Isolation of viral coat protein mutants with altered assembly and aggregation properties

A method was developed to screen bacteria for synthesis of mutant proteins with altered assembly and solubility properties using bacteriophage MS2 coat protein as a model self-associating protein. Colonies expressing coat protein from a plasmid were covered with an agarose overlay under conditions that caused the lysis of some of the cells in each colony. The proteins thus liberated diffused through the overlay at rates depending on their molecular sizes. After transfer of the proteins to a nitrocellulose membrane, probing with coat protein-specific antiserum revealed spots whose sizes and intensities were related to the aggregation state of coat protein. The method was employed in the isolation of assembly defective mutants and to find soluble variants of an aggregation-prone coat protein mutant.     

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.     

Mass characteristics of DNA obtained from chromosomes of a human carcinoma cell line

Sedimentation studies of DNA from chromosomes extracted from human mitotic cells showed that highmolecular-weight DNA can be obtained if cell hypotonic treatments and prolonged metaphase blocks are avoided. Two types of large double-stranded DNA were observed. One of these (M _r = 2.5×10⁸) appeared as a size class with characteristics reminiscent of the chromosomal DNA subunit hypothesis. However, this DNA is the decay product of larger molecules, whose minimum molecular weight is 6×10⁸.     

Human HepG2 and Rat Fao Hepatic-Derived Cell Lines Show Different Responses to Ciprofibrate, a Peroxisome Proliferator: Analysis by Flow Cytometry

Peroxisome proliferators, and especially hypolipidemic drugs such as ciprofibrate, are known to be hepatocarcinogens in rodents, but their effect in humans is controversial. In an attempt to investigate the effects of ciprofibrate at a cellular level, the analysis of individual whole cells was performed by flow cytometry on samples from two hepatic-derived cell lines: the rat Fao cell line and the human HepG2 cell line. The increase of light scatter signals in rat Fao cells treated for 3 days with ciprofibrate at 250 μMwas related to modifications of intrinsic cellular parameters, such as size and cytoplasmic granularity. Conversely, no variations appeared in human HepG2-treated cells. Moreover, the study of the cell cycle distribution of asynchronously growing cells showed an increase in the percentage of proliferative cells in Fao-treated cells, but not in HepG2-treated cells. In order to give a simultaneous assessment of changes in cellular parameters and cell metabolism, these flow cytometric experiments were completed with the measurements of the palmitoyl–CoA oxidase activity, used as a marker of peroxisome proliferation. The cellular modifications in the rat Fao cell line were accompanied by a great increase in this enzymatic activity, whereas the human HepG2 cell line, which failed to exhibit changes of cytometric data, presented no, or weak, increase in this oxidase activity. The cellular modifications observed in the rat Fao cell line may be related to the well-known hepatocarcinogenicity of ciprofibrate in rodents, whereas the absence of response of HepG2 cells is in favor of the noncarcinogenicity of this drug in humans. This report validates another methodological approach for the investigation of the safety of peroxisome proliferators in humans.     

The empirical question of thresholds and mechanisms of mate choice

Summary Theoretical discussions concerning how animals might best sample and select mates have suggested that individuals could base decisions either on a sample of mates (sampled-based decisions) or on a threshold of comparison (threshold-based decisions). Recent theoretical work demonstrates that threshold-based mating decisions generate higher expected fitness than sample-based mating decisions when search costs exist. Empirical results from most unmanipulated systems, however, either conclude that females make sample-based decisions or are inconclusive. A few experimental studies designed to detect mating thresholds purport to demonstrate threshold-based choice but an examination of these studies indicates such conclusions were premature. We believe that few examples of threshold-based choice exist because protocols designed to identify mating thresholds were often inconsistent with models of threshold choice. We suggest that future empirical work strive not to document mating thresholdsper se. Rather, future work might best reveal decision rules by manipulating the distribution of quality among potential mates; such manipulations predict uniquely how females using sample-based and threshold-based decision rules should behave.     

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota)

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac 1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Acl, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Acl, 3, 4. Correspondence to: D. Bhattacharya     

Observations of the mating behavior and dentition of the round stingray,Urolophus halleri

Synopsis The mating behavior and dentition ofUrolophus halleri, the round stingray was examined. Males frequently bite females during the mating period but most male biting does not result in copulation. In bites that do not lead to copulation, males bite the posterior (or occasionally the medial) portion of the females' disc but females often free themselves from these bites. In bites that precede copulation, males bite the anterior portion of the females' disc and females do not struggle to free themselves. Thus, females may exert some form of choice when they are bitten. Mature males have sexually dimorphic dentition that may aid in holding females. A principal component analysis (PCA) showed that in juvenile males, the relative size of the teeth decrease while the relative thickness of the disc increases as body size enlarges; adult males displayed no clear pattern. In adult females, there is a relative decrease in tooth size and increase in relative disc thickness as body size enlarges. The relative increase in females disc thickness in areas where they are bitten may function to minimize the amount of damage due to non-copulatory biting. There is no indication that biting functions to induce female receptivity but it may allow females and males to acquire information about potential mates. Thus, copulatory biting functions to maintain contact during copulation while the function of non-copulatory biting is less clear.     

The choline transporter resurfaces: new roles for synaptic vesicles?

Presynaptic choline uptake is vital to sustained neuronal acetylcholine (ACh) release; however, only with the recent cloning of choline transporters (CHTs) (i.e., SLC5A7), has a picture emerged of the regulatory pathways supporting CHT modulation. Studies arising from the development of CHT-specific antibodies reveal a large, intracellular reserve of CHT proteins, localized to ACh-containing, synaptic vesicles. The intersection of mechanisms supporting vesicular ACh release and choline uptake demonstrates an elegant mechanism for linking regulation of CHT membrane density to rates of ACh release. Furthermore, these studies point to control of the CHT endocytic process as an important target for novel therapeutics that could offset functional deficits in disorders bearing diminished cholinergic tone, including myasthenias and dementias.