Immunohistochemical localization of cyclic AMP and ultrastructural demonstration of adenylate cyclase activity in the testis of Esox lucius at time of spermiation

Summary In the testis of Esox lucius at the time of spermiation, activity of cyclic adenosine 3,5-monophosphate (cAMP) was immunocytochemically localized at the level of the Sertoli cells. In these cells adenylate cyclase activity was also ultracytochemically demonstrated by using adenylyl imidodiphosphate as a substrate. Reaction products of adenylate cyclase were primarily detectable on the basal and adluminal plasma membranes and on the surface of protrusions of the cell body into the lumen.     

Outbreak of hind limb paralysis in young CFW Swiss Webster mice

An outbreak of paralysis among 16- to 20-week-old CFW Swiss Webster sentinel mice developed in one of our barrier facilities. Two months after arrival and over a period of four weeks, six of 400 mice purchased from an approved vendor, developed progressive hind limb paralysis without other clinical signs of disease. On the basis of the histopathologic changes and negative serologic test results, lymphoblastic lymphoma causing compression of the spinal cord was diagnosed. There were two leading features to this outbreak: its unusual epidemiologic presentation, and the localization of the lesions principally in the lumbar muscles. A presumptive diagnosis of retroviral infection with Abelson's murine leukemia virus (A-MuLV) was established on the basis of histopathologic and immunohistochemical findings. Little is known about retroviral status in many commercial colonies, and few users report presence of spontaneous lymphomas. This report points out complications derived from commercially available animals that carry endogenous retroviruses. It also emphasizes the need of diagnosing and reporting clusters of hind limb paralysis or lymphomas in mice to assess the prevalence and relevance of retroviral infections in commercial colonies.     

The rat ErbB2 tyrosine kinase receptor produced in plants is immunogenic in mice and confers protective immunity against ErbB2+ mammary cancer

The rat ErbB2 (rErbB2) protein is a 185‐kDa glycoprotein belonging to the epidermal growth factor‐related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2‐pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ‐HT expression vector as 6X His tag fusions. All rErbB2 variants (72–74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2⁺ mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.     

Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K1 of 5.2 mM and a K2 of 0.81 min-1. Mg2+ afforded nearly complete protection with a Kd of 2.8 mM. Two moles of [14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 mM MgCl2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the beta subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum.     

Fast Two-Dimensional Bubble Analysis of Biopolymer Filamentous Networks Pore Size from Confocal Microscopy Thin Data Stacks

The average pore size ξ₀ of filamentous networks assembled from biological macromolecules is one of the most important physical parameters affecting their biological functions. Modern optical methods, such as confocal microscopy, can noninvasively image such networks, but extracting a quantitative estimate of ξ₀ is a nontrivial task. We present here a fast and simple method based on a two-dimensional bubble approach, which works by analyzing one by one the (thresholded) images of a series of three-dimensional thin data stacks. No skeletonization or reconstruction of the full geometry of the entire network is required. The method was validated by using many isotropic in silico generated networks of different structures, morphologies, and concentrations. For each type of network, the method provides accurate estimates (a few percent) of the average and the standard deviation of the three-dimensional distribution of the pore sizes, defined as the diameters of the largest spheres that can be fit into the pore zones of the entire gel volume. When applied to the analysis of real confocal microscopy images taken on fibrin gels, the method provides an estimate of ξ₀ consistent with results from elastic light scattering data.     

Selectivity of modification when latent and activated forms of the chloroplast F1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan

The characteristics and specificity of inactivation of the chloroplast F1-ATPase (CF1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg2+-ATPase activity of latent CF1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF1 with [14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the beta subunit. Treatment of the enzyme with [14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the beta subunit and substantial incorporation of 14C into the gamma subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF1. Incorporation of 14C into the gamma subunit is prevented by prior treatment of the latent CF1 or of the dithiothreitol-heat-activated CF1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF1 by Tyr-beta-328, which is homologous to Tyr-beta-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F1-ATPase. When CF1, modified at Tyr-beta-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [14C]Nbf-Cl are located in different beta subunits.     

Interactions between herbivorous fish guilds and their influence on algal succession on a coastal coral reef

Herbivory is an important mechanism affecting algal succession, particularly on coral reefs where the relationship between algae and corals is largely controlled by herbivores. However, different functional groups of herbivores may have contrasting effects on succession, which may explain different trajectories of coral reef recovery after disturbance. Here, the effects of different herbivore groups (roving herbivores = foragers and territorial damselfish = farmers) were isolated by a multi-factorial experiment carried out on a coastal coral reef with high macroalgal cover, high farmer densities and relatively low forager abundance. The effects of foragers and farmers were distinguished by monitoring algal succession on settlement tiles placed inside and outside exclusion cages, with orthogonal treatments established inside and outside damselfish territories (with appropriate cage controls). Within 12 months, algal assemblages on ungrazed tiles inside exclusion cages proceeded rapidly from fine filamentous turfs, to corticated algae, to tough erect (e.g. Amphiroa spp.) and foliose (e.g. Peyssonnellidae) calcified algae. Farmers had a dramatic impact on succession, essentially arresting the development of the algal community at a point where it was dominated by palatable filamentous algae of the genus Polysiphonia. Fleshy macroalgae such as Sargassum spp. were excluded from farmer territories. In contrast, foragers did not suppress fleshy macroalgae, but rather, appeared to decelerate succession and promote a relatively diverse assemblage. In contrast to forager-dominated reefs, farmer territories did not appear to function solely as forager exclusion areas or promote algal diversity as a result of intermediate grazing pressure. The relatively strong effects of farmers observed here may represent a future scenario for coral reefs that are increasingly subject to overfishing of large grazing fishes.