Cryo-electron tomography provides novel insights into nuclear pore architecture: implications for nucleocytoplasmic transport

To go beyond the current structural consensus model of the nuclear pore complex (NPC), we performed cryo-electron tomography of fully native NPCs from Xenopus oocyte nuclear envelopes (NEs). The cytoplasmic face of the NPC revealed distinct anchoring sites for the cytoplasmic filaments, whereas the nuclear face was topped with a massive distal ring positioned above the central pore with indications of the anchoring sites for the nuclear basket filaments and putative intranuclear filaments. The rather "spongy" central framework of the NPC was perforated by an elaborate channel and void system, and at the membrane pore interface it exhibited distinct "handles" protruding into the lumen of the NE. The most variable structural moiety of the NPC was a rather tenuous central plug partially obstructing the central pore. Its mobile character was documented by time-lapse atomic force microscopy. Taken together, the new insights we gained into NPC structure support the notion that the NPC acts as a constrained diffusion pore for molecules and particles without retention signal and as an affinity gate for signal-bearing cargoes.     

In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation,pronucleus formation and embryo development

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.     

Chronology of apoptosis in bovine embryos produced in vivo and in vitro

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.     

The proprotein convertase PC5A and a metalloprotease are involved in the proteolytic processing of the neural adhesion molecule L1

The transmembrane and multidomain neural adhesion molecule L1 plays important functional roles in the developing and adult nervous system. L1 is proteolytically processed at two distinct sites within the extracellular domain, leading to the generation of different fragments. In this report, we present evidence that the proprotein convertase PC5A is the protease that cleaves L1 in the third fibronectin type III domain, whereas the proprotein convertases furin, PC1, PC2, PACE4, and PC7 are not effective in cleaving L1. Analysis of mutations revealed Arg(845) to be the site of cleavage generating the N-terminal 140-kDa fragment. This fragment was present in the hippocampus, which expresses PC5A, but was not detectable in the cerebellum, which does not express PC5A. The 140-kDa L1 fragment was found to be tightly associated with the full-length 200-kDa L1 molecule. The complex dissociated from the membrane upon cleavage by a protease acting at a more membrane-proximal site of full-length L1. This proteolytic cleavage was inhibited by the metalloprotease inhibitor GM 6001 and enhanced by a calmodulin inhibitor. L1-dependent neurite outgrowth of cerebellar neurons was inhibited by GM 6001, suggesting that proteolytic processing of L1 by a metalloprotease is involved in neurite outgrowth.     

Isolation of the Promoters of Atlantic Salmon MHCII Genes

The major histocompatibility complex class II (MHCII) has a central role in the immune response of vertebrates with its function of presenting antigenic peptides to the T-cell receptors. We have isolated the promoters and intron 1 of MHCII and MHCII genes of Atlantic salmon. To isolate these promoters, we constructed an Atlantic salmon (Salmo salar) promoter finder kit (analogous to the commercially available human promoter finder kit). By nucleotide sequence alignment of known MHCII promoter regions, we identified the 3 conserved regulatory X, X2, and Y boxes in the salmon promoters. The W box was not found. In contrast, a salmon-specific putative W box was identified. Both of the isolated Atlantic salmon MHCII and promoters (included in patent applications by Genomar A/S, Oslo, Norway) were found to be functional since they both gave positive yellow fluorescence protein signal when inserted as promoters in the pEYFP-1 reporter plasmid and transfected into the salmon head kidney cell line (SHK-1).     

The nuclear pore complex: nucleocytoplasmic transport and beyond

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.     

