Carbonic anhydrase activity in the blood of adult sheep, fetal sheep and lambs

The activity of carbonic anhydrase (E.C.4.2.1.1) (CA) has been measured in the blood of adult and fetal sheep and lambs. The mean activity in adult sheep was 0.89 enzyme units (EU) per 100 micrograms of Hb. The activity in fetal sheep aged 90 days was just below 20% of this and in fetuses near full term was just under 40% of the mean adult level. The regression line gave an increase of CA activity (per 100 micrograms Hb) of 0.004 EU/day. The appearance of CA in fetal blood normally occurred before any detectable production of adult Hb. One aberrant fetus showed early development of the adult pattern in the red cells, having adult type Hb and adult levels of CA during the period of 116-128 days of fetal age. In the period after birth the CA level in the blood rose rapidly, reaching the adult level 30 days after birth. During this period activity per 100 micrograms HB increased by 0.014 EU/day, significantly faster than during fetal life.     

Virus protein changes and RNA termini alterations evolving during persistent infection

Cloned infectious vesicular stomatitis virus isolated following 5 years of persistent infection of BHK21 cells in vitro exhibits a number of peptide map changes in the G protein (spike glycoprotein), the M protein (membrane matrix protein) and the N protein (nucleocapsid structural protein). Only slight alterations have occurred in the peptide maps of the two VSV polymerase-associated proteins L and NS. Dideoxy sequencing of the 3′ ends of the cloned virus originally used to establish the persistent infection, and of the cloned virus recovered following 5 years of persistence, shows one base substitution in the three base junction between the 3′ leader sequence and the N protein-coding region. Repeated lytic passages of virus recovered from persistent infection led to no oligonucleotide map changes after 30 passages, but two map changes were present after 102 and remained after 133 lytic passages in BHK21 cells in vitro. Only one of these represented reversion to the original map position, and this “mutant” virus still exhibited a temperature-sensitive small plaque phenotype. Finally, the mutated virus recovered after more than $\text{5}\text{1}\text{2}$ years of persistent infection is now so slow-growing that it can establish persistent infection of BHK21 cells in the absence of DI particles (although DI particles are present constantly once the cells recover from the initial cytopathology).     

Selecting representative model micro-organisms

Background  

Micro-biological research relies on the use of model organisms that act as representatives of their species or subspecies, these are frequently well-characterized laboratory strains. However, it has often become apparent that the model strain initially chosen does not represent important features of the species. For micro-organisms, the diversity of their genomes is such that even the best possible choice of initial strain for sequencing may not assure that the genome obtained adequately represents the species. To acquire information about a species' genome as efficiently as possible, we require a method to choose strains for analysis on the basis of how well they represent the species.     

Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Red–blue plots for detecting clusters in count data

A new index and four new graphical displays, termed "red–blue" plots, are presented to study and measure clustering in spatially referenced count data. The index can detect clusters in the form of patches, comprising several nearby large counts, and in the form of gaps, comprising several nearby small counts. The new methods quantify the degree to which the count for each sample unit contributes towards the overall degree of clustering, either as part of a patch or as a gap; provide tests of nonrandomness to detect clustering; and facilitate a comprehensive definition of the size and dimension of a cluster. The methods are illustrated using aphid field data.     

Release of a chimeric protein into the medium from Escherichia coli using the C-terminal secretion signal of haemolysin.

Recently, we have identified a novel topogenic sequence at the C terminus of Escherichia coli haemolysin (HlyA) which is essential for its efficient secretion into the medium. This discovery has introduced the possibility of using this secretion system for the release of chimeric proteins from E. coli directly into the medium. We have now successfully fused this C-terminal signal to a hybrid protein containing a few residues of beta-galactosidase and the majority of the E. coli outer membrane porin OmpF lacking its own N-terminal signal sequence. We find that this chimeric protein is specifically translocated across the inner and outer membranes and is released into the medium. In addition, we have further localized the HlyA secretion signal to the final 113 amino acids of the C terminus. In fact, a specific secretion signal appears to reside at least in part within the last 27 amino acids of HlyA.     

Structure and order of the protein and carbohydrate domains of prothrombin fragment 1

The three-dimensional structure of prothrombin fragment 1 has been determined by X-ray crystallography at 3.8 A resolution. The fragment is composed of a number of structural units, some of which are ordered while others are disordered. The ordered part of the structure includes a compact kringle unit, a helical domain and a carbohydrate chain. The kringle structure is organized around a close pair of buried disulfide bridges. One of its carbohydrate chains, that attached to Asn 101, is fully ordered, but the carbohydrate chain attached to Asn 77 appears to be disordered. The calcium binding unit is composed of a disordered part containing all ten gamma-carboxyglutamic acid residues and an ordered part forming the helical domain. The highly conserved residues Phe 41, Trp 42 and Tyr 45, which form a hydrophobic cluster on the first helix, interact around a crystallographic two-fold axis with the equivalent residues in another molecule to form a dimer in the crystal.