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.     

Quantitative relationships between the measurable transport resistances of membrane carriers: theory and experiment

A theoretical analysis is made of the possible quantitative relationships between the transport resistances that characterise membrane carrier systems. It is shown that there exist only five possible patterns in which to rank the four transport resistances. Symbolising these as A, B, C and D, the five possible patterns are (i) A = B = C = D; (ii) A = B much greater than C, D; (iii) A = B much greater than C = D; (iv) A = B = 2C = 2D; (v) A = 2B = 2C much greater than D. A survey of the available experimental data shows that pattern (ii) is the most prevalent, pattern (v) is often found and pattern (iii) has been identified. None of the ten transport systems so far analysed experimentally failed to fit one of the predicted patterns.     

S-oxalylglutathione is a substrate for gamma-glutamyltransferase (GGT). Implications for the role of GGT in cell proliferation

With glycylglycine or water as acceptor, bovine kidney gamma-glutamyltransferase catalyzes reactions of the known mammalian metabolite, S-oxalylglutathione, at rates comparable to those of L-gamma-glutamyl-p-nitroanilide, a known good substrate. N-Oxalyl-cysteinylglycine is the eventual product of the former reaction. Since oxalyl thiolesters are implicated as important cell proliferation inhibitors, it is proposed that this reaction plays a major role in controlling cell proliferation.     

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     

Acetylcholine responses of identified neurons in Helix pomatia--III. Ionic composition of the depolarizing currents induced by acetylcholine

The ionic composition of the currents underlying the acetylcholine (ACh) depolarizations in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia was analysed. The equilibrium potential of the ACh responses was -2.8 +/- 0.6 mV (N = 49) and -4.0 +/- 0.7 mV (N = 79; mean +/- SEM) in the neurons B1 and B3, respectively. Replacement of NaCl in the bath solution by sucrose shifted the ACh equilibrium potential into the negative direction. A similar but less pronounced shift occurred when Ca2+ was substituted for Na+. Substitution of Cl- in the bath solution by propionate or an increase of the intracellular Cl- concentration did not affect the ACh equilibrium potential. Changes of K+ concentration in the bath between 1 and 50 mmol/l left the ACh equilibrium potential nearly unaffected when the Na+ concentration was at the control level. With a simultaneous reduction of extracellular Na+ an increase of K+ concentration shifted the ACh equilibrium potential towards more positive potentials. The findings are compatible with calculated K+ permeabilities if a K+ redistribution across the cell membrane is considered. In the neurons B1 and B3, channels operated by ACh are permeable for K+, Na+ and Ca2+, with the relative permeabilities 1.6:1.0:0.1.     

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.     

In vivo experimental modelling of the infectious process caused by stable L forms of the causative agent of typhoid

To simulate the infectious process and to study the persistence of L-forms, rabbits and guinea pigs were infected with S. typhi stable L-forms. The materials presented in this work indicate that both subconjunctival and intraperitoneal infection led to the development of the clinically indistinct, but morphologically pronounced pathological process with characteristic localization and typical changes in the gastrointestinal tract. The typical features of the process were the generalized immunomorphological reaction of the lymphoid apparatus with the appearance of light-colored reticulomacrophagal elements, the signs of the activation of humoral and cell-mediated immunity and the formation of small epitheloidocellular granulomas. The results of the investigation indicate that the stable cultures of S. typhi L-forms are highly pathogenic and capable of inducing the infectious process in experimental animals.