In situ studies of the phylogeny and physiology of filamentous bacteria with attached growth

Among the filamentous bacteria occasionally causing bulking problems in activated sludge treatment plants, three morphotypes with attached microbial growth are common, Eikelboom Type 0041, Type 1851 and Type 1701. A better knowledge of the phylogeny and physiology of these filamentous bacteria is necessary in order to develop control strategies for bulking. In this study we have used a combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR) to investigate the identity and in situ physiology of the Type 0041-morphotype and its attached bacteria in two wastewater treatment plants. Identification and enumeration of Type 0041 using group-specific 16S rRNA-targeted FISH probes revealed that approximately 15% of the filaments hybridized with a gene probe specific for the TM7 group, a recently recognized major lineage in the bacterial domain. All other filaments morphologically identified as Type 0041 only hybridized to the general bacterial EUB338-probe, indicating that they probably do not belong to commonly isolated bacterial phyla such as the Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, for which group-specific probes were used. The phylogenetic heterogeneity of Type 0041 again highlights the inadequacy of a morphology-based classification system. Like the filaments, most of the attached microbial cells were not identified beyond their affiliation to the Bacteria using the group-specific FISH probes. However, several different bacterial phyla were represented in the identified fraction suggesting that the attached microorganisms are phylogenetically diverse. The study of the in situ physiology of Type 0041 using MAR-FISH revealed that both the filaments and the attached bacteria on Type 0041 were versatile in the use of organic substrates and electron acceptors. It was observed that all Type 0041 could consume glucose, but none of the filaments were able to consume acetate under any conditions tested, in contrast to some of the attached bacteria. No significant physiological differences were found between TM7-positive and TM7-negative Type 0041 filaments, and only minor differences were observed between the two treatment plants tested. These are the first data on the physiology of the almost entirely uncharacterized TM7 phylum and show that TM7 filamentous bacteria can uptake carbon substrates under aerobic and anaerobic conditions.     

Online measurement of the viscosity in shake flasks enables monitoring of γ-PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate-starvation inducible promoter Ppst

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (V iscosity M onitoring O nline S ystem) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the P_pst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The P_pst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in g_γ-PGA/g_glucose of 190% could be achieved with the triple knockout mutant compared to the P_pst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.     

Antlers as biomonitors of environmental pollution by lead and fluoride: A review

Antlers are periodically cast and re-grown cranial appendages of deer. Both endochondral and intramembranous ossification are involved in the formation of antler bone. After velvet shedding, antlers are completely bony structures which are referred to as hard antlers. Growing antlers accumulate substances with an affinity to mineralized tissues. Among these substances are lead and fluoride. Due to the seasonally limited life span of antlers, the concentrations of lead and fluoride in hard antlers reflect uptake by the forming bone during a defined, species-specific period of some months. Antlers can thus be viewed as naturally standardized environmental samples that are well suited as biomonitors of environmental pollution by bone-seeking contaminants. Because hard antlers are collected by hunters as trophies and kept in private or public collections, material for study can be obtained rather easily. So far, lead concentrations in hard antlers have been reported only from Europe, whereas data for fluoride are available from both Europe and North America. Some studies compared contaminant concentrations in antlers from different regions, while others analyzed time-trends in contaminant levels in antlers from a single region. Using the latter approach, a pronounced drop of lead concentrations during recent decades has been reported for antlers from various European countries. This indicates a marked decline of environmental lead levels that can be attributed mainly to the phase-out of leaded gasoline and, in addition, to a reduction of lead emissions also from stationary sources. In Germany, a corresponding drop has also been recorded for fluoride concentrations in antlers, which is attributed to a decline of fluoride emissions from stationary sources due to improved emission-control measures. There is some evidence that exposure to higher levels of fluoride may lead to an impaired antler mineralization. Using antlers as biomonitors has been shown to be an efficient method for assessing environmental pollution by lead and fluoride at low cost. Further studies using this now well established approach are therefore encouraged.This revised version was published online in May 2005. The following errors were eliminated:1. In the chapter Antler growth and mineralization in the last sentence of the first paragraph a slash was missing between the words reindeer and caribou; 2. In the second paragraph of the same chapter the word reiudees was corrected to reindeer; 3. In the chapter Accumulation of lead and fluoride in growing antlers the word lightest was corrected to highest.