Control of Ste6 recycling by ubiquitination in the early endocytic pathway in yeast

We present evidence that ubiquitination controls sorting of the ABC-transporter Ste6 in the early endocytic pathway. The intracellular distribution of Ste6 variants with reduced ubiquitination was examined. In contrast to wild-type Ste6, which was mainly localized to internal structures, these variants accumulated at the cell surface in a polar manner. When endocytic recycling was blocked by Ypt6 inactivation, the ubiquitination deficient variants were trapped inside the cell. This indicates that the polar distribution is maintained dynamically through endocytic recycling and localized exocytosis ("kinetic polarization"). Ste6 does not appear to recycle through late endosomes, because recycling was not blocked in class E vps (vacuolar protein sorting) mutants (Deltavps4, Deltavps27), which are affected in late endosome function and in the retromer mutant Deltavps35. Instead, recycling was partially affected in the sorting nexin mutant Deltasnx4, which serves as an indication that Ste6 recycles through early endosomes. Enhanced recycling of wild-type Ste6 was observed in class D vps mutants (Deltapep12, Deltavps8, and Deltavps21). The identification of putative recycling signals in Ste6 suggests that recycling is a signal-mediated process. Endocytic recycling and localized exocytosis could be important for Ste6 polarization during the mating process.     

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.     

Solid-state fermentation production of tetracycline by <Emphasis Type="Italic">Streptomyces</Emphasis> strains using some agricultural wastes as substrate

The ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273 S. aureofaciens NRRL B2183 and S. vendagensis ATCC 25507 to produce tetracycline using some local agricultural wastes as solid state media, were assessed. The wastes employed include peanut (groundnut) shells, corncob, corn pomace and cassava peels. Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity. All the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the carbohydrate source. Streptomyces sp. OXC1 had the highest ability for tetracycline production with peanut shells as the substrate in solid fermentation (13.18 mg/g), followed by S. vendagensis ATCC 25507 (11.08 mg/g), S.rimosus NRRL B1679 (8.46 mg/g), S. alboflavus NRRL B1273 (7.59 mg/g), S. rimosus NRRL B2234 (6.37 mg/g), S. aureofaciens NRRL B2183 (4.27 mg/g). Peanut (groundnut) shells were the most effective substrate (4.36 mg/g) followed by corncob (2.64 mg/g), cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g). The composition of the peanut (groundnut) shell medium optimal for tetracycline production were peanut shells 100 g, organic nitrogen (peanut meal) 10 g, (NH ₄)₂ SO₄ 1 g, KH₂ PO ₄ 0.5 g, CaCO₃ > 0.5 g, NaCl 0.5 g, MgSO₄ · 7H₂ O 0.5 g, soluble starch 10 g, peanut oil 0.25 ml with initial moisture content of 65–68%, and initial pH 5.3–6.3. Substrate (1 g dry weight) was inoculated with 1.0 × 10 ⁸ conidia per ml and incubated at 28–31 °C for 5–7 days, producing 13.18 mg/g of total tetracycline. Tetracycline detection started on day 3 and attained its maximum level on day 5.     

Crystallization and preliminary X-ray diffraction studies of two myotoxic Lys49-phospholipases A2 complexed with alpha-tocopherol

BnSP-7 and BnSP-6, two Lys49-phospholipase A2 isolated from Bothrops neuwiedi pauloensis snake venom, were co-crystallized with alpha-tocopherol and X-ray diffraction data were collected for both complexes (2.2 and 2.6 A). A new "alternative" quaternary conformation for these two complexes compared with all other dimeric Lys49-PLA2 has been observed.     

Mitochondrial DNA of ancient Cumanians: culturally Asian steppe nomadic immigrants with substantially more western Eurasian mitochondrial DNA lineages

The Cumanians were originally Asian pastoral nomads who in the 13th century migrated to Hungary. We have examined mitochondrial DNA from members of the earliest Cumanian population in Hungary from two archeologically well-documented excavations and from 74 modern Hungarians from different rural locations in Hungary. Haplogroups were defined based on HVS I sequences and examinations of haplogroup-associated polymorphic sites of the protein coding region and of HVS II. To exclude contamination, some ancient DNA samples were cloned. A database was created from previously published mtDNA HVS I sequences (representing 2,615 individuals from different Asian and European populations) and 74 modem Hungarian sequences from the present study. This database was used to determine the relationships between the ancient Cumanians, modern Hungarians, and Eurasian populations and to estimate the genetic distances between these populations. We attempted to deduce the genetic trace of the migration of Cumanians. This study is the first ancient DNA characterization of an eastern pastoral nomad population that migrated into Europe. The results indicate that, while still possessing a Central Asian steppe culture, the Cumanians received a large admixture of maternal genes from more westerly populations before arriving in Hungary. A similar dilution of genetic, but not cultural, factors may have accompanied the settlement of other Asian nomads in Europe.     

