Centriole as microtubule-organizing centers for marginal bands of molluscan erythrocytes

The erythrocytes of blood clams (arcidae) are flattened, elliptical, and nucleated. They contain elliptical marginal bands (MBs) of microtubules, each physically associated with a pair of centrioles marginal bands (MBs) of microtubles, each physically associated with a pair of centrioles (Cohen, W., and I. Nemhauser, 1980, J. Cell Biol., 86:286-291). The MBs were found to be cold labile in living cells, disappearing within 1-2 h at 0 degrees C. After the cells had been rewarmed for 1-2 h, continuous MBs with associated centrioles were once again present. Time-course studies utilizing phase contrast, antitubulin immunofluorescence, and electron microscopy of cytoskeletons prepared during rewarming revealed structural evidence of centriole participation in MB reassembly. At the earliest stage of reassembly, a continuous MB was not present. Instead, relatively short and straight microtubules focused on a pointed centriolar “pole,” and none were present elsewhere in the cytoskeleton. Thin continuous MBs then formed, still pointed in the centriolar region. Subsequently, the MBs regained ellipticity, with their thickness gradually increasing but not reaching that of controls even after several hours of rewarming. At these later time points, microtubules still radiated from the centrioles and joined the MBs some distance away. In the presence of 0.1 mM colchicines, MB reassembly was arrested at the pointed stage. Electron microscopic observations indicate that pericentriolar material is involved in microtubule nucleation in this system, rather than the centriolar triplets directly. The results suggest a model in which the centrioles and associated material nucleate assembly and growth of microtubules in diverging directions around the cell periphery. Microtubules of opposite polarity would then pass each other at the end of the cell distal to the centrioles, with continued elongation eventually closing the MB ellipse behind the centriole pair.     

Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.     

Rates of synthesis of ribosomal protein and total ribonucleic acid through the cell cycle of the fission yeast Schizosaccharomyces pombe

The rates of synthesis of ribosomal proteins through the cell cycle of the fission yeast Schizosaccharomyces pombe have been examined by spec. act. estimations of isolated 80S ribosomes pulse-labelled with ³⁵S-sulphate. The spec. act. have minimum values at the beginning (0.0) and maxima between 0.6 and 0.9 of the cell cycle. This pattern in spec. act. is also shown by isolated 80S ribosomes pulse-labelled with ³H-uridine during synchronous cultures and is in marked contrast to the small, random variations in the spec. act. of isolated 80S ribosomes from control, asynchronous cultures pulse-labelled with ³⁵S-sulphate or ³H-uridine.A detailed examination of the rates of synthesis of total RNA through the cell cycle measured by the rates of incorporation of ³H-uridine and ³H-adenine shows a step in the rates of incorporation at the time of DNA synthesis. This step has further been shown to be independent both of the uridine concentration, over a range from 0.03 μM to 820 μM, and of pre-filling the adenine pool. This step thus appears to be independent of variations in rates of uptake of both purines and pyrimidines, or fluctuations in the pool size of the precursors and may be explained as a gene-dosage effect.The step in the pattern of synthesis of total RNA has been shown to yield a cyclic pattern in the spec. act. of the total RNA through the cell cycle. This pattern is similar to that of the spec, act. of RNA and of protein recovered from ribosomes. The variation exhibited by the ribosomal proteins is believed to be a consequence of the step in the pattern of RNA synthesis, with a concomitant fluctuation in the pool of ribosomal proteins synthesised continuously through the cell cycle.     

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.     

Occludin oligomeric assemblies at tight junctions of the blood–brain barrier are altered by hypoxia and reoxygenation stress

