An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

Summary We have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum. This work was supported by the Japanese Ministry of Education, Science and Culture and in part by grants from the National Institutes of Health. Editor's Statement This article reports a creative technical application of the author's previous work on lipid metabolism in lymphoid cells allowing an efficient, alternative selection procedure for isolation of hybridomas.     

Characterization of an amorphous and soluble hemagglutinin from Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis which were screened out depending on auto-agglutination and Ca2+ dependency, were examined for their production of hemagglutinin (HA), and its purification and characterization were performed. The HA with a broad reactivity with various mammalian erythrocytes was recovered from the culture supernatant of these strains grown at 37 C but not 25 C. HAs from two strains, R148R and T1040, were purified by salt precipitation, gel filtration and anion-exchange chromatography by HPLC. Both purified HAs were cysteine-deficient acidic protein with an apparent molecular weight in the range of 15,000 to 16,000. N-terminal amino acid sequences of the first 25 residues were found to share 12% identity with that of afimbrial adhesin from enterotoxigenic Escherichia coli 2230. Immunoelectron microscopy and immunodiffusion test with polyclonal antiserum raised against the purified R148RHA demonstrated that the HA was associated with the amorphous aggregates which were detached from bacteria. These results suggest that the HA of Y. pseudotuberculosis belongs to a third type of HA produced by the yersinial species.     

Isolation of influenza A virus from budgerigars and its serological characterization

A hemagglutinating agent was isolated from the respiratory organs of budgerigars suffering from diarrhea and malnutrition. This agent, possessing neuraminidase activity, was identified as influenza A virus by the double immunodiffusion test. The results of hemagglutination and neuraminidase-inhibition tests with monospecific antisera to the isolated surface antigens showed that the isolates possessed Hav4 hemagglutinin and Nav1 neuraminidase subunits both of which were closely related to the corresponding antigens of A/duck/Czech/56 (Hav4 Nav1).     

Oxytocin in the cerebrospinal fluid and plasma of pregnant and nonpregnant subjects

The oxytocin concentration in the cerebrospinal fluid (CSF) and plasma of pregnant women at term with and without labor pain were measured by radioimmunoassay and compared with those of non-pregnant women of matched age. The oxytocin concentrations in the CSF were 4.9 +/- 4.1 microU/ml (mean +/- S.D.) in pregnant women with labor pain, 4.1 +/- 2.4 microU/ml in those without labor pain and 4.0 +/- 2.8 microU/ml in nonpregnant women, and the oxytocin concentrations in the plasma of these subjects were 45.2 +/- 19.6, 17.1 +/- 22.2 and 7.0 +/- 5.3 microU/ml, respectively. Thus the oxytocin level in the CSF did not change appreciably even when the level in the plasma was raised in the pregnant women with labor pain. These findings suggest that oxytocin does not penetrate the blood-brain barrier, and that oxytocin in the CFS has little or no central role in parturition in women.     

Substrate-attached materials are enriched with tetraspanins and are analogous to the structures associated with rear-end retraction in migrating cells

Substrate-attached materials (SAMs) are cellular feet that remain on substrates after the treatment of adherent cells with EGTA. SAMs are thought to contain cell adhesion machineries, but their biochemical properties have not been addressed in detail. To gain insight into the molecular mechanisms operating in cell adhesions, we comprehensively identified the protein components of SAMs by liquid chromatography coupled with tandem mass spectrometry, followed by immunoblot analysis. We found that the tetraspanins CD9, CD81, and CD151 were enriched in SAMs along with other transmembrane proteins that are known to associate with tetraspanins. Notably, integrins were detected in SAMs, but the components of focal adhesions were scarcely detected. These observations are reminiscent of the “footprints” that remain on substrates when the retraction fibers at the rear of migrating cells are released, because such footprints have been reported to contain tetraspanins and integrins but not focal adhesion proteins. In support of this hypothesis, the formation of SAMs was attenuated by inhibitors of ROCK, myosin II and dynamin, all of which are known to participate in rear-end retraction in migrating cells. Furthermore, SAMs left on collagen-coated substrates were found by electron microscopy to be fewer and thinner than those on laminin-coated substrates, reflecting the thin and fragile retraction fibers of cells migrating on collagen. Collectively, these results indicate that SAMs closely resemble the footprints and retraction fibers of migrating cells in their protein components, and that they are yielded by similar mechanisms.     

Biotransformation of reticuline into coreximine,scoulerine, pallidine,and isoboldine with rat liver enzyme

(±)-Reticuline (1) was biotransformed into the protoberberine alkaloids, coreximine (12) and scoulerine (10), the morphinandienone alkaloid, pallidine (14), and the aporphine alkaloid, isoboldine (16). The transformation was stimulated by O₂ and the cofactor NAD, NADP, or NADPH, NADPH being more effective than the other cofactors. The N-methyl group of (±)-reticuline was not incorporated intact into protoberberines.     

Genetic divergence in <Emphasis Type="Italic">Ditrema</Emphasis><Emphasis Type="Italic">jordani</Emphasis> (Perciformes: Embiotocidae) from the Pacific coast of southern Japan,as inferred from mitochondrial DNA sequences

Genetic divergence in Ditrema jordani was investigated from sequence variations on the mitochondrial cytochrome b gene. Clear genetic differences were found between specimens collected from the Mie and Shizuoka prefectures (westward of Izu Peninsula) and those from the Kanagawa and Chiba prefectures (eastward of Izu Peninsula). The uncorrected genetic distance between the two groups, which may represent separate taxa, was much greater (3.1–3.7%) than that between D. temminckii and D. viride (1.1–2.4%), and between two subspecies of D. temminckii (0.8–1.3%), suggesting that the Izu Peninsula acts as a stable geographic barrier to gene flow between the D. jordani groups. The clear genetic divergence between the two geographic populations of D. jordani may be partly related to direct development (viviparity) and low dispersal ability in the genus.     

A new method for extracting conodonts and radiolarians from chert with NaOH solution

Microfossils are important components of sedimentary rocks used for palaeontological, biostratigraphic, palaeoenvironmental and palaeoclimatic investigations. They are usually extracted from rocks using an acid solution, which might vary depending on the embedding rock lithology. Here we propose a new method using common NaOH (sodium hydroxide; soda) to digest cherts (micro- and cryptocrystalline quartz) instead of the standard technique based on HF (hydrofluoric acid). This new method allows the collection of undamaged specimens of different kinds of microfossils, such as conodonts, radiolarians, teeth and dermal scales, the minerology of which is still preserved (e.g. biogenic apatite in conodonts). The use of soda is thus recommended, as it is less dangerous, less expensive, and it better preserves the extracted microfossils both in shape and mineralogy.     

Secondary contact in the Sea of Japan: the case of the <Emphasis Type="Italic">Careproctus rastrinus</Emphasis> species complex (Liparidae)

The Careproctus rastrinus species complex, widely known from the North Pacific, has been revealed recently to include nine genetically divergent groups on the basis of mitochondrial DNA (mtDNA) sequence variations. Herein we describe an AFLP analysis that focuses on three closely related groups in order to clarify the evolutionary history of the species complex in the Sea of Japan and off the Pacific coast of Japan. A principal coordinate analysis indicated the absence of nuclear divergence in two groups defined by mtDNA variations in the Sea of Japan, whereas another group from the Pacific coast of northern Japan was clearly distinct. This suggests extensive gene flow between two groups in the Sea of Japan as a result of secondary contact.