Functional overexpression of gamma-secretase reveals protease-independent trafficking functions and a critical role of lipids for protease activity

Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.     

Real-time polymerase chain reaction-based exponential sample amplification for microarray gene expression profiling

Conventional approaches to target labeling for gene expression analysis using microarray technology typically require relatively large amounts of RNA, a serious limitation when the available sample is limited. Here we describe an alternative exponential sample amplification method by using quantitative real-time polymerase chain reaction (QRT-PCR) to follow the amplification and eliminate the overamplified cDNA which could distort the quantitative ratio of the starting mRNA population. Probes generated from nonamplified, PCR-amplified, and real-time-PCR-amplified cDNA samples were generated from lipopolysaccharide-treated and nontreated mouse macrophages and hybridized to mouse cDNA microarrays. Signals obtained from the three protocols were compared. Reproducibility and reliability of the methods were determined. The Pearson correlation coefficients for replica experiments were r=0.927 and r=0.687 for QRT-PCR-amplification and PCR-overamplification protocols, respectively. Chi2 test showed that overamplification resulted in major biases in expression ratios, while these alterations could be eliminated by following the cycling status with QRT-PCR. Our exponential sample amplification protocol preserves the original expression ratios and allows unbiased gene expression analysis from minute amounts of starting material.     

Studies related to the synthesis of derivatives of 2,6-diamino-2,3,4,6-tetradeoxy-D-erythro-hexose (purpurosamine C), a component of gentamicin C12

Reduction of 1,6-anhydro-3,4-dideoxy-β-D-glycero-hex-3-enopyranos-2-ulose (levoglucosenone) with lithium aluminium hydride afforded principally 1,6-anhydro-3,4-dideoxy-β-D-threo-hex-3-enopyranose (3), which was converted into 3,4-dihydro-2(S)-hydroxymethyl-2H-pyran (8) following acid-catalysed methanolysis and reductive rearrangement of the resulting α-glycoside 4 with lithium aluminium hydride. 1,6-Anhydro-3,4-dideoxy-2-O-toluene-p-sulphonyl-β-D-threo-hexopyranose, prepared from 3, reacted slowly with sodium azide in hot dimethyl sulphoxide to give 1,6-anhydro-2-azido-2,3,4-trideoxy-β-D-erythro-hexopyranose, which was transformed into a mixture of methyl 2-acetamido-6-O-acetyl-2,3,4-trideoxy-α-D-erythro-hexopyranoside (10) and the corresponding β anomer following acid-catalysed methanolysis, catalytic reduction, and acetylation. Acid treatment of methyl 4,6-O-benzylidene-3-deoxy-α-D-erythro-hexopyranosid-2-ulose yielded the enone 15, which was readily transformed into methyl 6-O-acetyl-3,4-dideoxy-α-D-glycero-hexopyranosid-2-ulose (19). Procedures for the conversions of DL-8, 10, and 19 into methyl 2,6-diacetamido-2,3,4,6-tetradeoxy-α-D-erythro-hexopyranoside (methyl N,N′-di-acetyl-α-purpurosaminide C) have already been described.     

Developmental regulation of dUTPase in Drosophila melanogaster

dUTPase prevents uracil incorporation into DNA by strict regulation of the cellular dUTP:dTTP ratio. Lack of the enzyme initiates thymineless cell death, prompting studies on enzyme regulation. We investigated expression pattern and localization of Drosophila dUTPase. Similarly to human, two isoforms of the fly enzyme were identified at both mRNA and protein levels. During larval stages, a drastic decrease of dUTPase expression was demonstrated at the protein level. In contrast, dUTPase mRNAs display constitutive character throughout development. A putative nuclear localization signal was identified in one of the two isoforms. However, immunohistochemistry of ovaries and embryos did not show a clear correlation between the presence of this signal and subcellular localization of the protein, suggesting that the latter may be perturbed by additional factors. Results are in agreement with a multilevel regulation of dUTPase in the Drosophila proteome, possibly involving several interacting protein partners of the enzyme. Using independent approaches, the existence of such macromolecular partners was verified.     

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9- and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-A resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred.