Quick elucidation of cyclodepsipeptide sequence from sacoglossan Elysia grandifolia using electrospray ionisation-tandem mass spectrometry

Butanol fraction of sacoglossan Elysia grandifolia was investigated for identifying peptides using electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). Without prior isolation, the structural determination is achieved on the basis of mass fragmentation pattern and comparison with the previously established data. The ESI-MS of the fraction in the positive ion mode gave clusters of singly and doubly charged molecular ion peaks. The ESI-MS spectrum showed peaks for the presence of the peptides kahalalides F, G, R and S reported earlier. In addition, it also showed molecular ion peaks at m/z 1557.8 [M+H]+ and doubly charged ions at m/z 779.4 [M+2H]2+, 790.4 [M+Na]2+ and 796.4 [M+K]2+. The MS/MS at m/z 779.4 [M+2H]+2 at collision energy 40 V obtained series of b and y fragment ions. The MS/MS spectrum showed identical fragment ion y6 at m/z 643 which revealed that cyclic part is identical with kahalalide F, R and S. Careful examination of the fragment ions b1 to b7 with their corresponding y fragment ions y12 to y6, respectively and by comparison of MS/MS pattern of kahalalide S, established that proline can be replaced by tyrosine amino acid residue. The mass difference between b4 ( m/z 511) and b5 ( m/z 674) is equal to 163 which is equivalent to mass residue of tyrosine. Their y fragment ions also quickly helped in fixing the puzzle. This resulted in the identification of the peptide sequence cyclo-[Val-(5-MeHex-Val-Thr-Val-Val-Tyr-Lys-Ile)Thr-Ile-Val-Phe-Dhb)] for the new cyclodepsipeptide, kahalalide Z3. Thus, ESI-MS/MS has set a trend in quick identification of new marine molecules.

Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites

Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.

Identification of the Sex-Determining Region of the Ceratitis capitata Y Chromosome by Deletion Mapping

In the medfly Ceratitis capitata, the Y chromosome is responsible for determining the male sex. We have mapped the region containing the relevant factor through the analysis of Y-autosome translocations using fluorescence in situ hybridization with two different probes. One probe, the clone pY114, contains repetitive, Y-specific DNA sequences from C. capitata, while the second clone, pDh2-H8, consists of ribosomal DNA sequences from Drosophila hydei. Clone pY114 labeled most of the long arm and pDh2-H8 hybridizes to the short arm and the centromeric region of the long arm. In 12 of the analyzed 19 Y-autosome translocation strains, adjacent-1 segregation products survive to the late pupal or even adult stage and can, therefore, be sexed. This was correlated with the length of the Y fragment still present in these aberrant individuals and allowed us to map the male-determining factor to a region of the long arm representing ~15% of the entire Y chromosome. No additional factors, affecting for example fertility, were detected outside the male-determining region.

The anomeric effect in thio derivatives of benzocycloheptene

The conformational properties of six thio derivatives of benzocycloheptene were studied by 1H and 13C high-field NMR spectroscopy in solution at low temperature. Both parent compounds 2 and 6, with sulfur at positions 3 and 1, respectively, were found to exist in the chair conformation. The 2-substituted derivatives of 2 (3: Y = OCH3 and 4: Y = Cl) revealed the presence of the Ca form only. In contrast, the 2-substituted derivatives of 6 (7: Y = OCH3 and 8: Y = Cl), with sulfur at position 1, showed mixtures of three conformers, namely Ca:Ce:TB (Ca = axial chair, Ce = equatorial chair, and TB = twist-boat), whose populations in CHF2Cl were, respectively, 93:5:2 and 85:12:3. The predominance of the Ca form in both series argues in favour of a strong anomeric effect in spite of the large departure from coplanarity (~35°) in the:—S—C—Y fragment of both 7 and 8. This noncoplanarity appears to weaken only slightly the n–σ* overlap characteristic of the endo-anomeric effect, contrary to earlier observations for the:—O—C—Y fragment in 5. The differences between the effects of O and S are discussed.