Antibacterial peptides in stimulated human granulocytes: characterization of ubiquitinated histone H1A.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.     

4-Oxospiro[benzopyran-2,4'-piperidines] as selective alpha 1a-adrenergic receptor antagonists

The 4-oxospiro[benzopyran-2,4'-piperidine] ring system is contained within potent class III antiarrhythmic agents. We highlight how these agents can be chemically transformed into a new class of potent (< 1 nM) and selective (> 25-fold) alpha 1a-receptor subtype adrenergic antagonists.     

Expression of the Gm1-Species, [NeuN]-GM1, in a Case of Human Glioma

Altered glycosylation is a common feature in tumors of various kind and particular interest has been focused on the expression of tumor-associated gangliosides. We have previously identified some human glioma-associated gangliosides and in this study yet another, not previously described, ganglioside has been isolated. The ganglioside was prepared from human glioma tissue taken at autopsy. The new ganglioside bound cholera-toxin B-subunit and its structure was confirmed by fast atom bombardment—mass spectrometry to be NeuN-GM1 (II³NeuNH₂-GgOse₄Cer). In the dissected tumor specimen, the concentration of NeuN-GM1 was 0.1 mol/g wet weight and accounted for approximately 20% of the monosialoganglioside fraction. Normal human brain tissue specimens (n = 10) did not contain detectable (>0.5 nmol/g wet weight of tissue) amounts of NeuN-GM1, indicating that this ganglioside might be associated with human glioma. However, none of the 17 other tumour specimens reveal any detectable amounts of this ganglioside. In conclusion, NeuN GM1 is a glioma-associated ganglioside but its exceptional expression limits its relevance as a molecule involved in general tumor biology.     

Presenilin expression during induced differentiation of the human neuroblastoma SH-SY5Y cell line

Human neuroblastoma SH-SY5Y cells stably transfected with both wild-type and exon-9 deleted (deltaE9) presenilin constructs were used to study the role of the presenilin proteins during differentiation. Cells transfected with either wild-type or deltaE9 PS1, of which the latter abolishes normal endoproteolytic cleavage of the protein, showed no obvious differences in their ability to differentiate to a neuronal-like phenotype upon treatment with retinoic acid (RA). A defined pattern of PS1 expression was observed during differentiation with both RA and the phorbol ester TPA. Full-length PS1 was shown to increase dramatically within 5-24 h of RA treatment. TPA gave an earlier and longer lasting increase in full-length PS1 levels. The intracellular distribution pattern of PS1 was markedly altered following RA treatment. Within 24h PS1 was highly up-regulated throughout the cell body around the nucleus. Between 2 and 4 weeks PS1 staining appeared punctate and also localised to the nucleus. Increases in PS1 expression upon treatment with RA and TPA were blocked by treatment with cycloheximide, indicating a role of de-novo protein synthesis in this effect. PS2 expression remained unchanged during differentiation. Levels of full-length PS1 were also seen to increase during neurogenesis and neuronal differentiation in the forebrain of first trimester human foetuses between 6.5 and 11 weeks. These combined observations support the idea that PS1 is involved in neuronal differentiation by a mechanism likely independent of endoproteolysis of the protein.     

Extreme difference in rate of mitochondrial and nuclear DNA evolution in a large ectotherm, Galápagos tortoises

We sequenced approximately 4.5 kb of mtDNA from 161 individuals representing 11 named taxa of giant Galápagos tortoises (Geochelone nigra) and about 4 kb of non-coding nuclear DNA from fewer individuals of these same 11 taxa. In comparing mtDNA and nucDNA divergences, only silent substitutions (introns, ITS, mtDNA control region, and synonymous substitutions in coding sequences) were considered. mtDNA divergence was about 30 times greater than that for nucDNA. This rate discrepancy for mtDNA and nucDNA is the greatest yet documented and is particularly surprising for large ectothermic animals that are thought to have relatively low rates of mtDNA evolution. This observation may be due to the somewhat unusual reproductive biology and biogeographic history of these organisms. The implication is that the ratio of effective population size of nucDNA/mtDNA is much greater than the usually assumed four. The nearly neutral theory of molecular evolution predicts this would lead to a greater difference between rates of evolution.     

Characterization of Trichodesmium spp. by genetic techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.     

Gold-coated capillary based 2,4-dichlorophenoxyacetic acid chemi-luminescent assays: possibilities towards multianalysis

The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.