ml concentration by topical drops and subconjunctival injections, but was less useful at 5 mg/ ml. Amphotericin B was useful at 5 mg/ ml concentration by topical drops and less useful at 2 mg/ ml.No differences were found between the two concentrations by subconjunctival administration. Pimaricin was useful by topical drops at 50 mg/ ml concentration and less useful at 10 mg/ ml as well as by subconjunctival injections.     

Airborne grass pollen in Granada (Spain)

We have carried out a study on the annual and daily pollen concentrations from Gramineae over four consecutive years in the atmosphere of Granada (Spain). Samples of pollen grains were collected by the volumetric method with the aid of a Burkard sporetrap. Gramineae, according both to their high sensitizing capacity and to data from allergologists, are responsible for many pollinoses diagnosed in this area. In this work, daily pollen levels from April to July are monitored and the variations identified are interpreted in relation to meteorological conditions. Results indicated that the highest airborne concentrations of Gramineae pollen were found in May and June, although the beginning and intensity of pollination have been variable during these 4 years.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Analysis of Actinobacillus pleuropneumoniae and related organisms by DNA-DNA hybridization and restriction endonuclease fingerprinting.

The objective of this study was to determine the degree of genetic relatedness of Actinobacillus pleuropneumoniae to selected members of the family Pasteurellaceae, with particular emphasis on species commonly associated with swine. Free-solution DNA-DNA hybridization studies revealed that representative strains of all 12 serotypes of A. pleuropneumoniae formed a homogeneous group, sharing 74 to 90% sequence homology with A. pleuropneumoniae serotype 1. All serotypes of A. pleuropneumoniae tested demonstrated a high degree of genetic relatedness (66 to 79%) to the type species of the genus Actinobacillus, A. lignieresii. Little homology (less than 20%) was detected between A. pleuropneumoniae strains and selected Haemophilus spp. and Pasteurella spp. Since free-solution hybridization methods are technically demanding and require large amounts of highly purified DNA, restriction endonuclease fingerprinting (REF) was examined to determine whether it could be a useful taxonomic tool for classification of members of the family Pasteurellaceae. REF profiles were compared, and the degree of similarity between organisms was quantitated by calculating Jaccard similarity coefficients. There was a significant positive relationship between the REF Jaccard coefficients and the DNA homology values determined from free-solution hybridization experiments.     

Insights on the amino acid side-chain interactions of a synthetic T-cell determinant.

The effect of single amino acid substitutions at positions 18 and 20 on the T-cell determinant (TD) character of peptide p12-26 from lambda repressor protein and on its recognition by a monoclonal antibody was studied by means of 40 synthetic peptides of a length of 15 amino acids. ELISA competition experiments showed that the identity of amino acid at position 20 is very important for antibody recognition, whereas that of amino acid at position 18 is much less important. In contrast, both Leu 18 and Ala 20 are important residues in defining the TD character of peptide p12-26. The most tolerated replacements, ordered in increasing disrupting power are: Ala 20 by Cys, Ser or Gly and Leu 18 by Ile or Val. Any other amino acid replacement completely abolishes the TD capacity of peptide p12-26. The peptides used in this study were synthesized using a multiple solid-phase peptide synthesizer newly designed. Their purity was very high as shown by amino acid sequence experiments.     

Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells

To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3'-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase Go and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.