Three-dimensional models of chick embryo somites obtained by mathematical methods applied to ultrastructural data

Summary A model of a thoracolumbar somite of a chick embryo at the 53rd incubation hour was obtained by mathematical methods, after identification of somite cell types by means of electron microscopy.Each specific district occupied by the cell types was precisely determined.On the basis of these observations, the somite was three-dimensionally reconstructed and the spatial positions of the primitive myotome, dermatome, sclerotome, undifferentiated mesoderm and myocele were precisely identified.     

On the fungitoxicity of some new thiocyanatopyrazole derivatives: Electron microscopical study in trichophyton mentagrophytes

Four thiocyanatopyrazole derivatives were synthesized and their fungistatic activity was demonstrated in vitro against a number of dermatophytic fungi. In Trichophyton mentagrophytes, the most active compound induced an unusual increase of the plasma membrane with production of intra and extracytoplasmic complexes, a deterioration of nuclear and mitochondrial membranes and a formation of autophagic-like vacuoles. Plasmolysis, accompanied by an almost complete disorganization of cytoplasmic structures, seemed to be the final event. A possible mechanism of action of the compounds was discussed.Investigation supported by a grant from Consiglio Nazionale delle Ricerche of Italy (Contract No. 7500536).     

Fluorogenic detection of primary amines in plant histochemistry with fluorescamine: A comparative study on the effects of coagulant and non-coagulant fixatives

Summary The new highly sensitive method of fluorescamine reaction for the topochemical detection of primary amino groups was studied as a substitude of ninhydrin-Schiff's reaction for the localisation of total proteins in plant tissues. The influence of various coagulant and non-coagulatn fixatives on the induction of fluorescamine fluorescence was examined: ethanol, formaldehyde gas and solution, glutaraldehyde, acrolein, osmium tetroxide, Bouin, Rossman, Clarke and Zenker's fluids and FMA were employed. It was found that the use of the fluorogenic method is conditioned by the fixative ability to keep the amino groups disposable and by its capability to reduce the natural autofluorescence of plant material. A detailed account of the fixation methodology demonstrated that non-coagulant acrolein and coagulant mercuric chloride are the most promising fixatives for the use of the fluorescamine reaction in plant histochemistry.     

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by an stain for primary and secondary amino group (e.g. ninydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in higly yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.     

Glycoalkaloid Profile in Potato Haploids Derived from Solanum tuberosum–S. bulbocastanum Somatic Hybrids

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GA) that may affect either human health or biotic stress resistance. Therefore, GA composition must be a major criterion in the evaluation of breeding products when species genomes are merged and/or manipulated. This work reports the results of GA analysis performed on unique haploid (2n=2x=24) plants obtained from tetraploid (2n=4x=48) Solanum bulbocastanum–S. tuberosum hybrids through in vitro anther culture. Glycoalkaloids were extracted from tubers and analyzed by HPLC. Haploids generally showed the occurrence of parental GA. However, in several cases loss of parental GA and gain of new GA lacking in the parents was observed. It may be hypothesized that new GA profiles of our haploids is the result of either genetic recombination or combinatorial biochemistry events. To highlight differences between haploids and parents, soluble proteins and antioxidant activities were also determined. Both were always higher in haploids compared to their parents. The nature of the newly formed GAs will be further investigated, because they may represent new metabolites that can be used against pest and diseases, or are useful for human health.     

Plasmid transformation of Ruminococcus albus by means of high-voltage electroporation

To apply recombinant DNA techniques to the genetic manipulation of cellulolytic ruminal bacteria, a plasmid vector transformation system must be available. The objective of this work was to develop a system for plasmid transformation of Ruminococcus albus. Using high voltage electrotransformation, pSC22 and pCK17 plasmid vectors, derived from lactic acid bacteria plasmids and replicating via single-stranded DNA intermediate, were successfully introduced into three freshly isolated R. albus strains and into R. albus type strain ATCC 27210. The optimization of the electrotransformation condition raised the electroporation efficiency up to 3 x 10(5) transformants per microgram of pSC22 plasmid.     

Effects of 50 Hz magnetic fields on mouse spermatogenesis monitored by flow cytometric analysis

Flow cytometry (FCM) was performed to monitor the cellular effects of extremely-low-frequency magnetic field on mouse spermatogenesis. Groups of five male hybrid F1 mice aged 8–10 weeks were exposed to 50 Hz magnetic field. The strength of the magnetic field was 1.7 mT. Exposure times of 2 and 4 h were chosen. FCM measurements were performed 7, 14, 21, 28, 35, and 42 days after treatment. For each experimental point, a sham-treated group was used as a control. The possible effects were studied by analyzing the DNA content distribution of the different cell types involved in spermatogenesis and using the elongated spermatids as the reference population. The relative frequencies of the various testicular cell types were calculated using specific software. In groups exposed for 2 h, no effects were observed. In groups exposed for 4 h, a statistically significant (P < 0.001) decrease in elongated spermatids was observed at 28 days after treatment. This change suggests a possible cytotoxic and/or cytostatic effect on differentiating spermatogonia. However, further studies are being carried out to investigate the effects of longer exposure times. © 1995 Wiley-Liss, Inc.     

Tissue-selective regulation of protein homeostasis and unfolded protein response signalling in sporadic ALS

Amyotrophic lateral sclerosis (ALS) is a disorder that affects motor neurons in motor cortex and spinal cord, and the degeneration of both neuronal populations is a critical feature of the disease. Abnormalities in protein homeostasis (proteostasis) are well established in ALS. However, they have been investigated mostly in spinal cord but less so in motor cortex. Herein, we monitored the unfolded protein (UPR) and heat shock response (HSR), two major proteostasis regulatory pathways, in human post-mortem tissue derived from the motor cortex of sporadic ALS (SALS) and compared them to those occurring in spinal cord. Although the UPR was activated in both tissues, specific expression of select UPR target genes, such as PDIs, was observed in motor cortex of SALS cases strongly correlating with oligodendrocyte markers. Moreover, we found that endoplasmic reticulum-associated degradation (ERAD) and HSR genes, which were activated predominately in spinal cord, correlated with the expression of neuronal markers. Our results indicate that proteostasis is strongly and selectively activated in SALS motor cortex and spinal cord where subsets of these genes are associated with specific cell type. This study expands our understanding of convergent molecular mechanisms occurring in motor cortex and spinal cord and highlights cell type–specific contributions.