Aggregation of tobacco mosaic virus by sodium chondroitin sulfate

The precipitation of tobacco mosaic virus by sodium chondroitin sulfate in an aqueous solution was investigated kinetically by means of turbidimetry. The virus solution became turbid after the addition of chondroitin sulfate. A threshold concentration of chondroitin, 1.33 mg/ml, was required for virus precipitation, irrespective of the virus concentration. The precipitation resulted from a mutual spatial exclusion phenomenon, leading to the separation of the virus as a crystalline phase. The dimension of chondroitin sulfate calculated at the threshold concentration agreed well with that obtained by other methods. The initial slopes and the aggregation half-times of the virus aggregates depended on both chondroitin and virus concentrations and the former increased with the increase in concentration of each. Above the threshold concentration of chondroitin sulfate, tobacco mosaic virus aggregation was a rapid-aggregation process and ended within 100 sec.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Physiological evaluation of temperature effect on the growth processes of the mysid, Neomysis intermedia Czerniawsky

Ingestion, respiration, and molting loss rates were measuredover the 3 – 29°C range in Neomysis intermedia. Weightspecific rates of these physiological processes ranged from2 to 140% body C day^–1 for ingestion, from 2 to 15% bodyC day^–1 for respiration, and from 0.1 to 5% body C day^–1for molting loss. All weight-specific rates showed a logarithmicdecrease with a logarithmic increase in body weight, and a logarithmicincrease with a linear increase in temperature below 20 or 25°C.The effect of temperature, however, was different between thephysiological rates, with a large temperature dependency foringestion (Q₁₀ = 2.6 –3.9) and molting loss (Q¹⁰ = 2.9– 3.6) and a moderate temperature dependency for respiration(Q₁₀ = 1.9 – 2.1). Calculated assimilation efficiencychanged with body size, but was constant over the temperaturerange examined. Allocation of assimilated materials varied witha change in temperature, reflecting the different temperaturedependence between physiological processes. It was deduced thatthe strong temperature dependency of the growth rate in N. intermediaobserved in the previous studies resulted from the large temperatureeffect on ingestion and assimilation rates, superimposed bythe different allocation of assimilated materials. ¹Present address: Department of Botany, University of Tokyo,Hongo, Tokyo 113, Japan     

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography