Sperm competition and last-male precedence in the honeybee

Five microsatellite loci were used to determine paternities in six Apis mellifera colonies headed by naturally mated queens. The last inseminating males were identified by collecting and genotyping the mating sign left in the genital tract of each queen. Significant differences in paternity frequencies were observed between males, but the proportion of worker and queen offspring sired by the last inseminating drone did not differ significantly from those of other drones. Each male kept his rank of precedence for the different cohorts, although the variance in subfamily proportions decreased over time, most notably in the colony displaying the lowest level of polyandry. These results suggest that, if sperm competition exists in the honeybee, it does not significantly increase the fitness of the last inseminating drone. The spermatozoa of the different inseminating drones are not totally mixed before they reach the spermatheca, in particular when only few males mate with the queen. The weak difference in the subfamily proportions observed between queen and worker samples confirms that nepotistic interactions are rare. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.     

Stable isotope records (O, C) of Jurassic aragonitic shells from England and NW Poland: palaeoecologic and environmental implications

Shells of fully marine Middle to Upper Jurassic molluscs from England and north-western Poland were analysed with respect to their stable isotope (δ¹⁸O, δ¹³C) compositions, and palaeoecological and environmental life conditions of these molluscs were inferred from them. Light microscopical and SEM inspection and an analysis of the minor element content (Fe, Mn, Mg, Sr) suggest rather unaltered isotope signals. The δ¹⁸O and δ¹³C values show a characteristic distribution among three groups of co-occurring organisms. Benthic (adult) bivalves generally preserved higher δ¹⁸O and δ¹³C values than ammonites, whereas planktic bivalve larvae tend to possess the lowest δ¹⁸O but higher δ¹³C than adult bivalves. As this distribution pattern is found in numerous horizons and sections of Bathonian to Kimmeridgian age in NW Poland and England, it is thought to reflect real palaeoenvironmental parameters. All observations can be incorporated in a single model that assumes (i) seasonally induced temperature stratification of the water column, (ii) a correlation between phytoplankton blooms and reproduction season of planktic-planktotrophic bivalves, and (iii) insignificant vital effects with respect to the δ¹³C in bivalves, but strong biological control in ammonites. In addition, the δ¹⁸O evolution suggests that the Late Bajocian to Middle/Late Bathonian and Early Oxfordian to Late Kimmeridgian were considerably warmer than the latest Bathonian to Late Callovian time interval. The oxygen isotopic records from other European regions indicate a similar pattern of long-term palaeotemperature evolution. The comparatively high water temperatures during the Callovian to Oxfordian of the Isle of Skye (NW Scotland) are enigmatic, however. In the Early Oxfordian, sea surface and bottom temperatures began to rise in continental Europe and England. These changes coincide with a south-westward drift of the West European crustal plate, but a causal relationship remains to be demonstrated.     

Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.     

Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish

Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.     

Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel

The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals. Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion. We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences. Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component.     

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity

C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.     

Identification and characterization of a putative homolog of a spliceosome component,precursor RNA processing 3 in Drosophila melanogaster

Nuclear pre-mRNA splicing occurs in a large RNA-protein complex that contains four small nuclear ribonucleoprotein particles (snRNPs) as well as many protein factors. The Precursor RNA processing 3 (Prp3) is a U4/U6-associated splicing factor. A putative homologue of Prp3, which showed a 45% identity to the human Prp3 in an amino acid sequence, was identified in Drosophila melanogaster (dPrp3). A full-length cDNA clone was isolated and sequenced from the embryonic cDNA library. This gene consisted of 2 exons and contained an open-reading frame that encoded 550 amino acid residues. A Northern blot analysis showed that dPrp3 is expressed both maternally and zygotically. Immunostaining revealed that dPrp3 was localized to the nuclei of nurse cells and follicle cells in early embryos, which is consistent with its role as a component of spliceosome.     

Kinetic analysis of the inhibition of phenylalanine ammonia-lyase by 2-aminoindan-2-phosphonic acid and other phenylalanine analogues

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.