Protease gene families in Populus and Arabidopsis

Background  

Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution.     

Retrograde degeneration of basal forebrain cholinergic neurons after neurotoxin lesions of the neocortex: application of ganglioside GM1

Injections of the neurotoxin kainic acid were made unilaterally at multiple loci in the cerebral cortex of the rat in an attempt to reproduce aspects of the central pathology of Alzheimer's disease. Neurochemical markers of cholinergic and GABAergic function in the cortex and basal forebrain, determined after various intervals, suggested that subsequent to initial destruction of cortical neuronal cell bodies, trans-synaptic retrograde degeneration of cholinergic neurons occurred in the nucleus basalis magnocellularis (NBM) projecting to the cortex.

Contrary to the situation noted after devascularizing cortical lesions, there was no spontaneous recovery from this effect of kainic acid in the ipsilateral NBM. Similarly, these retrograde effects could not be prevented by the administration of the ganglioside GM₁. These observations suggest that kainic acid compromises the plastic capacity of this cholinergic projection, perhaps by affecting the production of endogenous trophic factors. This may be of relevance in developing the use of neurotoxins for models of neurodegenerative disease.     

Chloroplast polypeptides synthesized by leaf segments and isolated chloroplasts during senescence in barley.

Starting from senescent barley (Hordeum vulgare L. cv Hassan) leaf segments receiving light and hormone treatments affecting senescence, the plastid polypeptides synthesized by isolated chloroplasts and by leaf segments were analyzed by radiolabelling followed SDS-PAGE and fluorography. Among 20 to 30 polypeptides detected, a few were specifically synthesized (by chloroplasts and/or leaf segments) after each senescence treatment. Apparently, the polypeptides labelled in assays with isolated chloroplasts are truly synthesized in vivo, because most of them were also labelled in assays with leaf segments. The comparison of polypeptide profiles, for every senescence treatment, after labelling with isolated chloroplasts or leaf segments, suggests that most plastid polypeptides synthesized during senescence are coded in plastid DNA.     

Development and application of a monoclonal rat peroxidase antiperoxidase (PAP) immunocytochemical reagent

Summary Monoclonal antibodies are being increasingly used in immunocytochemistry but their localisation by the peroxidase antiperoxidase (PAP) procedure requires the use of rat or mouse PAP. In this paper we describe the development and application of a monoclonal rat PAP. This reagent has been used successfully for immunocytochemistry at light and electron microscopy level in combination with rat monoclonal antibodies against serotonin (5-HT), substance P and somatostatin. The monoclonal rat PAP has several advantages over conventional polyclonal rat PAP and is likely to be a valuable developing reagent in immunocytochemistry using rat monoclonal antibodies.     

Hemicholinium mustard derivatives: Preliminary assesment of cholinergic neurotoxicity

We have attempted to design novel neurotoxins based on the use of hemicholinium derivatives. Three compounds were tested for their neurochemical effects on cholinergic, gabaergic and catecholaminergic markers in the hippocampus, striatum and cortex following intracerebroventricular administration. The effects were compared with those of the non-specific alkylating agent (nitrogen mustard) and the previously reported ethylcholine mustard aziridinium ion (AF 64A). The results indicate that only one of these derivatives (HcM-9) exhibits comparable neurotoxic effects on cholinergic markers with a similar pattern of specificity to that of AF 64A. In addition, HcM-9 showed less overall toxicity, this being reflected in a higher survival rate. The present results indicate that hemicholinium derivatives could be good substrates for further molecular modifications, thus a step towards the design of a more specific cholinergic neurotoxin.Special Issue dedicated to Prof. Eduardo De Robertis.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.