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 effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Hyperoxia causes increased albumin permeability of cultured endothelial monolayers

The effect of phasic eye movement activity on ventilation during rapid-eye-movement (REM) sleep was studied in seven healthy young adults by use of the respiratory inductive plethysmograph. Mean ventilation (VE) and ventilatory components during REM sleep were not significantly different from that seen in either stages 1-2 or 3-4 sleep. The percent of rib cage contribution to ventilation in REM sleep, 29.3 +/- 5.1%, was reduced compared with 54.4 +/- 5.8% in stage 1-2 and 52.2 +/- 4.3% in stage 3-4 sleep (P less than 0.005). When one separated breaths by the degree of associated phasic eye movement activity, it became apparent that breathing during REM sleep is very heterogeneous. Increasing eye movement activity was associated with inhibition of ventilation with a reduction in VE from 5.1 +/- 0.3 to 3.8 +/- 0.3 l/min. Tidal volume and frequency both fell, whereas inspiratory duration was unchanged. Compartmental ventilation was also affected, with the fall in the rib cage contribution from 37.8 +/- 6.4 to 15.3 +/- 5.6%. Chest wall and abdominal movement became more asynchronous as phasic-eye-movement activity increased and frank paradoxical breathing was seen.     

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.     

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.     

Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative deamination of L-lysine, exerts an inhibitory effect on DNA, RNA and protein synthesis in human cells of carcinoma ovarius (CaOv) in vitro.     

Modification of testicular and thyroid function by chronic exposure to short photoperiod: a comparison in four rodent species

Exposure of male Syrian hamsters (Mesocricetus auratus) for 10 weeks to short photoperiod (SP) providing 10 hr light: 14 hr darkness (10:14 LD) produced a significant reduction in the weights of the reproductive organs, plasma thyroxine (T4) levels and free T4 index (FT4I) compared to the values of animals exposed to long photoperiod (LP, 14:10 LD). C57bl male house mice (Mus musculus) kept in SP (10:14 LD) had reproductive organ weights equivalent to those of mice kept in long days (14:10 LD) and lower T3 uptake (T3U) values. Male gerbils (Meriones unguiculatus) exposed to 13 weeks of SP (10:14 LD) had lower body weights, testes and seminal vesicle weights and higher T3U values compared to LP (14:10 LD) controls. However, no effect was seen on plasma T4 and triiodothyronine (T3) values nor the FT4I and free T3 index (FT3I). White-footed male mice (Peromyscus leucopus) exposed to SP (8:16 LD) had significantly lower testes and seminal vesicle weights while plasma T4 and T3 levels were unaffected. Snell strain house mice (Mus musculus) exposed to SP (8:16 LD) had normal reproductive organ weights compared to the values of LP-exposed (16:8 LD) control animals. However, there was a significant depression in T3 and in the FT3I in the SP animals.