Hypoxic (low oxygen) and reperfusion (post‐hypoxic reoxygenation) phases of stroke promote an increase in microvascular permeability at tight junctions (TJs) of the blood–brain barrier (BBB) that may lead to cerebral edema. To investigate the effect of hypoxia (Hx) and reoxygenation on oligomeric assemblies of the transmembrane TJ protein occludin, rats were subjected to either normoxia (Nx, 21% O₂, 60 min), Hx (6% O₂, 60 min), or hypoxia/reoxygenation (H/R, 6% O₂, 60 min followed by 21% O₂, 10 min). After treatment, cerebral microvessels were isolated, fractionated by detergent‐free density gradient centrifugation, and occludin oligomeric assemblies associated with plasma membrane lipid rafts were solubilized by perfluoro‐octanoic acid (PFO) exclusively as high molecular weight protein complexes. Analysis by non‐reducing and reducing sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis/western blot of PFO‐solubilized occludin revealed that occludin oligomeric assemblies co‐localizing with ‘TJ‐associated’ raft domains contained a high molecular weight ‘structural core’ that was resistant to disassembly by either SDS or a hydrophilic reducing agent ex vivo, and by Hx and H/R conditions in vivo. However, exposure of PFO‐solubilized occludin oligomeric assemblies to SDS ex vivo revealed the non‐covalent association of a significant amount of dimeric and monomeric occludin isoforms to the disulfide‐bonded inner core, and dispersal of these non‐covalently attached occludin subunits to lipid rafts of higher density in vivo was differentially promoted by Hx and H/R. Our data suggest a model of isoform interaction within occludin oligomeric assemblies at the BBB that enables occludin to simultaneously perform a structural role in inhibiting paracellular diffusion, and a signaling role involving interactions of dimeric and monomeric occludin isoforms with a variety of regulatory molecules within different plasma membrane lipid raft domains.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

The function of a Drosophila glypican does not depend entirely on heparan sulfate modification

Division abnormally delayed (Dally) is one of two glycosylphosphatidylinositol (GPI)-linked heparan sulfate proteoglycans in Drosophila. Numerous studies have shown that it influences Decapentaplegic (Dpp) and Wingless signaling. It has been generally assumed that Dally affects signaling by directly interacting with these growth factors, primarily through its heparan sulfate (HS) chains. To understand the functional contributions of HS chains and protein core we have (1) assessed the growth factor binding properties of purified Dally using surface plasmon resonance, (2) generated a form of Dally that is not HS modified and evaluated its signaling capacity in vivo. Purified Dally binds directly to FGF2, FGF10, and the functional Dpp homolog BMP4. FGF binding is abolished by preincubation with HS, but BMP4 association is partially HS-resistant, suggesting the Dally protein core contributes to binding. Cell binding and co-immunoprecipitation studies suggest that non-HS-modified Dally retains some ability to bind Dpp or BMP4. Expression of HS-deficient Dally in vivo showed it does not promote signaling as well as wild-type Dally, yet it can rescue several dally mutant phenotypes. These data reveal that heparan sulfate modification of Dally is not required for all in vivo activities and that significant functional capacity resides in the protein core.     

The question of uniqueness of ancient bacteria

Microorganisms are associated with a variety of ancient geological materials. However, conclusive proof that these organisms are as old as the geological material and not more recent introductions has generally been lacking. Over the years, numerous reports of the isolation of ancient bacteria from geological materials have appeared. Most of these have suffered from the fact that the protocol for the surface sterilization of the sample was either poorly defined, inadequate or rarely included data to validate the overall effectiveness of the sterilization protocol. With proper sterility validation and isolation protocol, a legitimate claim for the isolation of an ancient microbe can be made. Biochemical, physiological, or morphological data indicate that these ancient microbes are not significantly different from modern isolates. As the role (decomposition) of modern and ancient microbes has not changed over time, it is probably unreasonable to expect these organisms to be vastly different. A discussion on the reasons for the homogeneity of ancient and modern microbes is presented. Journal of Industrial Microbiology & Biotechnology (2002) 28, 32–41 DOI: 10.1038/sj/jim/7000174 Received 20 May 2001/ Accepted in revised form 16 June 2001     

Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex

Recent studies have shown that the platelet membrane glycoprotein Ia-IIa (VLA-2) complex mediates the Mg(++)-dependent adhesion of platelets to collagen and that this adhesion is inhibited by Ca++ in a simple, linear, noncompetitive manner. These findings suggested that separate binding sites for Mg++ and Ca++ stabilize different divalent cation-dependent structures within the receptor complex. To provide evidence for the existence of such structures purified platelet Ia-IIa complex was subjected to limited proteolytic digestion in the presence of Mg++, Ca++, Mg++ and Ca++, or EDTA and the resulting peptides mapped by SDS-PAGE using both one and two-dimensional techniques. Unique patterns of tryptic peptides were produced under each of the conditions. The results indicate that Mg++ and Ca++ stabilize different structures within the Ia-IIa (VLA-2) complex and that these structures influence both the collagen binding activity and proteolytic susceptibility of the complex.