The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.     

The phosphorylation of adrenal proteins in response to adrenocorticotropic hormone

The phosphorylation of rat adrenal protein components in response to adrenocorticotropin has been studied in adrenal quarters, isolated cells, and in vivo. In adrenal quarters, adrenocorticotropic hormone (ACTH)-stimulated phosphorylation or dephosphorylation of proteins was not affected by the presence of protein synthesis inhibitors despite a total inhibition of steroidogenesis. (The term dephosphorylation refers to an apparent decrease in the labeling of a particular protein with 32P at various times after the addition of ACTH. This may be due to enzymatic removal of phosphate or protein degradation or complexation of this protein with another cellular component.) Studies with isolated cell preparations identified several proteins that are phosphorylated or dephosphorylated in response to hormone. These changes in phosphorylation were also observed in adrenal quarters and correlated well with ACTH-stimulated steroidogenesis as determined by temporal analysis and dose-response studies of corticosterone production. In vivo injection of male hypophysectomized rats with [32P]phosphate and ACTH demonstrated changes in the labeling of six adrenal proteins. Many of the proteins phosphorylated in vivo were also demonstrated to be phosphorylated in both in vitro systems. Finally, the injection of a physiological dose of ACTH appeared to selectively activate the type I cAMP-dependent protein kinase within the microsomal fraction as determined by the binding of a photoaffinity-labeled reagent. These results suggest that alterations in phosphorylation of adrenal proteins in response to ACTH is proximal to or independent of the obligatory role of protein synthesis in acute steroidogenesis.     

Absorption of light in photoreceptors: Effect of waveguiding property

The effect of waveguiding property (i.e., the intensity distribution) of the photoreceptor on the number of photons absorbed in a photoreceptor has been studied. It has been found that the effect is significant only for large values of the exposure and the maximum effect is less than 11% in the case of human rod photoreceptor. In the analysis, the funnelling effect, which follows from the coupling between the interior and exterior fields, has not been considered.Work partially supported by the Department of Science and Technology (India)B. D. Gupta is associated with the School of Bioscience Studies     

Widespread occurrence of AP in amyloidotic tissues. An immunohistochemical observation

Plasma (P)-component of amyloid (AP or SAP), while not an integral part of the amyloid fibril, has been considered to be intimately associated with virtually every different type of amyloid. In the present study, we evaluated the distribution of AP in the organs frequently involved in two forms of human systemic amyloidosis (AA and AF) and in mouse AA amyloidosis, by use of immunohistochemistry with anti-AP. Although the amyloid deposits generally showed moderate reactions with anti-AP, they were not always clearly distinguished from the surrounding non-amyloid tissue elements which often stained as well. The basement membrane often showed even stronger reaction to anti-AP than the adjacent amyloid deposits, and liver sections demonstrated such a high overall reaction to anti-AP that the anti-AP reaction on the amyloid deposits was often obscurred. The present results suggest that the binding between AP and the amyloid fibril may not be monospecific, that AP by this technique occurs rather widely throughout the body, and therefore that anti-AP may not be considered as specific a marker for amyloid deposits in immunohistochemical and perhaps other studies as well.     

Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M.

The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and analysis of monoclonal antibodies against various gangliosides

Female BALB/c mice were immunized with human melanoma (Mewo) cells containing ganglioside GD3 as a surface antigen. Immune splenocytes were fused with syngeneic P3-X63.Ag 8 myeloma cells. Antibodies produced by hybrid clones were analyzed by solid phase immunoassay. B, C, D and Q clones producing antibodies against Raja clavata brain gangliosides were obtained. Monoclonal B and C antibodies bound monosialogangliosides. Monoclonal D antibody bound a number of gangliosides but reacted predominantly with GD1a. Monoclonal Q antibody reacted selectively with GQ1c. It is assumed that ganglioside GQ1c is expressed on the melanoma cell surface and may be found only in the early stage of ontogenesis of high vertebrates.     

Variation in serotiny of three Banksia species along a climatic gradient

The degree of serotiny (i.e. the proportion of follicles remaining closed in each year's crop of cones since the last fire) was measured in Bank-sia attenuata, B. menziesii and B. prionotes at five sites along a climatic gradient extending 500 km north of Perth, Western Australia. The decrease in annual rainfall and increase in average temperature along the gradient paralleled a decrease in plant height and an increase in the degree of serotiny of all species. Extreme serotiny was recorded in the scrub-heath at the xeric end of the gradient whereas two species were essentially non-serotinous in the low woodland at the most mesic site. It is concluded that degree of serotiny is related to the fire characteristics of the site which depend on plant height. In xeric scrub-heath, the entire canopies of the Banksia spp. are consumed by fire which promotes massive release of seed. This facilitates recruitment in an otherwise unpredictable and unreliable seedbed. In mesic woodland, where cones rarely come into contact With flames, seeds are released spontaneously and site conditions are more conducive to recruitment in the inter-fire period.