Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

Laser-assisted microinjection into targeted animal cells

A pulsed (17 nanoseconds) Nd:YAG laser (1064 nm) was used to inject impermeable dyes (propidium iodide andiodide and merocyanine 540) and a plasmid (pEGFP-N1) encoding green fluorescent protein (GFP) into human breast adenocarcinoma cells (MCF-7). The cell membrane integrity and viability were fully preserved in this laser-assisted transfer.     

Controlled rotation of biological microscopic objects using optical line tweezers

Controlled, continuous rotation of cells or intracellular objects was achieved using optical tweezers with an elliptic beam profile (line tweezers), which was generated by placing a cylindrical lens in the path of the trapping beam. By rotating the cylindrical lens, rotation of the elliptic trapping beam and hence of the object trapped therein was achieved. Compared to previously reported techniques for rotation of microscopic objects, this approach is much simpler, gives better utilization of available laser power and also allows much easier control of the trap beam profile. We have used this approach for rotation of biological objects varying in size from 2 to 40 m. At 25 mW trapping beam power at the object plane E. coli bacteria could be rotated at speeds approaching 10 Hz and an intracellular object (presumably a calcium oxalate crystal) trapped inside Elodea densa plant cell could be rotated with speeds of up to 4 Hz. To our knowledge, this is the first report for rotation of an intracellular object.     

Global organellar proteomics

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.     

Characterisation of senescence-induced changes in light harvesting complex II and photosystem I complex of thylakoids of Cucumis sativus cotyledons: age induced association of LHCII with photosystem I

Structure and function of chloroplasts are known to after during senescence. The senescence-induced specific changes in light harvesting antenna of photosystem II (PSII) and photosystem I (PSI) were investigated in Cucumis cotyledons. Purified light harvesting complex II (LHCII) and photosystem I complex were isolated from 6-day non-senescing and 27-day senescing Cucumis cotyledons. The chlorophyll a/b ratio of LHCII obtained from 6-day-old control cotyledons and their absorption, chlorophyll a fluorescence emission and the circular dichroism (CD) spectral properties were comparable to the LHCII preparations from other plants such as pea and spinach. The purified LHCII obtained from 27-day senescing cotyledons had a Chl a/b ratio of 1.25 instead of 1.2 as with 6-day LHCII and also exhibited significant changes in the visible CD spectrum compared to that of 6-day LHCII, indicating some specific alterations in the organisation of chlorophylls of LHCII. The light harvesting antenna of photosystems are likely to be altered due to aging. The room temperature absorption spectrum of LHCII obtained from 27-day senescing cotyledons showed changes in the peak positions. Similarly, comparison of 77K chlorophyll a fluorescence emission characteristics of LHCII preparation from senescing cotyledons with that of control showed a small shift in the peak position and the alteration in the emission profile, which is suggestive of possible changes in energy transfer within LHCII chlorophylls. Further, the salt induced aggregation of LHCII samples was lower, resulting in lower yields of LHCII from 27-day cotyledons than from normal cotyledons. Moreover, the PSI preparations of 6-day cotyledons showed Chl a/b ratios of 5 to 5.5, where as the PSI sample of 27-day cotyledons had a Chl a/b ratio of 2.9 suggesting LHCII association with PSI. The absorption, fluorescence emission and visible CD spectral measurements as well as the polypeptide profiles of 27-day cotyledon-PSI complexes indicated age-induced association of LHCII of PSII with PSI obtained from 27-day cotyledons. We modified our isolation protocols by increasing the duration of detergent Triton X-100 treatment for preparing the PSI and LHCII complexes from 27-day cotyledons. However, the PSI complexes isolated from senescing samples invariably proved to have significantly low Chl a/b ratio suggesting an age induced lateral movement and possible association of LHCII with PSI complexes. The analyses of polypeptide compositions of LHCII and PSI holocomplexes isolated from 6-day control and 27-day senescing cotyledons showed distinctive differences in their profiles. The presence of 26-28 kDa polypeptide in PSI complexes from 27-day cotyledons, but not in 6-day control PSI complexes is in agreement with the notion that senescence induced migration of LHCII to stroma lamellae and its possible association with PSI. We suggest that the migration of LHCII to the stroma lamellae region and its possible association with PSI might cause the destacking and flattening of grana structure during senescence of the chloroplasts. Such structural changes in light harvesting antenna are likely to alter energy transfer between two photosystems. The nature of aging induced migration and association of LHCII with PSI and its existence in other senescing systems need to be estimated in the future.     

Senescence induced structural reorganization of thylakoid membranes in Cucumis sativus cotyledons; LHC II involvement in reorganization of thylakoid membranes

We report the formation and appearance of loosely stacked extended grana like structures along with plastoglobuli in the chloroplasts isolated from 27-day old senescing cucumber cotyledons. The origin and the nature of these extended grana structures have not been elucidated earlier. We isolated Photosystem I complexes from 6-day-old control and 27-day-old senescing cotyledons. The chlorophyll a/b ratio of the isolated Photosystem I complex obtained from 6-day cotyledons was 5–5.5 as against a ratio of 2.9 was found in Photosystem I complexes obtained from 27-day-old senescing cotyledons. We also found that the presence of LHC II in the Photosystem I complexes isolated from 27-day cotyledonary chloroplasts. The presence of LHC II in Photosystem I complexes in senescing and not in control samples, clearly suggest the detachment and diffusion of LHC II complexes from stacked grana region to Photosystem I enriched stroma lamellar region thereby, forming loose disorganized extended grana structures seen in the transmission electron microscope. Furthermore, we show that under in vitro condition the senescing cotyledon chloroplasts exhibited lower extent of light induced phosphorylation of LHC II than the control samples suggesting a possible irreversible phosphorylation and diffusion of LHC II in vivo during the progress of senescence in Cucumis cotyledons. From these findings, we suggest that the senescence induced phosphorylation of LHC II and its migration towards Photosystem I may be a programmed one some how causing the destruction of the thylakoid membrane. The released membrane components may be stored in the plastoglobuli prior to their mobilization to the younger plant parts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acardiac anomaly spectrum

BACKGROUND: Acardiac anomaly spectrum is a rare congenital malformation found in monozygotic twin pregnancy. Besides the absence of heart, the condition is associated with variable grades of developmental disruption. Thus, no two cases are similar. METHODS: This case report is based on physical examination and autopsy findings. RESULTS: The twin had acardia and partial development of head and face. There was complete absence of upper extremities. CONCLUSIONS: The twin reversed arterial perfusion (TRAP) theory is the most accepted etiology of the disorder. Normally, the cephalic pole is the most severely affected, being most distal to the retrograde perfusion. In acardia, partial development of head, face, and brain is usually associated with the development of the upper extremities. However, in the present case, there was extensive cephalic development in the absence of upper extremity development.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.