The localization of enzymes in tissue sections by immuno-histochemistry. Conventional antibody and mixed aggregation techniques

Synopsis Methods for detecting enzymes in tissue sections by antibody techniques are reviewed. In all these techniques, sections are first incubated with antibody. The bound antibody is visualized in one of four ways: identifying a label such as fluorescein linked to the antibody; using a labelled anti-antibody; employing complement and labelled anti-complement; or making use of a mixed aggregation immuno-cytochemical method.The last technique consists of three steps. A section is first incubated with antiserum, and secondly with the soluble enzyme under investigation. Thirdly the desired enzyme is stained using a conventional cytochemical method. The method is specific since, for example, the soluble enzyme used in the second step can bind only to antigenic determinants which are identical to those of the enzyme localized in the tissue. Thus purification of antigen and antibody sources is simplified, and chemical modifications of the antigen and antibody are avoided.Antibody also acts as a selective fixative for tissue antigen. It will inhibit the catalytic activity of its antigen and, in this way, permit the enzyme activity arising after the reaction of tissue enzyme-antibody complex with soluble enzyme to be amplified selectively. The mixed aggregation immuno-cytochemical technique has been used successfully with membrane-bound enzymes and cytoplasmic enzymes and for the demonstration of catalytically inactive enzyme precursors.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Potential pathogenic factors produced by a clinical nontoxigenicVibrio cholerae O1

A clinical isolate of nontoxigenicVibrio cholerae O1 that caused intestinal fluid accumulation (FA) in adult mice produced proteolytic, hemolytic, and cytotoxic activities in in vitro assays. The linkage of these secreted factors to the FA activity was studied by transposon (TnphoA) mutagenesis. Ten of the 12 TnphoA insertion mutants that were defective for proteolytic activity produced FA, hemolytic and cytotoxic activities; the remaining two mutants lost these latter three activities. These results indicate that FA activity is independent of proteolytic activity but closely associated with cytotoxic and hemolytic activities. Our results with the adult mouse model and a nontoxigenicV. cholerae O1 are in general agreement with previous studies that demonstrated linkage of cytotoxin and hemolysin of toxigenicV. cholerae O1 and non-O1 with FA activity in rabbit ileal loops.     

Tubulin Dimers Oligomerize before Their Incorporation into Microtubules

In the presence of GTP, purified dimers of α- and β-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.     

Genome organization: balancing stability and plasticity

The cell needs to stably maintain its genome and protect it from uncontrolled modifications that would compromise its function. At the same time, the genome has to be a plastic structure that can dynamically (re)organize to allow the cell to adopt different functional states. These dynamics occur on the nanometer to micrometer length scale, i.e. ranging from the level of single proteins up to that of whole chromosomes, and on a microsecond to hour time scale. Here, we review different contributions to the dynamic features of the genome, describe how they are determined experimentally, and discuss the results of these measurements in terms of how the requirements for stability and plasticity are accommodated with specific activities in the nucleus.     

Tandem fluorescent protein timers for in vivo analysis of protein dynamics

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.     

Conclusions about aminopeptidase in tissue sections from studies of amino acid naphthylamide hydrolysis.

Catalytic properties (KM, Vmax) of aminopeptidase in pig kidney sections, in isolated membranes and in a solubilized purified form were investigated using amino acid 2-naphthylamides and 4-methoxy-2-naphthylamides. In the first case these properties were estimated on the basis of the stain intensity resulting from the coupling of product with Fast Blue B, in the latter two cases they were measured fluorometrically. The following observations were made: (1) In all three cases the substrate turnover was shown to be a direct function of time and enzyme concentration. (2) The values obtained for the solubilized and the membrane bound form were practically identical but differed from those found in tissue sections. (3) Each amino acid derivative had defined constants, but these were difficult to obtain in sections, especially if it was necessary, on account of poor solubilities, to use low substrate concentrations. (4) Hydrophilic amino acid derivatives were adsorbed to tissue membranes much less than hydrophobic ones. (5) Fast Blue B caused a non-competitive inhibition of enzymic activity. (6) Binding of antibody against pure aminopeptidase caused inhibition of the enzymic hydrolysis of all the naphthylamides. Thus, histochemical stain intensities per time and area derived from one substrate at a defined concentration are suitable for the determination of enzyme concentrations. However, no conclusions regarding the homogeneity of the enzyme in sections can be drawn by comparing the stain intensities obtained with different substrates in contrast to data from the inhibition of substrate hydrolysis by antibody.     

Climate Change and Structural Vulnerability of a Metropolitan Energy System

In this article, we present methodology and results of a vulnerability assessment of the energy system of the metropolitan region Bremen‐Oldenburg in Northwest Germany. This work is part of the regional climate adaptation project “nordwest2050” aiming at innovative solutions toward a climate‐proof and resilient region. Methodologically, we extended the established vulnerability assessment based on climate change impacts by a structural analysis, highlighting general weaknesses of the metropolitan energy system. Our findings indicate that the structural vulnerabilities of the energy system around Bremen‐Oldenburg pose a greater threat to maintaining the system's services than climate change itself. Climate‐change–based vulnerabilities, however, aggravate many of the structural vulnerabilities and therefore demand attention in their own right. The structural vulnerabilities mainly originate from political and regulatory uncertainties, turbulent market conditions, conflicts along the supply chains, and the current dynamics in the energy sector induced by increased climate mitigation efforts. One of our main conclusions is thus that the metropolitan energy system's capabilities to handle turbulence, perturbations, and surprises must be improved. This will also help in reducing the climate‐change vulnerabilities, because such a system is better equipped when facing currently hard‐to‐predict changes in climate parameters. The results of the assessment described here will be used as the starting point to find options for innovations toward a climate‐proof and resilient energy system for the region in the course of the remaining project.     

Molecular diffusion and binding analyzed with FRAP

Intracellular molecular transport and localization are crucial for cells (plant cells as much as mammalian cells) to proliferate and to adapt to diverse environmental conditions. Here, some aspects of the microscopy-based method of fluorescence recovery after photobleaching (FRAP) are introduced. In the course of the last years, this has become a very powerful tool to study dynamic processes in living cells and tissue, and it is expected to experience further increasing demand because quantitative information on biological systems becomes more and more important. This review introduces the FRAP methodology, including some theoretical background, describes challenges and pitfalls, and presents some recent advanced applications.