Ecological aspects of the evolutionary processes

Darwin in his On the Origin of species made it clear that evolutionary change depends on the combined action of two different causes, the first being the origin of genetically based phenotypic variation in the individual organisms comprising the population and the second being the action of selective agents of the external environment placing demands on the individual organisms. For over a century following Darwin, most evolutionists focused on the origin of inherited variation and its transmission; many workers continue to regard genetics to be the core of evolutionary theory. Far less attention has been given to the exact nature of the selective agents with most evolutionists still treating this cause imprecisely to the detriment of our understanding of both nomological and historical evolutionary theory. Darwin was vague in the meaning of his new concept of "Natural Selection," using it interchangeably as one of the causes for evolutionary change and as the final outcome (= evolutionary change). In 1930, natural selection was defined clearly as "non-random, differential reproduction of genes" by R. Fisher and J.B.S. Haldane which is a statement of the outcome of evolutionary process and which omits mention of the causes bringing about this change. Evolutionists quickly accepted this outcome definition of natural selection, and have used interchangeably selection both as a cause and as the result of evolutionary change, causing great confusion. Herein, the details will be discussed of how the external environment (i.e., the environment-phenotype interaction) serves as selective agents and exerts demands on the phenotypic organisms. Included are the concepts of fitness and of the components of fitness (= adaptations) which are respectively (a) survival, (b) direct reproductive and (c) indirect reproductive features. Finally, it will be argued that historical-narrative analyses of organisms, including classification and phylogenetic history, are possible only with a full understanding of nomological evolutionary theory and with functional/adaptive studies of the employed taxonomic features in addition to the standard comparative investigations.     

Enantioseparation of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidones on chiral stationary phases based on 3,5-dimethylanilides of N-(4-alkylamino-3,5-dinitro)benzoyl L-alpha-amino acids

Three novel chiral packing materials for high-performance liquid chromatography were prepared by covalently binding of (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]propan-amide (7), (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]-4-methylpentanamide (8), and (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonyl-amino]-2-phenylacetamide (9) to aminopropyl silica. The resulting chiral stationary phases (CSPs 1-3) proved effective for the resolution of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidone derivatives (TR 1-14). The mechanism of their enantioselection, supported by the elution order of (S)-TR 13 and (R)-TR 13 and molecular modeling of the complex of the slower running (S)-TR 13 with CSP 1 is discussed.     

All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases

Mucin-type linkages (GalNAcalpha1-O-Ser/Thr) are initiated by a family of glycosyltransferases known as the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases, EC 2.4.1.41). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues, forming an alpha anomeric linkage. Despite the seeming simplicity of ppGaNTase catalytic function, it is estimated on the basis of in silico analysis that there are 24 unique ppGaNTase human genes. ppGaNTase isoforms display tissue-specific expression in adult mammals as well as unique spatial and temporal patterns of expression during murine development. In vitro assays suggest that a subset of the ppGaNTases have overlapping substrate specificities, but at least two ppGaNTases (ppGaNTase-T7 and -T9 [now designated -T10]) appear to require the prior addition of GalNAc to a synthetic peptide before they can catalyze sugar transfer to this substrate. Site-specific O-glycosylation by several ppGaNTases is influenced by the position and structure of previously added O-glycans. Collectively, these observations argue in favor of a hierarchical addition of core GalNAc residues to the apomucin. Various forms of O-glycan pathobiology may be reexamined in light of the existence of an extensive ppGaNTase family of enzymes. Recent work has demonstrated that at least one ppGaNTase isoform is required for normal development in Drosophila melanogaster. Structural insights will no doubt lead to the development of isoform-specific inhibitors. Such tools will prove valuable to furthering our understanding of the functional roles played by O-glycans.     

TEL1 from Saccharomyces cerevisiae suppresses chromosome aberrations induced by ionizing radiation in ataxia-telangiectasia cells without affecting cell cycle checkpoints

The TEL1 gene from Saccharomyces cere- visiae has been shown to be the closest sequence homologue to ATM, the gene mutated in ataxia-telangiectasia (A-T) patients. Functional homology shared between the ATM and Tel1 proteins has recently been demonstrated based on heterologous expression of the TEL1 gene in human cells derived from A-T patients. TEL1 expression complemented specific cellular A-T deficiencies, i.e. increased radiation-induced apoptosis, telomere shortening and spontaneous hyperrecombination. The mechanism of cellular A-T complementation by TEL1 appears to be independent of p53-dependent signaling cascades, since the deficiency of A-T cells to properly induce p53 upon ionizing radiation was not corrected by TEL1. We now find that the basic number of chromosome aberrations is increased and the number of radiation-induced chromosome aberrations is suppressed in A-T cells upon TEL1 expression. In cell cycle analyses, we find no changes in basic cell cycle distribution or in radiation-induced cell cycle checkpoints following TEL1 expression. We conclude that the radioprotective function of the Tel1 protein includes suppression of apoptosis and suppression of chromosome aberrations, and that both cellular endpoints can be uncoupled from ionizing radiation-induced cell cycle checkpoints. Received: 6 November 2000 / Accepted: 1 October 2001     

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.     

Tracking chromaffin granules on their way through the actin cortex

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a ∼300-nm (1/e²) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D ⁽³⁾). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D ⁽³⁾ was about 10,000 times lower than expected for free diffusion. Granules located ∼60 nm beneath the plasma membrane moved on random tracks (D ⁽³⁾≈10⁻¹⁰ cm² s⁻¹) with several reversals in direction before they approached their docking site at angles larger than 45^∘. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D ⁽³⁾ of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex. Received: 18 November 1999 / Revised version: 26 January 2000 / Accepted: 2 February 2000     

Igf2 imprinting in development and disease

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.     

Using MT(-/-) mice to study metallothionein and oxidative stress

Mice with null mutations for metallothionein genes MT-1 and MT-2 were used to study the role that metallothionein plays in protecting cellular targets in vivo from oxidative stress. Wild-type (MT(+/+)) and MT-null (MT(-/-)) mice were treated with either saline or zinc and exposed to two types of oxidative stress: gamma-irradiation or 2-nitropropane. There was no alteration in the antioxidant defense system (superoxide dismutase, catalase, or glutathione peroxidase and glutathione levels) to compensate for the lack of the metallothionein in the MT(-/-) mice. The amount of oxidative damage to liver DNA, lipids, and proteins were similar for the MT(-/-) and MT(+/+) mice even though the levels of metallothionein in the livers of the saline- or zinc-pretreated MT(+/+) mice were 5- to 100-fold greater than found in the MT(-/-) mice. To determine if metallothionein can protect mice from the lethal effects of ionizing radiation, the mean survivals of MT(-/-) and MT(+/+) mice exposed to whole body gamma-irradiation were measured and found to be similar. However, the mean survival increased significantly after zinc pretreatment for both the MT(-/-) and MT(+/+) mice. These results demonstrate that tissue levels of metallothionein do not protect mice in vivo against oxidative stress.     

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

Solid-state spectral editing techniques have been used by others to simplify ¹³C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance ¹³C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with -elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical ¹³C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.