An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modeling Lanthanide Complexes: Towards the Theoretical Design of Light Conversion Molecular Devices

Theoretical techniques have been developed and/or improved to predict the molecular structure of lanthanide complexes which were used to calculate their electronic properties, in particular, their electronic spectra and energy levels necessary to calculate the rates of energy transfer from the ligands to the metal ion. The molecular structure has been obtained by the SMLC/AM1 (Sparkle Model for the Calculation of Lanthanide Complexes – Austin Model 1) model where the lanthanide ion is simulated by a sparkle implemented into the AM1 Hamiltonian used to perform a HF-SCF (Hartree-Fock Self-Consistent Field) calculation. The previous implementation of the SMLC/AM1 model (sparkle/1) involving only two parameters has been generalized to be consistent with the AM1 Hamiltonian and the new model (sparkle/2) significantly improved the prediction of molecular structures of Eu(III) complexes. For the electronic spectra and energy level calculations of the lanthanide complexes the model replaces the metal ion by a point charge with the ligands held in their positions as determined by the SMLC/AM1 model, and uses a INDO/S-CI (intermediate neglect of differential overlap/spectroscopic-configuration interaction) model. A preliminary study of the solvent effects on the absorption spectra of the free ligand is also presented. For the ligand-lanthanide ion energy transfer Fermi's golden rule is used with the multipolar and exchange mechanisms being implemented and tested for several complexes. These theoretical techniques have been applied to several complexes yielding very good results when compared to experimental data as well as predictions for the molecular and electronic structures and the relative contributions of the mechanisms for the energy transfer rates. This revised version was published online in June 2006 with corrections to the Cover Date.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia -derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia , and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia . This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia . The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.     

Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A₁₂) and globular hydrophilic and amphiphilic (G^H₄, G^A₄, G^A₂, and G^A₁) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G^H₄ and G^A₄ decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A₁₂ AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G^A₄ AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G^A₄ forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.     

Cryptic coloration and choice of escape microhabitats by grasshoppers (Orthoptera: Acrididae)

Cryptic coloration is found in many Orthoptera, especially in Acrididae, showing a great variety of forms. In a grasshopper assemblage in southeastern Brazil, preferences for escape places were detected; grasshoppers tended to escape to backgrounds in which they seem to be more cryptic. Coloration was measured using the Simpson diversity index, to quantify 'aspect diversity' (diversity of colours and shapes of patches along the insect's body). A weak positive correlation was found between grasshoppers' aspect diversity and diversification in use of escape places (use of many backgrounds to escape). Grasshoppers with similar colour patterns tended to use the same structures (leaves, sandy soil, stones) to escape.     

Photochemical reaction of 9-cis-retro-γ-rhodopsin at low temperatures

9-cis-Retro-γ;rhodopsin (λ_max = 420 nm) was prepared from 9-cis-retro-γ-retinal and cattle opsin. After cooling to liquid nitrogen temperature (77 K), the pigment was irradiated with light at 380 nm. The spectrum shifted to the longer wavelengths, owing to formation of a batho product. This fact indicates that the conjugated double bond system from C-5 to C-8 of the chromophoric retinal in rhodopsin was not necessary for formation of bathorhodopsin. Reirradiation of the batho product with light at wavelengths longer than 520 nm yielded a mixture composed of presumably 9- or 11-cis forms of retro-γ-rhodopsin. These three isomers are interconvertible by light at liquid nitrogen temperature. Thus the retro-γ-rhodopsin system is similar in photochemical reaction at 77 K to cattle rhodopsin system. Each system has its own batho product. Based on these results, it was infered that the formation of bathorhodopsin is due to photoisomerization of the chromophoric retinal of rhodopsin and is not due to translocation of a proton on the ring or on the side chain from C-6 to C-8 of the chromophoric retinal to the Schiff-base nitrogen.     

A method for the sequential study of synaptonemal complexes by light and electron microscopy

Summary A method is described for the sequential study of synaptonemal complexes by light and electron microscopy. The method is easy, permits one to determine the geometry of chromosome pairing, and should become a routine procedure in the diagnosis of human male subfertility. It should also be useful to establish the risk of recurrence of chromosome aberrations in the progeny of carriers of chromosome rearrangements.This paper is dedicated to the memory of Prof. Gerónimo Forteza Bover, the pioneer of human genetics in Spain, an excellant teacher and a good friend     

Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter.The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter.Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge.Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed.A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.