New peptide conjugates with 9-aminoacridine: synthesis and binding to DNA.

New peptides-9-aminoacridine conjugates with an ethylene diamine linker-have been synthesized (both solution and solid phase methods were used) and their interactions with DNA have been studied. The affinity of H-Phe-Gln-Gly-Ile(2)-NHCH(2)CH(2)NH-Acr conjugate and of its extended analogue containing 6-aminohexanoic acid to DNA were lower than that of a standard H-Gly-NHCH(2)CH(2)NH-Acr conjugate. The results fit well into our concept of peptide conjugates with lowered binding activity to DNA, which could be capable of unlimited extravascular distribution. Moreover, new structures could be potentially useful as the mild tuners of DNA interaction with strong bis-acridine binders.     

Neural networks and robotic microneedles enable autonomous extraction of plant metabolites

Plant metabolites comprise a wide range of extremely important chemicals. In many cases, like savory spices, they combine distinctive functional properties—deterrence against herbivory—with an unmistakable flavor. Others have remarkable therapeutic qualities, for instance, the malaria drug artemisinin, or mechanical properties, for example natural rubber. We present a breakthrough in plant metabolite extraction technology. Using a neural network, we teach a computer how to recognize metabolite-rich cells of the herbal plant rosemary (Rosmarinus officinalis) and automatically extract the chemicals using a microrobot while leaving the rest of the plant undisturbed. Our approach obviates the need for chemical and mechanical separation and enables the extraction of plant metabolites that currently lack proper methods for efficient biomass use. Computer code required to train the neural network, identify regions of interest, and control the micromanipulator is available as part of the Supplementary Material.

A method for autonomous extraction from individual cells using a micromanipulator is developed and applied to glandular trichomes.     

Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ³², was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.     

Uptake of the β-Lactam Precursor α-Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease

External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (K_m values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.     

Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd²⁺ or Zn²⁺ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.     

Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.     

Molecular and Clinical Characteristics of MSH6 Variants: An Analysis of 25 Index Carriers of a Germline Variant

The MSH6 gene is one of the mismatch-repair genes involved in hereditary nonpolyposis colorectal cancer (HNPCC). Three hundred sixteen individuals who were known or suspected to have HNPCC were analyzed for MSH6 germline mutations. For 25 index patients and 8 relatives with MSH6 variants, molecular and clinical features are described. For analysis of microsatellite instability (MSI), the five consensus markers were used. Immunohistochemical analysis of the MLH1, MSH2, and MSH6 proteins was performed. Five truncating MSH6 mutations, of which one was detected seven times, were found in 12 index patients, and 10 MSH6 variants with unknown pathogenicity were found in 13 index patients. Fourteen (54%) of 26 colorectal cancers (CRCs) and endometrial cancers showed no, or only weak, MSI. Twelve of 18 tumors of truncating-mutation carriers and 3 of 17 tumors of missense-mutation carriers showed loss of MSH6 staining. Six of the families that we studied fulfilled the original Amsterdam criteria; most families with MSH6, however, were only suspected to have HNPCC. In families that did not fulfill the revised Amsterdam criteria, the prevalence of MSH6 variants is about the same as the prevalence of those in MLH1/MSH2. Endometrial cancer and/or atypical hyperplasia were diagnosed in 8 of 12 female carriers of MSH6 truncating mutations. Most CRCs were localized distally in the colon. Although, molecularly, missense variants are labeled as doubtfully pathogenic, clinical data disclose a great resemblance between missense-variant carriers and truncating-mutation carriers. We conclude that, in all patients suspected to have HNPCC, MSH6-mutation analysis should be considered. Neither MSI nor immunohistochemistry should be a definitive selection criterion for MSH6-mutation analysis.     

Familiarity and flexible mating strategies of a solitary rodent, Dipodomys ingens

Because mating is a product of individual reproductive strategies that may vary with changing conditions, we predicted variable mating behaviour in an arid-adapted, territorial rodent, the giant kangaroo rat, Dipodomys ingens. We also predicted that familiarity would facilitate nonaggressive courtship and mating in this solitary rodent. Through direct observations in the field, we found that mating varied from exclusive to multiple partners. Where densities were low, and on nights when multiple females were in oestrus, each animal mated with one member of the opposite sex. In conditions where the operational sex ratio was skewed towards multiple males, males footdrummed and competed for females. Males were able to mate with one or two females in adjacent territories, and they successfully competed for these same females throughout the breeding season. Females that mated exclusively with one male had more pups emerge from the burrow compared with females that experienced male competition. Females allowed nearest neighbour males to enter their burrows, and they engaged in more nonaggressive contact with close neighbours than with other males. Paired encounters in the field showed less aggression towards neighbours than strangers. In laboratory tests, females were less aggressive towards and allowed more contact with familiar than unfamiliar males. These results show that D. ingens can alter mating strategies as conditions change. Familiarity is an important factor in nonaggressive interactions between males and females and may be important to mate preferences in females during reproduction. The less aggressive behaviour to neighbours than to strangers (‘dear enemy’ phenomenon) is consistent with other solitary animals that defend multipurpose territories. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.     

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.