Normal mode refinement: crystallographic refinement of protein dynamic structure. II. Application to human lysozyme.

The dynamic structure of a protein, human lysozyme, is determined by the normal mode refinement of X-ray crystal structure. This method uses the normal modes of both internal and external motions to distinguish the real internal dynamics from the external terms such as lattice disorder, and gives an anisotropic and concerted picture of atomic fluctuations. The refinement is carried out with diffraction data of 5.0 to 1.8 A resolution, which are collected on an imaging plate. The results of the refinement show: (1) Debye-Waller factor consists of two parts, highly anisotropic internal fluctuations and almost isotropic external terms. The former is smaller than the latter by a factor of 0.72 in the scale of B-factor. Therefore, the internal dynamics cannot be recognized directly from the apparent electron density distribution. (2) The internal fluctuations show basically similar features as those predicted by the normal mode analysis, with almost the same amplitude and a similar level of anisotropy. (3) Correlations of fluctuations are detected between two lobes forming the active site cleft, which move simultaneously in opposite directions. This corresponds to the hinge-bending motion of lysozyme.     

International Commission for Protection Against Environmental Mutagens and Carcinogens. A method for combining and comparing short-term genotoxicity test data: preface. A report from ICPEMC Committee 1.

The ability to repair 'mis-instructive', O6-methylguanine, and 'non-instructive', AP sites, DNA lesions in Fischer 344 rat livers at various ages was determined. Different behaviours were observed. While the AP-endodesoxyribonuclease enzymes displayed a high constant level throughout the animals' lifetime, the O6-methylguanine-DNA methyltransferase activity presented a stepwise modulation (DNA normalisation of results): the O6-MT activity significantly increased within the first month of animal life and enhanced again after 6 months reaching a maximum plateau in the 12-18-month-old animals. Thereafter a net significant decrease of O6-MT enzyme was detected in the 24-month-old group. While the repair of the widely formed AP sites appeared uniformly efficient like 'house keeping' functions, the removal of the rare precancerous O6-methylguanine is age-dependent indicating a decreased protection of the youngest and oldest animals against this 'mis-instructive' damage. However, any extrapolation of the age-associated cancer risk needs further assessment.     

Calcium-dependent binding of phosphorylated human pre interleukin 1 alpha to phospholipids

The effect of phosphorylation of pre interleukin 1 alpha (IL 1 alpha) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1 alpha (residues 64 to 271) and phosphorylated this pre IL 1 alpha in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1 alpha selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1 alpha to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100 microM was required. Unphosphorylated pre IL 1 alpha did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1 alpha did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1 alpha bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1 alpha binds to the inner surface of plasma membrane in a Ca2(+)- and phospholipid-dependent manner.     

Population genetics of Japanese monkeys: II. Blood protein polymorphisms and population structure

Genetic variability in individual troops of the Japanese macaque (Macaca fuscata fuscata) was quantified by the proportion of polymorphic loci and the average heterozygosity per individual from the results of starch-gel electrophoreses of blood proteins controlled by 32 independent genetic loci. The former averaged 9.2% and the latter 1.3%, the values being remarkably lower than those estimated for other animal populations. Geographical distribution of the genetic variations was not uniform in the whole species but the variants occurred only in limited areas. Assuming the selective neutrality of segregating alleles and the two-dimensional stepping-stone model of population structure, the genetic migration rate between the local demes per generation could be estimated to average less than inverse of average effective deme size. Here, the local deme is not a troop itself, but it consists of several troops tightly connected with each other by frequent exchanges of reproductive males. Analyses of correlation between geographic and genetic distances between troops revealed that the gene constitutions of two troops apart more than 100 km on an island could be regarded as practically independent of each other. These results suggest that the population structure of the Japanese macaque species has a tendency to split into a number of local subpopulations in which the effect of random genetic drift is prevailing.     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)