An experimental test of the relationship between temporal variability of feeding opportunities and baseline levels of corticosterone in a shorebird

In this study, we tested the hypothesis that baseline corticosterone levels increase with a change from constant to variable feeding schedules. Captive red knots, Calidris canutus, were presented with food that was either available during the same time each day (constant) or starting at variable times during the day. Food intake rates, frequency of aggressive interactions, and baseline levels of corticosterone were measured. In the majority of cases, red knots showed higher plasma corticosterone concentrations during feeding schedules that were irregular than when food was available at consistent times. These findings are supported by a previous study that showed that red knots take a long time to adjust to the newly offered, predictable conditions of their aviary environment. The frequency of conflicts in the different groups and (size-corrected) body mass were not correlated with average corticosterone level. The results are examined in the light of literature showing that increases in corticosterone in response to acute, unpredictable events mediate behavioral responses such as increased explorative behavior and memory. For red knots that have to find their food on the temperate-zone mudflats in Western Europe, an increased circulating corticosterone level may be adaptive during periods when the patchily distribution of buried bivalves and the burying behavior of such prey presents them with a variable and unpredictable food supply.     

Origin of human immunodeficiency virus type 1 quasispecies emerging after antiretroviral treatment interruption in patients with therapeutic failure

The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.     

The new bilaterally pedicled V-Y advancement flap for face reconstruction

A new bilaterally pedicled V-Y advancement flap based on two subcutaneous pedicles that vascularize the skin island through subdermal plexus lateral bridges is described for face reconstruction. It differs from traditional V-Y advancement flaps in that it does not rely on the classic subcutaneous "vertical" pedicle that is sectioned from top to bottom to improve advancement of the skin island. This technique had predictable results for 12 years in 425 consecutive patients, with infection occurring in 2.8 percent of the cases and complete necrosis in less than 1 percent of the flaps.     

Analysis of mouse Rad54 expression and its implications for homologous recombination

Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs). Important proteins in this pathway are Rad51 and Rad54. Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54. We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules. Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation. We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion. The models differ in whether Rad54 functions locally or globally. In the first model, Rad54 acts in cis relative to the site of strand invasion. Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament. Subsequently, it promotes duplex DNA unwinding. In the second model, Rad54 acts in trans relative to the site of strand invasion. Rad54 binds duplex DNA distant from the site that will be unwound. Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.     

Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells

The bioluminescent Ca²⁺-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca^{2 +}-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC₅₀ value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca^{2 +}-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I. Revised version received: 12 September 2001 Electronic Publication     

Automated identification of diploid reference cells in cervical smears using image analysis

BACKGROUND: Acquisition of DNA ploidy histograms by image analysis may yield important information regarding the behavior of premalignant cervical lesions. Accurate selection of nuclei for DNA measurement is an important prerequisite for obtaining reliable data. Traditionally, manual selection of nuclei of diagnostic and reference cells is performed by an experienced cytotechnologist. In the present study, a method for the fully automated identification of nuclei of diploid epithelial reference cells in Feulgen- restained Papanicolaou (PAP) smears is described. METHODS: The automated procedure consists of a decision tree implemented on the measurement device, containing nodes with feature threshold values and multivariate discriminant functions. Nodes were constructed to recognize debris and inflammatory cells, as well as diploid and nondiploid epithelial cells of the uterine cervix. Evaluation of the classifier was performed by comparing resulting diploid integrated optical densities with those from manually selected reference cells. RESULTS AND CONCLUSION: On average, automatically acquired values deviated 2.4% from manually acquired values, indicating that the method described in this paper may be useful in cytometric practice.     

Crystal structure of a stress inducible protein from Mycoplasma pneumoniae at 2.85 Å resolution

Journal of Structural and Functional Genomics -     

Multiplex PCR screening of soil isolates for novel Bacillus-related lineages

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze-heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.