Nucleotide Sequence, Genomic Organization, and Chromosomal Localization of Genes Encoding the Human NMDA Receptor Subunits NR3A and NR3B

The N-methyl-D-aspartate (NMDA) receptors are glutamate-regulated ion channels that are critically involved in important physiological and pathological functions of the mammalian central nervous system. We have identified and characterized the gene encoding the human NMDA receptor subunit NR3A (GRIN3A), as well as the gene (GRIN3B) encoding an entirely novel subunit that we named NR3B, as it is most closely related to NR3A (57.4% identity). GRIN3A localizes to chromosome 9q34, in the region 13-34, and consists of nine coding exons. The deduced protein contains 1115 amino acids and shows 92.7% identity to rat NR3A. GRIN3B localizes to chromosome 19p13.3 and contains, as does the mouse NR3B gene (Grin3b), eight coding exons. The deduced proteins of human and mouse NR3B contain 901 and 900 amino acid residues, respectively (81.6% identity). In situ hybridization shows a widespread distribution of Grin3b mRNA in the brain of the adult rat.     

Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc-Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the alpha6 and beta1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-beta. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of beta1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc-Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment.     

Untangling anthropogenic and climatic influence on riverine forest in the Kruger National Park, South Africa

Understanding the interplay between climatic and anthropogenic factors is a major challenge in palaeo-ecology. In particular, it is often difficult to distinguish anthropogenic and “natural” fire in the charcoal record. In this paper, analysis of fossil pollen, charcoal, diatoms and isotopic evidence from Mapimbi, a small lake in the Kruger National Park, South Africa, suggests that for most of the past ca. 700 years, the riverine gallery forests surrounding Mapimbi were primarily influenced by climate, and benefited during warmer, wetter periods. The transitions between four, statistically different phases in the time-series data coincide with regional climate records previously constructed from speleothem data, and are consistent with the transition from the medieval warm period ending in the 14th century a.d. to the cooler, drier conditions prevailing during the little ice age of ca. a.d. 1400–1800. The data also suggest a period of significant, anthropogenic influence after a.d. 1800, when maize was grown and the incidence of localised fires increased. An increase in woody cover in recent decades may be associated with the management of the area by Kruger National Park. A decline in cultivation occurred in the end of the 20th century linked with changes in socio-political organisation.     

Pine embryogenesis: Many licences to kill for a new life

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes.Key words: conifer, developmental cell death, embryogenesis, megagametophyte, necrotic cell death, pine, seed development     

Is there a relationship between crop farming and the Alnus decline in the eastern Baltic region?

Data from 59 sequences studied through pollen analysis were used to examine the decline in Alnus in Estonia during the Iron Age. Between a.d. 300 and 1300, the Alnus pollen frequency declined markedly in 30 records distributed evenly across the investigated area. The beginning of the decline was time transgressive, coincidental with the start of extensive cultivation, and was frequently connected with the commencement of rye cultivation and the availability of land suitable for cultivation. The greatest reduction in Alnus abundance occurred during the Late Iron Age between a.d. 900 and 1000. This spatially random asynchrony suggests that one or more factors affected Alnus populations across the whole northern region. Human impact is discussed as a plausible cause of the decline. To determine the initiation of extensive crop farming in the eastern Baltic area, pollen diagrams from Latvia, Lithuania and the Novgorod region were also examined.     

Detection of tyrosine phosphorylated proteins by combination of immunoaffinity enrichment, two-dimensional difference gel electrophoresis and fluorescent Western blotting

We describe fluorescence-based 2-D gel electrophoresis methods for visualization of low abundant, cancer relevant tyrosine phosphorylated (pTyr) proteins. The methods investigated were fluorescent Western blotting and two-dimensional difference gel electrophoresis (2-D DIGE) for detection of non-enriched and immunoaffinity enriched pTyr protein patterns. The same anti-phosphotyrosine specific antibody, 4G10, was used for both approaches. The results from fluorescent Western blotting of total proteins and from enriched CyDye DIGE pre-labeled pTyr proteins showed similar down regulation of phosphorylation upon treating of cells from a cancer model system (K562 chronic myeloid leukemia cells) with imatinib. This treatment introduced a known perturbation of phosphorylation that enabled testing of these new approaches to analyze variations in tyrosine phosphorylation levels. Enrichment of pTyr proteins was found highly advantageous for the outcome. Out of a simplified 2-D DIGE experiment of immunoaffinity enriched control and treated pTyr proteins, differential analysis as well as protein identification by mass spectrometry (MS) was possible.     

Interactions of the Males and Females of Three Sympatric Drosophila virilis-Group Species, D. montana, D. littoralis, and D. lummei, (Diptera: Drosophilidae) in Intra- and Interspecific Courtships in the Wild and in the Laboratory

Four species of the Drosophila virilis group, D. montana, D. littoralis, D. lummei, and D. ezoana, occur sympatrically in several locations in northern Europe. Courtship interactions between the flies of the three first-mentioned species were observed at malt baits in Kemi, northern Finland, to find out how the flies of different species recognize conspecific individuals and how interspecific courtships differ from intraspecific ones in the wild. Intraspecific courtships (including females of different reproductive stages) and interspecific courtships were also videotaped and analyzed in laboratory. In the wild the males courted both conspecific and allospecific females, even though the species varied in how much the males were attracted to females of different species. Interspecific courtships usually broke off when the male touched the female or when the male and/or the female vibrated his/her wings, producing acoustic cues. In the laboratory males courted conspecific females irrespective of the reproductive stage of the female, even though the courtships directed toward immature and fertilized females usually included only orienting and touching (no licking and singing). D. littoralis, and very rarely D. montana and D. lummei, males courted also allospecific females. In the few interspecific courtships between these three species, where the male proceeded to singing, females responded to male singing by vibrating their wings. This ended the courtship. It is suggested that both the chemical cues affecting female attractivity and the acoustic signals of males and females, which are produced by wing vibration, function in maintaining sexual isolation between these three species.     

CRITERIA OF FEMALE MATE CHOICE IN DROSOPHILA LITTORALIS,D. MONTANA,AND D. EZOANA

We examined sexual selection by Drosophila littoralis, D. montana, and D. ezoana females on male courtship sounds to determine whether the females use absolute or relative criteria when choosing their mates. Behavior of the females was observed, when they were courted by a single male producing normal sounds, or by a single wing-manipulated male producing abnormal sounds; and when they were courted by one or both of these males in a choice situation. The females usually accepted short-winged (but not wingless) males producing abnormal sounds, if they had no alternatives. However, if they heard the sound produced by a normal male, they rejected the deficient male. Drosophila littoralis and D. ezoana females selected between two wing-manipulated males with different wing areas. Our results suggest that the females choose their mates on the basis of relative criteria if the signals emitted by the courting males are within the range of acceptable cues.     

Large-scale purification and preliminary X-ray diffraction studies of human aspartylglucosaminidase

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous polypeptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 Å and are thus suitable for determination of the crystal structure of AGA.