Aid for the Choice of Buffer Concentration

The peptic hydrolysis of bovine β-lactoglobulin (β-Lg) was performed to establish the basis for producing a low-phenylalanine peptide rather than a free amino acid mixture for use in the dietetics of phenylketonuria. A 1% β-Lg solution (pH 1.5) was incubated with 0.01% pepsin at 37°C for 24 hr. The peptides produced were fractionated by high-performance liquid chromatograhy to analyze their constituent amino acids. Most of the major peptides were identified in the light of the primary structure of α-Lg to assign 31 cutting points in their protein molecule. These included cutting points at the carboxyl side of Phe-82, Phe-105 and Phe-136. This result suggests that further hydrolysis of the peptic hydrolysate of β-Lg with an exopeptidase, particularly with a carboxypeptidase, would be effective in liberating phenylalanine to produce a low-phenylalanine peptide mixture.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Effects of an inhomogeneous magnetic field on flowing erythrocytes

Effects of an inhomogeneous magnetic field on narrow erythrocyte streams in a wide and transparent laminar buffer flow were studied. The stream line of erythrocytes containing paramagnetic hemoglobin showed distinct displacement toward the stronger magnetic field. The displacement increased in the order, oxygenated erythrocytes (no displacement), erythrocytes containing cyanomethemoglobin, deoxygenated erythrocytes, erythrocytes containing methemoglobin in the high spin state; more precisely the displacement was proportional to the square of the paramagnetic moment of hemoglobin contained in the erythrocytes. In addition, the displacement was proportional to the product of the magnetic flux density and its gradient, and approximately proportional to the hematocrit of the flowing-erythrocyte suspension, and was much larger than that calculated for a single erythrocyte. These phenomena could be successfully interpreted by the interaction of paramagnetic erythrocytes with the inhomogeneous magnetic field, the resistance force (Stokes Law) from the bulk water, and the hydrodynamic interaction between erythrocytes.     

Natural development time of Eodiaptomus japonicus (Copepoda: Calanoida) in Lake Biwa

Seasonal and ontogenetic changes in natural development timewere studied for Eodiaptomus japonicus in Lake Biwa in 1986and 1987. Wild individuals in a certain developmental stagewere collected, fed on natural food and examined until theyhad moulted twice. Natural development times fluctuated irrespectiveof temperature from May to October. Food deficiency delayeddevelopment in all feeding stages, and food availability probablydetermined natural development time. Serious food limitationraised mortality in copepodid stage I. In November developmentwas delayed even with enough food. The development of E.japonicuswas almost isochronal except for a short prefeeding naupliarstage I and a long copepodid stage V.     

Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

In this study, we have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenecity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library we found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, we have found perfect overlapping of the alphoid DNA sites with the centromere antigen sites in both metaphase chromosomes and nuclei at various stages in the cell cycle. We have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.     

High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe.

We describe a highly efficient alkali cation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of fission yeast Schizosaccharomyces pombe mutants. cDNA libraries constructed with the pcD or pcD2 vector are transduced into yeast by cotransfection with a linearized vector, which allows an enhanced homologous recombination between the yeast vector and the library plasmid leading to the efficient formation of concatemers containing pcD molecules. The transformation frequencies obtained by the method are 10(6) colonies per 10(8) cells transfected with 2 micrograms of library and 1 microgram of vector, 50-60% of which contain pcD molecules. The high-efficiency alkali cation method circumvents many of the shortcomings of the spheroplast method generally used for Schiz. pombe transfection. The vectors are maximized for the efficiency of library transduction and minimized for the rearrangements of pcD molecules during propagation in yeast. This system allows rapid screening of multi-million cDNA clone libraries for rare cDNAs in a routine scale of experiments. Using this system, various mammalian cDNAs that are extremely difficult, time-consuming, or unclonable to clone by other methods have been cloned.     

Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction

The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)