Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Two Drosophila beta tubulin isoforms are not functionally equivalent

We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Progressive determination of cell fates along the dorsoventral axis in the sea urchin Heliocidaris erythrogramma

In the direct-developing sea urchin Heliocidaris erythrogramma the first cleavage division bisects the dorsoventral axis of the developing embryo along a frontal plane. In the two-celled embryo one of the blastomeres, the ventral cell (V), gives rise to all pigmented mesenchyme, as well as to the vestibule of the echinus rudiment. Upon isolation, however, the dorsal blastomere (D) displays some regulation, and is able to form a small number of pigmented mesenchyme cells and even a vestibule. We have examined the spatial and temporal determination of cell fates along the dorsoventral axis during subsequent development. We demonstrate that the dorsoventral axis is resident within both cells of the two-celled embryo, but only the ventral pole of this axis has a rigidly fixed identity this early in development. The polarity of this axis remains the same in half-embryos developing from isolated ventral (V) blastomeres, but it can flip 180° in half-embryos developing from isolated dorsal (D) blastomeres. We find that cell fates are progressively determined along the dorsoventral axis up to the time of gastrulation. The ability of dorsal half-embryos to differentiate ventral cell fates diminishes as they are isolated at progressively later stages of development. These results suggest that the determination of cell fates along the dorsoventral axis in H. erythrogramma is regulated via inductive interactions organized by cells within the ventral half of the embryo.     

USE OF SENSORY EVALUATION FOR MEASURING DEODORIZING EFFECTS OF AN AIR CONDITIONER

The application of sensory methodology for measuring deodorizing effect of an air conditioner equipped with electric plasma was introduced. Deodorizing effect was measured using chemical and sensory methods at different time (0, 30 and 60 min) and mode (control, blowing and cooling) of an air conditioner. Smoke from a roll of cigarette in a closed room was used as a source of odor and the concentrations of acetic acid and ammonia were measured as odorous chemical components. As one of the sensory methods triangle test was used and as a first step to obtain deodorizing effects by triangle test, the threshold of each panelist was obtained as the log dilution ratio of odor concentration at which the difference from odorless air was detected. The odor concentration at each time and mode was calculated using the threshold of the panel and the deodorizing effect was obtained on the basis of the odor concentration. In addition to a triangle test, scaling methods such as category scaling or magnitude estimation were used to measure deodorizing effect of an air conditioner. Deodorizing effects by scaling methods were calculated based on odor intensity with time at each mode. The regression analysis was done between the efficacy of deodorizing effect by sensory test and those by acetic acid and ammonia, the R² values of the regression equations for triangle test, category scale, and magnitude estimation were 0.84, 0.72 and 0.69, respectively. Deodorizing effect by triangle test explained the decrease of acetic acid and ammonia better than those by category scaling or magnitude estimation while high cost and time consuming labor involved in triangle tests reduced the merit. The results of this study demonstrated that various sensory methods could be used to measure deodorizing effect of air conditioners and further researches on fast and reliable methods are needed to establish the official procedures.     

Fluorophore-labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.     

Studies on the generation of B lymphocytes in fetal liver and bone marrow.

With the use of immunofluorescence techniques, cells containing cytoplasmic IgM (cIgM+), but lacking detectable surface IgM (sIgM+), have been identified in mouse fetal liver and adult bone marrow as a distinct cell population to sIgM+ B lymphocytes. We have shown that there is a considerable difference in the rate of entry of cIgM+ and sIgM+ cells into DNA synthesis in these locations. Moreover, within the cIgM+ population, the largest cells are the main group entering DNA synthesis. Our results are compatible with the notion that a pool of rapidly proliferating, large cIgM+ cells is present in fetal liver and adult bone marrow and that these cells give rise to populations of smaller cIgM+ cells, which move out of cell cycle, and convert to sIgM+ B lymphocytes. However, we recognize that this interpretation is speculative. Finally, we have shown that fetal bone marrow is a site of generation of sIgM+ B lymphocytes, but the question as to whether these cells are derived from Ig- precursors within marrow itself remains open.