Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S-labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high-sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer-assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S-labelled antibody, the detection of TH could be performed at the cellular level.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

A novel stimulator of protein phosphorylation in Neurospora crassa

Summary An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100000 × g supernatants of Neurospora crassa mycelial extracts. This 38 000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin in vitro phosphorylation.The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.Abbreviations PK protein kinase - MES 2-N-Morpholino ethane-sulfonic acid - PMSF phenylmethylsulfonyl fluoride - S₁₀₀ 100000 × g Supernatant     

Growth hormone-releasing factor (GRF)-like immunoreactivity in sensory ganglia of the rat

Summary Growth hormone-releasing factor (GRF)-like immunoreactivity has been demonstrated in the trigeminal and spinal ganglia of fetal, young and adult rats by use of peroxidase-antiperoxidase immunohistochemistry. GRF-like-immunoreactive cells first appear during the second half of embryonic life, as early as day 17. In untreated animals the GRF-immunoreactive elements form approximately 1% of all ganglion cells in the trigeminal and spinal ganglia; their numbers do not change significantly during development. The granular immunoreaction product is confined to perikarya, especially to the perinuclear region. Nerve fibers displaying GRF-like immunoreactivity were found neither in the ganglia, nor in the corresponding central and peripheral areas of termination. The possible role of GRF in sensory ganglia is discussed.     

IgM response and resistance to ascites tumor growth

Summary Antibody response and protection against Ehrlich ascites tumor (EAT) was studied in eight EAT-immunized strains of mice (AL/N, BALB/C, C57BL/6J, F1(C57BL/6×BALB/C), C57BL/10J, B10.BR, CBA/Ca, SW). The results showed a close association between IgM response and resistance to subsequent tumor challenge. Thus, protection was only achieved in those animals giving a measurable IgM response against EAT cell surface antigens, i.e., all inbred strains of mice tested, except CBA/Ca, and some outbred SW mice. The lack of IgM response to these antigens in CBA/Ca was not linked to the strain H-2 haplotype. Resistance could be passively transferred to nonimmunized mice by means of serum, or purified IgM, from protected immune animals. Moreover, complement depletion by cobra venom factor treatment did not modify the protection afforded to those mice. IgM reactivity to EAT cells was completely abolished by previous cell trypsinization. Trypsin removed but did not destroy the antigen(s) recognized by the IgM, since all its activity could be absorbed with the supernatant of the EAT cell trypsinization. Absorption assays with this supernatant treated with different agents, showed that lipids, simple peptides and nucleic acids were not important components of the antigenic determinants. On the contrary, its susceptibility to -galactosidase and particularly to a mild periodate oxidation, suggested that determinants recognized by the IgM against the EAT cell surface are carbohydrate in nature.     

Factors influencing bacterial production of inducers of settlement behavior of larvae of the oysterCrassostrea gigas

Dissolved chemical inducers of settlement behavior of veliger larvae of the oysterCrassostrea gigas are found in supernatants of both pigmented species of bacteria (Alteromonas colwelliana, Vibrio cholerae strain HTX) as well as nonpigmented bacteria (Excherichia coli, Vibrio cholerae strain 596-B). Usually less than 10% of veligers exhibited settlement behavior in response to supernatants from the early bacterial growth phases, whereas 30–90% of larvae responded when exposed to supernatant from late-log and stationary phase cultures. Percentages of larvae exhibiting settlement behavior were inversely correlated with oxygen levels in the culture. Furthermore, the behavioral response decreased with pigment formation, suggesting that quantities of noxious compounds such as quinones may build up in the supernatants of cultures of pigmented bacteria. Tyrosinase, an enzyme that converts L-tyrosine to L-DOPA in the first step of melanogenesis, was detected both in the bacterial pellet and the supernatant during growth of the pigmented species. The enzyme is not required for the production of settlement inducer as the nonpigmented speciesE. coli andV. cholerae (596-B) also released inducer into the supernatant and had no detectable tyrosinase. The data suggest either that there is more than one inducer of settlement behavior found in bacterial supernatants or that the inducer is not L-DOPA or an L-DOPA-mimetic associated with the melanin biochemical pathway.     

Identification and immunohistochemical localization of various bovine pancreatic trypsin inhibitor-isoforms in bovine pituitary gland

Summary Three isoinhibitors of bovine pancreatic trypsin inhibitor (BPTI) have been identified and isolated from bovine pituitary gland. The results of the purification process by affinity chromatography on immobilized trypsin, the electrophoretic mobility in non-denaturing conditions, the antiproteolytic activity and the immunochemical reactions indicate that these inhibitors correspond to those previously isolated from bovine spleen and lung. In addition, immunohistochemical experiments show that the isoinhibitors and BPTI are exclusively localized in the mast cells, and not in the endocrine cells, of the pars intermedia and posterior lobe (neurohypophysis) of the pituitary gland. The physiological implications of these findings are discussed.     

Non-invasive assessment of superficial soft tissue local displacements during movement: A feasibility study

In the movement analysts community, the assessment of the displacement of skin photogrammetric markers relative to the underlying bone (soft tissue displacement, STD) is considered to be a priority. The aim of this study is to present a non-invasive method that allows for the characterization of STD for any marker location, subject, and motor task. In particular, this method provides an estimate of the STD vector in a bone-embedded frame. The body segment under analysis is endowed with the largest possible number of skin markers located over all areas of interest. Any given STD vector is observed from all the marker cluster frames that can be built by suitably combining all the available markers. A subset of the latter frames is identified that is made of frames endowed with uncorrelated local movements. The estimate of a given STD vector is determined through the coherent average of the vectors reconstructed using the above-mentioned independent frames. This estimate is affected by a 180° phase indeterminacy.The proposed method and the underlying hypotheses were validated using markers located on the thighs of two female subjects treated for a total knee replacement. The relevant STD estimates, STD_m, were compared with those directly observed using photogrammetry combined with 2D fluoroscopic projections and the prosthesis CAD model (STD_f). Recordings were made while the volunteers performed step up/down motor tasks.The root mean square value of STD_m was found in the range 2.5–23.0 mm and was consistent with the RMS values of STD_f and with other results reported in the literature and obtained in similarly unconstrained conditions. Moreover, STD_m and STD_f showed a pattern similarity measured by a correlation coefficient equal to 0.83 (±0.13) and by a normalised root mean square distance equal to 27% (±16%).The described estimate of the STD pattern and magnitude, even with the above-mentioned indeterminacies, constitutes valuable information when aiming at optimal marker placement and is an indispensable prerequisite for bone pose estimator design and assessment.     

Antitumor activity and COMPARE analysis of bis-indole derivatives

This paper reports the synthesis of new derivatives (formed by two indole systems separated by a central moiety) analogous of potent antitumor agents previously described. The activity of the bis-indoles bearing a pyridine core confirms the good result described in the previous paper and compound 4c was chosen for the first in vivo experiment (Hollow Fiber Assay). COMPARE analysis and structure–activity relationships were also considered. Contrary to data reported by other Authors, no correlations were found between antitumor activity and NQO1 induction.