Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

Acetamide Agar Medium Selective for Pseudomonas aeruginosa

A synthetic base of acetamide and salts in agar permitted isolation of small numbers of Pseudomonas aeruginosa from swarming Proteus and other gram-negative species.     

Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V

The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsCl-ethidium bromide density gradients, which corresponded to supercoiled and to relaxed circular and linear DNA, respectively. Neutral CsCl equilibrium centrifugation indicated that the Hz-1V DNA had a buoyant density of 1.7024 g/ml, which corresponded to a guanine-plus-cytosine (G+C) content of 43%. Thermal denaturation indicated a high G+C domain(s) in the Hz-1V genomic DNA. The domain(s), which included about 11% of the total genomic DNA, exhibited a T(m) of 97 degrees C. The remaining portion (89%) of the DNA had a T(m) of 86.5 degrees C. The T(m)s corresponded to G+C contents of 42 and 67%, respectively. The mean genetic complexity of Hz-1V DNA determined by DNA reassociation kinetic analysis was found to be 152 x 10(6). A possible rapidly reassociating component comprising approximately 13% of the genome was observed. The mean molecular weights from restriction endonuclease digests were 159 x 10(6) for both HindIII and EcoRI. Genomic heterogeneity was found in both the wild-type Hz-1V stock and in two plaque isolates. Of 12 single-plaque isolates, 3 basic restriction endonuclease DNA fragment patterns were observed. The molecular size estimates from electron microscopic contour lengths of uncloned viral DNA ranged from 70 to 158 megadaltons, and the mode was the 130- to 140-megadalton class.     

Influence of spinal cord transection on the presence and axonal transport of CGRP-, chromogranin A-, VIP-, synapsin I-, and synaptophysin-like immunoreactivities in rat motor nerve

Using immunofluorescence and cytofluorimetric scanning (CFS), we investigated the short-term (1-7 days) influence of lower thoracic spinal cord transection on lumbar motor neurons. The content of calcitonin gene-related peptide- (CGRP) like immunoreactivity (LI), chromogranin A (Chr A) -LI, vasoactive intestinal polypeptide (VIP)-LI, Syn I-LI, and synaptophysin (p38)-LI in motor perikarya, and the anterograde and retrograde axonal transport of these substances in the sciatic nerve, were studied in nerve crush (6 h) experiments. During the week after transection, CGRP-LI in perikarya decreased, whereas Chr A-LI increased. VIP-LI, co-localized with Chr A-LI in motor perikarya, did not change after transection. The antero- and retrograde transport of CGRP-LI in the sciatic nerve, occurring in both motor and sensory axons, appeared unchanged in cytofluorimetric scanning (CFS) graphs, but the microscopical picture clearly showed that large motor axons had a decreased content of CGRP-LI at 3 and 7 days posttransection, whereas thinner axons were unchanged in fluorescence intensity. The anterograde transport of Chr A-LI, present in both motor and postganglionic adrenergic axons, was decreased 1 and 3 days after lesion, but returned to control by day 7. There was a marked decrease in anterograde transport of VIP-LI, present mainly in postganglionic sympathetic axons, at day 3, but at 7 days transport was normal. The amounts of transported p38, the synaptic vesicle marker, were in the normal range during the whole period. Syn I-LI accumulation anterogradely was somewhat decreased at 3 and 7 days posttransection, and at 1 day the retrograde accumulation was significantly increased. The results suggest that removal of supraspinal input to intact lower motor neurons causes alterations in metabolism and axonal transport of organelle-associated substances, partly probably related to the complex pattern of transmitter leakage from degenerating, descending nerve terminals. These alterations appear to take place also in postganglionic sympathetic neurons in the sciatic nerve, that originate in the lumbar sympathetic chain. © 1992 John Wiley & Sons, Inc.     

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.     

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.     

Genetic biodiversity in the Baltic Sea: species-specific patterns challenge management

Information on spatial and temporal patterns of genetic diversity is a prerequisite to understanding the demography of populations, and is fundamental to successful management and conservation of species. In the sea, it has been observed that oceanographic and other physical forces can constitute barriers to gene flow that may result in similar population genetic structures in different species. Such similarities among species would greatly simplify management of genetic biodiversity. Here, we tested for shared genetic patterns in a complex marine area, the Baltic Sea. We assessed spatial patterns of intraspecific genetic diversity and differentiation in seven ecologically important species of the Baltic ecosystem—Atlantic herring (Clupea harengus), northern pike (Esox lucius), European whitefish (Coregonus lavaretus), three-spined stickleback (Gasterosteus aculeatus), nine-spined stickleback (Pungitius pungitius), blue mussel (Mytilus spp.), and bladderwrack (Fucus vesiculosus). We used nuclear genetic data of putatively neutral microsatellite and SNP loci from samples collected from seven regions throughout the Baltic Sea, and reference samples from North Atlantic areas. Overall, patterns of genetic diversity and differentiation among sampling regions were unique for each species, although all six species with Atlantic samples indicated strong resistence to Atlantic-Baltic gene-flow. Major genetic barriers were not shared among species within the Baltic Sea; most species show genetic heterogeneity, but significant isolation by distance was only detected in pike and whitefish. These species-specific patterns of genetic structure preclude generalizations and emphasize the need to undertake genetic surveys for species separately, and to design management plans taking into consideration the specific structures of each species.     

Protein conformational exchange measured by 1H R1ρ relaxation dispersion of methyl groups

Activated dynamics plays a central role in protein function, where transitions between distinct conformations often underlie the switching between active and inactive states. The characteristic time scales of these transitions typically fall in the microsecond to millisecond range, which is amenable to investigations by NMR relaxation dispersion experiments. Processes at the faster end of this range are more challenging to study, because higher RF field strengths are required to achieve refocusing of the exchanging magnetization. Here we describe a rotating-frame relaxation dispersion experiment for ¹H spins in methyl ¹³CHD₂ groups, which improves the characterization of fast exchange processes. The influence of ¹H–¹H rotating-frame nuclear Overhauser effects (ROE) is shown to be negligible, based on a comparison of R _1ρ relaxation data acquired with tilt angles of 90° and 35°, in which the ROE is maximal and minimal, respectively, and on samples containing different ¹H densities surrounding the monitored methyl groups. The method was applied to ubiquitin and the apo form of calmodulin. We find that ubiquitin does not exhibit any ¹H relaxation dispersion of its methyl groups at 10 or 25 °C. By contrast, calmodulin shows significant conformational exchange of the methionine methyl groups in its C-terminal domain, as previously demonstrated by ¹H and ¹³C CPMG experiments. The present R _1ρ experiment extends the relaxation dispersion profile towards higher refocusing frequencies, which improves the definition of the exchange correlation time, compared to previous results.     

Fish larvae distribution among different habitats in coastal East Africa

Fish larvae abundances, diversity and trophic position across shallow seagrass, coral reef and open water habitats were examined to characterize their distribution in coastal East Africa. Larvae were identified to family and analysed for abundance differences between sites and habitats, trophic level using stable-isotope analysis and parental spawning mode. Abundances differed greatly between sites with the highest numbers of larvae occurring in the open-water and seagrass habitats. Larval fish diversity was high across habitats with 51 families identified with small differences between sites and among habitats. Notably, larvae of abundant large herbivorous fishes present in reef and seagrass habitats were almost completely absent at all sampling locations. In the seagrass, demersal spawned larvae were more abundant compared with the reef and open-water habitats. Stable-isotope analysis revealed that fish larvae have a varied diet, occupying trophic level two to three and utilizing planktonic prey. This study offers new insights into distributional aspects of fish larvae along the East African coast where such information is sparse.