Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Processing of multi-dimensional NMR data with the new software PROSA

Summary The new program PROSA is an efficient implementation of the common data-processing steps for multi-dimensional NMR spectra. PROSA performs linear prediction, digital filtering, Fourier transformation, automatic phase correction, and baseline correction. High efficiency is achieved by avoiding disk storage of intermediate data and by the absence of any graphics display, which enables calculation in the batch mode and facilitates porting PROSA on a variety of different computer systems; including supercomputers. Furthermore, all time-consuming routines are completely vectorized. The elimination of a graphics display was made possible by the use of a new, reliable automatic phase-correction routine. CPU times for complete processing of a typical heteronuclear three-dimensional NMR data set of a protein vary between less than 1 min on a NEC SX3 supercomputer and 40 min on a Sun-4 computer system.     

Processing of behaviorally relevant temporal parameters of acoustic stimuli by single neurons in the superior olivary nucleus of the leopard frog

Summary Response characteristics of 130 single neurons in the superior olivary nucleus of the northern leopard frog (Rana pipiens pipiens) were examined to determine their selectivity to various behaviorally relevant temporal parameters [rise-fall time, duration, and amplitude modulation (AM) rate of acoustic signals. Response functions were constructed with respect to each of these variables. Neurons with different temporal firing patterns such as tonic, phasic or phasic-burst firing patterns, participated in time domain analysis in specific manners. Phasic neurons manifested preferences for signals with short rise-fall times, thus possessing low-pass response functions with respect to this stimulus parameter; conversely, tonic and phasic-burst units were non-selective and possessed all-pass response functions. A distinction between temporal firing patterns was also observed for duration coding. Whereas phasic units showed no change in the mean spike count with a change in stimulus duration (i.e., all-pass duration response functions), tonic and phasic-burst units gave higher mean spike counts with an increase in stimulus duration (i.e., primary-like high-pass response functions). Phasic units manifested greater response selectivity for AM rate than did tonic or phasic-burst units, and many phasic units were tuned to a narrow range of modulation rates (i.e., band-pass). The results suggest that SON neurons play an important role in the processing of complex acoustic patterns; they perform extensive computations on AM rate as well as other temporal parameters of complex sounds. Moreover, the response selectivities for rise-fall time, duration, and AM rate could often be shown to contribute to the differential responses to complex synthetic and natural sounds.Abbreviations SON superior olivary nucleus - DMN dorsal medullary nucleus - TS torus semicircularis - FTC frequency threshold curve - BF best excitatory frequency - PAM pulsatile amplitude modulation - SAM sinusoidal amplitude modulation - SQAM square-wave amplitude modulation - MTF modulation transfer function - PSTH peri-stimulus time histogram     

The equine protease inhibitory system (Pi): Abnormal expressions of PiF,PiL, and PiS

Three cases of abnormal expression of the equine protease inhibitory alleles, Pi F, L, and S₁, were observed following the examination of 30,000 plasma samples by one-dimensional acid (pH 4.6) polyacrylamide gel electrophoresis. Characterization of the abnormal proteins in terms of isoelectric point, molecular mass, inhibitory spectra, and sialic acid content was performed using one- and two-dimensional electrophoretic techniques. The Pi F and S₁ abnormalities were postulated to be the result of amino acid substitutions causing alterations in the processing of the carbohydrate side chains. No explanation could be offered for the Pi L abnormality other than a charge shift mutation. Abnormal types, F*, L*, and S*₁ behaved as alleles but the distribution of L* in offspring from one stallion (present in only 6 of 83 offspring) differed significantly from expectation.This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, N.S.W. 2031.     

Neural mechanisms of auditory information processing by identified interneurones in Orthoptera

Three qualities of sound—the directional, the temporal and the spectral—are important for intraspecific communication in Orthoptera. The neural mechanisms employed by identified interneurones for encoding these sound qualities are illustrated by examples of physiological processes found at different levels of the CNS. Discussed are: (1) the creation of directional information by local interneurones in the thorax, and the use of time-intensity trading in sound location; (2) mechanisms for encoding the temporal parameters of sound by interneurones ascending to the brain; (3) frequency-dependent neural filtering of auditory information by local interneurones.     

Detection of TRH extended peptides in rat hypothalamus using an antibody raised to pGluHisProGlyLys

An antibody was raised to the synthetic pentapeptide pGluHisProGlyLys which, in radioimmunoassay (RIA), could detect the pentapeptide at a level of 10 fmole per tube and exhibited <0.5 per cent cross reactivity with a series of related peptides. The RIA was used to demonstrate the presence of C-terminally extended forms of thyrotropin releasing hormone (TRH) in rat hypothalamus. After extraction, the endogenous peptides were resolved by gel exclusion chromatography and TRH-extended peptides were revealed by trypsin digestion to release the pentapeptide. The TRH extended peptides occurred in substantial quantity, approximately 11 pmoles/g, indicating that only partial processing of the gene duplicated prohormone takes place.     

In vitro studies on the subcellular location of glucosidase I and glucosidase II in dog pancreas

When programmed with yeast prepro--factor mRNA, the heterologous reticulocyte/dog pancreas translation system synthesizes two pheromone related polypeptides, a cytosolically located primary translation product (pp--F_cyt, 21 kDa) and a membrane-specific and multiply glycosylated e-factor precursor (pp--F₃, 27.5 kDa). Glycosylation of the membrane specific pp--F₃ species is competitively inhibited by synthetic peptides containing the consensus sequence Asn-Xaa-Thr as indicated by a shift of its molecular mass from 27.5 kDa to about 19.5 kDa (pp--F₀) , whereas the primary translation product pp--Fcyt is not affected. Likewise, only the glycosylated pp--F₃ structure is digested by Endo H yielding a polypeptide with a molecular mass between PP--F₀ and pp--Fcyt. These observations strongly suggest that the primary translation product is proteolytically processed during/on its translocation into the lumen of the microsomal vesicles. We believe that this proteolytic processing is due to the cleavage of a signal sequence from the pp--Fcyt species, although this interpretation contradicts previous data from other groups. The distinct effect exerted by various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-dNM, 1-deoxymannojirimycin) on the electrophoretic mobility of the pp--F₃ polypeptide indicates that its oligosaccharide chains are processed to presumbly Man₉-GlcNAc₂ structures under thein vitro conditions of translation. This oligosaccharide processing is most likely to involve the action of glucosidase I and glucosidase II as follows from the specificity of the glycosidase inhibitors applied and the differences of the molecular mass observed in their presence. In addition, several arguments suggest that both trimming enzymes are located in the lumen of the microsomal vesicles derived from endoplasmic reticulum membranes.Abbreviations dNM 1-deoxynojirimycin - N-Me-dNM N-methyl-dNM - dMM 1-deoxymannojirimycin - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Computer-aided three-dimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules

Summary With the use of a digital image-processing method three-dimensional reconstructions of the arrangement of spermatocytes in human seminiferous tubules were performed. With this method it was possible to investigate the cellular distribution in the tubule in nearly any given perspective and projection. In addition, by means of simple mathematical procedures, such as by transformation of Cartesian coordinates into cylindrical coordinates, it was possible to vary the shape of a reconstruction, i.e., to convert the cylindrical image of a tubular portion into a right-angled r--z-representation.The present work not only confirms the existence of a complex helical plan of organization of the human seminiferous epithelium but also provides further aspects of the phenomenon of physiological germ-cell loss and its integration into the kinetics of spermatogenesis.Dedicated to Prof. E.C. Roosen-Runge, Seattle, on the occasion of his 75th birthday