Collective motions in proteins investigated by X-ray diffuse scattering

We have developed theoretical models for analysis of X-ray diffuse scattering from protein crystals. A series of models are proposed to be used for experimental data with different degrees of precision. First, we propose the normal mode model, where conformational dynamics of a protein is assumed to occur mostly in a limited conformational subspace spanned by a small number of low-frequency normal modes in the protein. When high precision data are available, variances and covariances of the normal mode variables can be determined from experimental data using this model. For experimental data with lower degrees of precision, we introduce a series of simpler models. These models express the covariance matrix using relatively simple empirical correlation functions by assuming the correlation between a pair of atoms to be isotropic. As an application of these simpler models, we calculate diffuse-scattering patterns from a human lysozyme crystal to examine how each adjustable parameter in the models affects general features of the resulting patterns. The results of the calculation are summarized as follows. (1) The higher order scattering makes a significant contribution at high resolutions. (2) The resulting simulated patterns are sensitive to changes in correlation lengths of about 1 Å, as well as to changes of the functional form of the correlation function. (3) But only the “average” value of the intra- and intermolecular correlation lengths seems to determine the gross features of the pattern. (4) The effect of the atom-dependent amplitude of fluctuations is difficult to observe. © 1994 John Wiley & Sons, Inc.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.     

Hepatocellular carcinoma associated with chronic Schistosoma mansoni infection in a chimpanzee

Hepatocellular carcinoma associated with chronic Schistosoma mansoni infection in a chimpanzee, estimated to be a 12-year-old and born in West Africa, is reported. The hepatic tumor appeared as a solitary firm nodule, and histological examination revealed well-differentiated hepatocellular carcinoma with a trabecular pattern. Hepatitis B virus and hepatitis C virus infections were excluded by serological testing in that animal. This is the first report of hepatocellular carcinoma in the chimpanzee with schistosomiasis.     

The kinetics of end-product inhibition of l-lactate production from xylose and glucose by Lactococcus lactis IO-1

Summary The relative contributions of lactate inhibition and the generation of sterile (undividing) cells to the low xylose utilisation rate of Lactococcus lactis IO-1 was investigated. The lactate inhibition constant of xylose grown cells was shown to be 9.3 times more than that of glucose grown cells. However, the sterile cell production rate and LDH inactivation rate of the xylose cultures were at least 10 times less than the glucose cultures. Thus, it is suggested that the slower substrate consumption rate in xylose medium is caused mainly by the large inhibition constant for the end product.     

Mating and diapause in hibernating adults ofMenida scotti puton (Heteroptera: Pentatomidae)

InMenida scotti aduts which mate during hibernation, we examined the effect of photoperiod on diapause induction in terms of the developmental degree of reproductive organs and corpora allata. In a hibernating population, mating season and physiological changes related to dipause were investigated. When newly emerged adults were reared under long- or short-day conditions, reservoir of the ectodermal accessory gland and corpora allata in males developed, while the developments of ovary and corpora allata in females were suppressed under both conditions. This suggests that diapause of this species is induced only in females independently photoperiodic conditions during adult stage. In a hibernating population, mating was observed from early to late November and from late March to early May. Observations of the development of reproductive organs and corpora allata suggest that diapause is induced only in females in the former period, while both sexes are not in diapause in the latter period.     

Methenamine-Silver Staining: a Simple and Sensitive Staining Method for Senile Plaques and Neurofibrillary Tangles

An improved methenamine-silver impregnation method is presented which exhibits sensitivity for amyloid substances comparable to that of anti-β protein immunostaining. In optimally treated sections, this technique stained both β-amyloid deposits and neurofibrillary tangles, which are known to have a β-pleated structure. This simple procedure allows a large number of sections to be stained for routine examination.     

Divergence pattern and selective mode in protein evolution: The example of vertebrate myoglobins and hemoglobin chains

Summary The evolutionary relation of vertebrate myoglobin and the hemoglobin chains including the agnathan hemoglobin chain is investigated on the basis of a new view of amino acid changes that is developed by canonical discriminant analysis of amino acid residues at individual sites. In contrast to the clear discrimination of amino acid residues between myoglobin, hemoglobin a chain, and hemoglobin chain in warm-blood vertebrates, the three types of globins in the lower class of vertebrates show so much variation that they are not well discriminated. This is seen particularly at the sites that are ascertained in mammals to carry the amino acid residues participating in stabilizing the monomeric structure in myoglobin and the residues forming the subunit contacts in hemoglobin. At these sites, agnathan hemoglobin chains are evaluated to be intermediate between the myoglobin and hemoglobin chains of gnathostomes. The variation in the phylogenetically lower class of globins is also seen in the internal region; there the amino acid residues of myoglobin and hemoglobin chains in the phylogenetically higher class exhibit an example of parallel evolution at the molecular level. New quantities, the distance of sequence property between discriminated groups and the variation within each group, are derived from the values of discriminant functions along the peptide chain, and this set of quantities simply describes an overall feature of globins such that the distinction between the three types of globins has been clearer as the vertebrates have evolved to become jawed, landed, and warm-blooded. This result strongly suggests that the functional constraint on the amino acid sequence of a protein is changed by living conditions and that severe conditions constitute a driving force that creates a distinctive protein from a less-constrained protein.Offprint requests to: J. Otsuka