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.     

Flavones are inhibitors of HIV-1 proteinase.

Substituted gamma-chromones were found to weakly inhibit HIV-1 proteinase, an important enzyme in the replication and processing of the AIDS virus. Chromones bearing hydroxyl substituents and a phenolic group at the 2-position (flavones) were the most active compounds and structure-activity relationships for a limited series of flavone inhibitors are presented. Dixon plots are reported and a possible mechanism for flavone-induced inhibition is proposed. The results are also compared with those for some structurally related non-peptidic inhibitors of HIV-1 proteinase. Since some flavonoid compounds have already been shown to have antiviral activity against AIDS, the present observations of anti-HIV-1 proteinase activity may be particularly significant.     

Inhibition of HIV-1 proteinase by metal ions.

1. Certain metal ions have been identified as inhibitors (IC50 1-20 microM) of the aspartic proteinase of Human Immunodeficiency Virus Type 1 (HIV-PR). 2. By contrast most simple metal ions do not inhibit this enzyme. 3. Those that did inhibit have in common a high charge/size ratio or "hard" acidic nature, preferring to combine covalently with oxygen donor ligands. 4. Some evidence from independent X-ray crystal structure determinations suggests that the metalloinhibitors identified here may bind in the active site of the enzyme via coordination to the carboxylate side chains of the essential active site residues Asp 25 and 125. 5. Although the measured inhibition is only microM, very few enzyme-inhibitor interactions can be taking place and so more complex metalloinhibitors with ligands that can also bind to peptide side chains of the enzyme might be significantly more potent inhibitors of HIV-PR and of viral replication.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol)

A panel of six na?ve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.     

A fusion protein system for the recombinant production of short disulfide-containing peptides

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.     

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMRsolution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide Abeta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native Abeta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length Abeta peptides Abeta(1-40) and Abeta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which Abeta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of Abeta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation.     

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.