Characterization of different oligomeric forms of CRISP2 in the perinuclear theca versus the fibrous tail structures of boar spermatozoa†.

Mammalian sperm carry a variety of highly condensed insoluble protein structures such as the perinuclear theca, the fibrous sheath and the outer dense fibers, which are essential to sperm function. We studied the role of cysteine rich secretory protein 2 (CRISP2); a known inducer of non-pathological protein amyloids, in pig sperm with a variety of techniques. CRISP2, which is synthesized during spermatogenesis, was localized by confocal immunofluorescent imaging in the tail and in the post-acrosomal region of the sperm head. High-resolution localization by immunogold labeling electron microscopy of ultrathin cryosections revealed that CRISP2 was present in the perinuclear theca and neck region of the sperm head, as well as in the outer dense fibers and the fibrous sheath of the sperm tail. Interestingly, we found that under native, non-reducing conditions CRISP2 formed oligomers both in the tail and the head but with different molecular weights and different biochemical properties. The tail oligomers were insensitive to reducing conditions but nearly complete dissociated into monomers under 8 M urea treatment, while the head 250 kDa CRISP2 positive oligomer completely dissociated into CRISP2 monomers under reducing conditions. The head specific dissociation of CRISP2 oligomer is likely a result of the reduction of various sulfhydryl groups in the cysteine rich domain of this protein. The sperm head CRISP2 shared typical solubilization characteristics with other perinuclear theca proteins as was shown with sequential detergent and salt treatments. Thus, CRISP2 is likely to participate in the formation of functional protein complexes in both the sperm tail and sperm head, but with differing oligomeric organization and biochemical properties. Future studies will be devoted to the understand the role of CRISP2 in sperm protein complexes formation and how this contributes to the fertilization processes.

Cognitive effects of lacosamide as adjunctive therapy in refractory epilepsy.

BACKGROUND: Lacosamide (LCM) is a novel antiepileptic drug (AED) with potential benefit as adjunctive treatment in patients with partial-onset seizures. As yet, limited information on cognitive effects of LCM is available, especially in real-life settings. AIMS: In this open clinical prospective study, the cognitive effects of LCM were evaluated when used as adjunctive antiepileptic therapy in patients with refractory epilepsy. METHODS: We included 33 patients aged between 16 and 74 years (mean: 37 years). All patients had a localization-related epilepsy. Patients were assessed at baseline before starting LCM treatment and during follow-up when the optimal clinical dose was achieved. MATERIALS: Subjective complaints were evaluated using the SIDAED; effects on cognition were evaluated using the computerized visual searching task (CVST). RESULTS: The CVST showed significant faster information processing reaction times at the second evaluation (P = 0.013), which was not correlated with seizure control, type of epilepsy, age, gender, drug load, number of concomitant drugs, dose or duration of LCM treatment. On the SIDAED, patients complained more about their cognitive function at the second evaluation (P = 0.005). For the SIDAED, a positive correlation at follow-up was found between the total severity score and higher age (r = 0.375, P = 0.031), but not with epilepsy factors or treatment characteristics. DISCUSSION/CONLUSION: Screening of the cognitive effects of LCM showed that LCM does not have negative effects on information processing speed. As this is the most sensitive function for cognitive side effects of AEDs, LCM does not seem to induce the common negative cognitive effects. Remarkably, patients complained more, especially about their cognitive function, which is possible the 'doing better, feeling worse phenomenon'.

Differences in the endosomal distributions of the two mannose 6-phosphate receptors.

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.

Extent of linkage disequilibrium in chicken.

Many of the economically important traits in chicken are multifactorial and governed by multiple genes located at different quantitative trait loci (QTLs). The optimal marker density to identify these QTLs in linkage and association studies is largely determined by the extent of linkage disequilibrium (LD) around them. In this study, we investigated the extent of LD on two chromosomes in a white layer and two broiler chicken breeds. Pairwise levels of LD were calculated for 33 and 36 markers on chromosomes 10 and 28, respectively. We found that useful LD (i.e. an r(2) value higher than 0.3) in Nutreco chicken breed E5 (inbred) can extend to around 1 cM on chromosomes 10 and 28, although in a second region on chromosome 28 it extends to about 2.5 cM. The extent in breed Nutreco E3 (outbred) was very short in chromosome 10 (15 kb) but very much larger on chromosome 28, particularly in one region of depressed heterozygosity. The layer breed E2 (inbred) showed an extent of useful LD up to 4 cM on chromosome 10; the extent on chromosome 28 could not be assessed due to an erratic pattern of LD on that chromosome, although in one region LD appears to be in the order of 0.8 cM. This indicates that there may be very large differences in patterns of LD between different chicken breeds and different genomic regions.

Immunogold determination of amylase concentrations in pancreatic subcellular compartments.

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.

Integration of chicken genomic resources to enable whole-genome sequencing.

Different genomic resources in chicken were integrated through the Wageningen chicken BAC library. First, a BAC anchor map was created by screening this library with two sets of markers: microsatellite markers from the consensus linkage map and markers created from BAC end sequencing in chromosome walking experiments. Second, HINdIII digestion fingerprints were created for all BACs of the Wageningen chicken BAC library. Third, cytogenetic positions of BACs were assigned by FISH. These integrated resources will facilitate further chromosome-walking experiments and whole-genome sequencing.

Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung.

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.

Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice.

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.

Chromosomal assignment of chicken clone contigs by extending the consensus linkage map.

