Human hybrid immunoglobulin M containing immunoglobulin A

Summary Some hybridoma clones made by fusion of a human lymphoblastoid cell line, HO323 with human B lymphocytes, secreted not only IgA but also IgM-like immunoglobulin molecules. The IgM-like immunoglobulin had a molecular size of 900 K which corresponded to that of IgM. Immunochemical analyses revealed that the IgM-like immunoglobulin contained two monomeric IgA and three monomeric IgM molecules. In the IgA moieties, half of original light chains were replaced withx chains derived from the IgM, and vice versa.     

Centrifugal bisection of starfish oocytes

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.     

Adaptation of mouse-human hybridomas to a protein free medium

Summary Five mouse-human hybridoma cell lines secreting human IgM class monoclonal antibodies were examined for possible adaptation to a protein free medium. Four were successfully adapted to the medium. The adapted cell lines could be cultured for more than two months without any changes in their cell growth abilities.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

Tremorgenic Mycotoxin from Penicillium paraherquei

A tremorgenic mycotoxin was isolated from Penicillium paraherquei Abe ex G. Smith and identified as verruculogen. It was produced at the rate of approximately 1 mg/g of the dried fungal mycelium cultured on peptone-enriched Czapek-Dox medium at 28°C.     

Ca2+-induced lateral phase separations in phosphatidic acid-phosphatidylcholine membranes

The effects of Ca²⁺ on phosphatidic acid-phosphatidylcholine membranes have been studied using phospholipid spin labels. ESR spectra of spin-labeled phosphatidic acid-phosphatidylcholine membranes and phosphatidic acid-spin-labeled phosphatidylcholine membranes are exchange-broadened immediately upon addition of CaCl₂. These changes directly and conclusively indicate Ca²⁺-induced clustering of spin-labeled phosphatidylcholine and aggregation of spin-labeled phosphatidic acid bridged by Ca²⁺-chelation in the binary phopholipid membranes. In the Ca²⁺-chelated aggregates, the motions of the alkyl chains of phosphatidic acid are greatly reduced and the lipid molecules are more closely packed. The clusters and aggregates are formed in patches and the sizes are dependent on the fractions. Ba²⁺ and Sr²⁺ induce the lateral phase separations to the same extent as Ca²⁺. Mg²⁺ is also effective but to a lesser extent. In acid solutions (pH 5.5), the Ca²⁺-induced lateral phase separations are of slightly lesser extent than in alkaline solution (pH 7.9). These results are compared with those for phosphatidylserine-phosphatidylcholine membranes reported previously and necessary conditions for the lateral phase separations are discussed.     

Production of Pentameric Hybrid Immunoglobulins Consisting of IgA and IgM

The amylomaltase from Escherichia coli IFO 3806 was purified to homogeneity seen by SDS- polyacrylamide gel electrophoresis after DEAE-Sephadex, Ultrogel AcA 44, hydroxylapatite, and 1,6- hexane-diamine-Sepharose 4B column chromatographies. The molecular weight of the purified enzyme was 93,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.5 and at 35°C, and stable up to 45°C at pH 7.0 and from pH 6.0 —7.3 at 40°C on 30min incubation. The enzyme acted on maltotetraitol, maltopentaitol, and maltosylsucrose besides maltooligosaccharides, but did not act on maltitol, maltotriitol, glucosylsucrose, isomaltose, panose, isopanose, or isomaltosyl- maltose. This enzyme did not catalyze hydrolytic action on maltotetraitol, maltopentaitol, or maltosylsucrose.     

Studies on Molecular Properties of αs1-κ-Casein Complex by the Hydrodynamical Methods

Some molecular properties of α_s1-κ-casein complex, α_s1- and κ-casein polymers were examined by gel filtration, ultracentrifugation, and viscometry at pH 7.1. The Stoke’s radii of α_s1-κ-casein complex, α_s1- and κ-casein polymers were 99, 44 and 108 Å, respectively. The molecular weight of the above proteins were approximately 45 × 10⁴, 10 × 10⁴ and 80 × 10⁴, respectively. The stokes radius of α_s1-κ casein complex reduced compared with that of κ-casein polymer and the molecular weight of the complex was about half that of κ-casein polymer. These results suggest that κ-casein polymer dissociates into 4 smaller particles when α_s1-κ-casein complex is formed. The frictional coefficient and Scheraga-Mandelkern constants for each protein suggest that the molecular shape of α_s1-casein polymer is globular and that of α_s1-κ-casein complex and κ-casein polymer is rod-like.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.