Effect of Haemopoietic Stem-Cell Proliferation Regulators On Early and Late Spleen Colony-Forming Cells

An inhibitor and stimulator of CFU-s proliferation can be obtained from haemopoietic tissue containing, respectively, relatively quiescent CFU-s (e.g. normal bone marrow) and proliferating CFU-s (e.g. regenerating bone marrow). Their effects on the proliferative behaviour of steady-state and regenerating marrow CFU-s, which produce colonies 7, 10 and 12 days post-transplantation have been investigated. The results demonstrate changing sensitivities of CFU-s to inhibitor and stimulator as they progress through a developmental age structure. ‘Older’ CFU-s (producing early spleen colonies) are more sensitive to stimulator, ‘Younger’ CFU-s (producing late spleen colonies) are more sensitive to inhibitor.     

Primary structure of wheat germ agglutinin isolectin 2. Peptide order deduced from X-ray structure

The complete amino acid sequence of wheat germ agglutinin isolectin 2 has been determined by the method of sequential Edman degradation and with the aid of the three-dimensional structure known from X-ray crystallography. Peptides ranging from 2 to 18 residues in length were obtained by thermolysin digestion of the S-carboxymethylated protein and purified by gel filtration and high-performance liquid chromatography. The peptide order was established primarily by matching (carboxymethyl)cysteines with the clearly defined half-cystine positions in the X-ray structure, thereby satisfying the disulfide repeat pattern observed in all four isostructural domains (A, B, C, and D) of wheat germ agglutinin, and by examination of amino acid compositions and terminal sequences of ten tryptic peptides. The unique assignment of peptides to these domains was consistent with all invariant half-cystines and glycines, as well as the single tryptophan, the two closely spaced histidines, and a number of other residues clearly identified in the X-ray structure analysis. Discrepancies between the chemical and X-ray sequences lie exclusively in poorly defined regions of the electron density map, at the N- and C-termini, and at the first intercystine loop of each domain. The latter loop was found to be eight instead of six residues in length, thus extending the size of domains A, B, and C from 41 to 43 residues and that of domain D to 42 residues. Regions of extensive interdomain homology, in addition to that of the half-cystines, are clustered at the central portion of each domain fold and are likely to be important for the integrity of the three-dimensional structure of the dimer molecule.     

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation- defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.     

Critical spatial requirement within the origin of simian virus 40 DNA replication.

We inserted a single base pair into the center of a 27-base-pair palindrome within the replication origin of simian virus 40. The mutation did not directly alter the symmetry of the palindrome or the protein-binding sequences within the palindrome. DNA binding studies showed that subunits of the simian virus 40 A protein (T antigen) bound to each of the four recognition pentanucleotides in the origin palindrome but did so with reduced affinity in comparison with wild-type origins. The mutant origin cloned in a plasmid DNA failed to replicate in COS cells. Thus, precise spatial interactions among subunits of A protein are necessary for stable origin binding and are crucial for subsequent steps in the initiation of DNA replication. Furthermore, any possible functional interactions of the simian virus 40 A protein with cellular DNA would require a great fidelity of protein binding arrangements to initiate cellular DNA replication.     

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.     

Ordination of vegetation change in Guadalupe Mountains,New Mexico,USA

Vegetation change over a nine-year period was studied in Guadalupe Mountains, New Mexico. Permanent transects in desert shrub vegetation were sampled in 1972 and 1980. Emphasis was given to shrubs because of their importance to big game diets. Univariate paired t-tests and reciprocal averaging ordination were used to detect and display coordinated changes in species composition over time. Despite apparently less browsing pressure in desert shrub vegetation in 1980 there were few significant changes in species composition. In addition, preferred forage species showed reduced reproduction while species of intermediate and poor forage value dis-played increased reproduction during this time. These data do not support traditional rangeland succession theory which states that enhanced reproduction of preferred species should follow grazing or browsing pressure reduction.     

Changes in quantity, composition, and surface activity of alveolar surfactant at birth

We hypothesized that when the lung makes the transition from the fluid- to the air-filled state at birth, there are changes in physical and functional properties of the alveolar surfactant. To test this hypothesis, newborn rabbits were killed at different times in the first 24 h of life, their lungs lavaged with ice-cold saline, and the lavage fluid subfractionated by differential centrifugation. The phospholipid and protein content and composition and the kinetics of surface tension lowering of the subfractions were examined. We found that with the onset of breathing, shifts occur in the distribution of surfactant subfractions as a surfactant apoprotein-free phospholipid fraction is generated. The ratio of rapidly sedimentable apoprotein-rich to slowly sedimentable, apoprotein-free fractions decreases from 31 at birth to 4 at 24 h of life. Concurrently, rates of surface tension lowering by the subfractions increase with time. The results suggest that the adult pattern of pool sizes and surface activity of alveolar surfactant is not present at birth but evolves slowly over the 1st day of life.     

Regulation of mycotoxin production by Fusarium graminearum: Complementation effects between two mutant types

Summary Starting from anileu auxotroph ofFusarium graminearum producing high levels of the mycotoxin zearalenone, selection after UV irradiation gave low-producing mutants of essentially normal morphology,zea,ileu. Heterokaryons betweenzea,ileu strains and an auxotrophic strainlz,inos derived from the lazy morphological mutant ofFusarium graminearum, which has abnormal morphology and also produces little or n zearalenone, produced significant levels (over 50% of the wild-type level) of mycotoxin. The observation confirms views as to the regulatory nature of thelazy mutation.     

1H NMR studies of heme pocket conformation in zinc-substituted leghemoglobin, a diamagnetic analog of deoxyleghemoglobin

Reconstitution of apoleghemoglobin with zinc protoporphyrin IX is reported. NMR spectra show that the reconstitution is orientation specific and that there is no detectable heme isomerism or conformational heterogeneity. Resonances of heme substituents and distal and proximal amino acid protons have been assigned. Only minor differences in porphyrin-protein packing occur between zinc leghemoglobin and the CO complex of ferrous leghemoglobin. The zinc is five-coordinate and is ligated by the proximal histidine. Comparisons with diamagnetic six-coordinate complexes show that the distal His-61 and Leu-65 side chains move away from the binding site upon coordination of exogenous ligands. Conformational changes are minimal when the ligand is O2.