The bacterial artificial clone-based physical map for chicken plays an important role in the integration of the consensus linkage map and the whole-genome shotgun sequence. It also provides a valuable resource for clone selection within applications such as fluorescent in situ hybridization and positional cloning. However, a substantial number of clone contigs have not yet been assigned to a chromosomal location or have an ambiguous chromosome assignment. In this study, 86 single nucleotide polymorphism markers derived from 86 clones were mapped on the genetic map. These markers added anchoring information for 56 clone contigs and 13 individual clones, covering a total of 57,145 clones.

Genetic mapping of quantitative trait loci affecting susceptibility in chicken to develop pulmonary hypertension syndrome.

Pulmonary hypertension syndrome (PHS), also referred to as ascites syndrome, is a growth-related disorder of chickens frequently observed in fast-growing broilers with insufficient pulmonary vascular capacity at low temperature and/or at high altitude. A cross between two genetically different broiler dam lines that originated from the White Plymouth Rock breed was used to produce a three-generation population. This population was used for the detection and localization of quantitative trait loci (QTL) affecting PHS-related traits. Ten full-sib families consisting of 456 G2 birds were typed with 420 microsatellite markers covering 24 autosomal chromosomes. Phenotypic observations were collected on 4202 G3 birds and a full-sib across family regression interval mapping approach was used to identify QTL. There was statistical evidence for QTL on chicken chromosome 2 (GGA2), GGA4 and GGA6. Suggestive QTL were found on chromosomes 5, 8, 10, 27 and 28. The most significant QTL were located on GGA2 for right and total ventricular weight as percentage of body weight (%RV and %TV respectively). A related trait, the ratio of right ventricular weight as percentage to total ventricular weight (RATIO), reached the suggestive threshold on this chromosome. All three QTL effects identified on GGA2 had their maximum test statistic in the region flanked by markers MCW0185 and MCW0245 (335-421 cM).

Polymorphic microsatellites developed by cross-species amplifications in common pheasant breeds.

Genetic variability was analysed in two common breeds of pheasant (Phasianus colchicus L. 1758) by means of cross-species amplifications of microsatellite loci: 154 chicken, Gallus gallus and 32 turkey, Meleagris gallopavo, primers were tested for amplification of pheasant DNA. Thirty-six primers (25 specific for chicken and 11 for turkey) amplified pheasant DNA. Fifteen markers yielded specific products and were tested for polymorphism. Eight of them (55%) were polymorphic, with an average polymorphism of two alleles per locus. Specific polymerase chain reaction (PCR) products were sequenced; repeats were found in 11 of the 15 markers, although only two loci showed the same repeat and could be homologous to chicken ones.

Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment.

The hydrophobic surfactant protein B (SP-B) is synthesized in alveolar type II cells as a 40-kDa precursor protein that is processed via 39- and 23-kDa intermediates to the mature 8-kDa size. To determine the site of SP-B processing, the subcellular distribution of the precursor and mature forms of SP-B was investigated on ultrathin cryosections of human lung using two polyclonal antibodies that discriminate between precursor forms and the mature form of SP-B. An antibody against the human lysosomal membrane glycoprotein CD63 was used to identify organelles in the endosomal/lysosomal pathway. Precursor SP-B was present in the endoplasmic reticulum, the Golgi complex, and multivesicular bodies but was absent from lamellar bodies and plasma membrane. Mature SP-B was present in multivesicular bodies and lamellar bodies but almost completely absent in the endoplasmic reticulum and the Golgi complex. Semiquantitative evaluation of the immunogold labeling by counting the gold particles representing precursor or mature SP-B in the different compartments showed that the mature-to-precursor ratio was low in the endoplasmic reticulum and the Golgi complex (0.13 and 0.11, respectively), increased in the multivesicular bodies to 3.4, and was very high (65) in lamellar bodies. Multivesicular bodies and lamellar bodies contain the lysosomal membrane marker CD63 and are therefore part of the lysosomal pathway. These data strongly suggest that precursor SP-B is proteolytically processed to its mature 8-kDa form intracellularly in an endosomal/lysosomal compartment, most probably in multivesicular bodies.

The gene orders on human chromosome 15 and chicken chromosome 10 reveal multiple inter- and intrachromosomal rearrangements.

Comparative mapping between the human and chicken genomes has revealed a striking conservation of synteny between the genomes of these two species, but the results have been based on low-resolution comparative maps. To address this conserved synteny in much more detail, a high-resolution human-chicken comparative map was constructed from human chromosome 15. Mapping, sequencing, and ordering of specific chicken bacterial artificial chromosomes has improved the comparative map of chromosome 15 (Hsa15) and the homologous regions in chicken with almost 100 new genes and/or expressed sequence tags. A comparison of Hsa15 with chicken identified seven conserved chromosomal segments between the two species. In chicken, these were on chromosome 1 (Gga1; two segments), Gga5 (two segments), and Gga10 (three segments). Although four conserved segments were also observed between Hsa15 and mouse, only one of the underlying rearrangement breakpoints was located at the same position as in chicken, indicating that the rearrangements generating the other three breakpoints occurred after the divergence of the rodent and the primate lineages. A high-resolution comparison of Gga10 with Hsa15 identified 19 conserved blocks, indicating the presence of at least 16 intrachromosomal rearrangement breakpoints in the bird lineage after the separation of birds and mammals. These results improve our knowledge of the evolution and dynamics of the vertebrate genomes and will aid in the clarification of the mechanisms that underlie the differentiation between the vertebrate species.