Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Older males signal more reliably

The hypothesis that females prefer older males because they have higher mean fitness than younger males has been the centre of recent controversy. These discussions have focused on the success of a female who prefers males of a particular age class when age cues, but not quality cues, are available. Thus, if the distribution of male quality changes with age, such that older males have on average genotypes with higher fitness than younger males, then a female who mates with older males has fitter offspring, which allows the female preference to spread through a genetic correlation. We develop a general model for male display in a species with multiple reproductive bouts that allows us to identify the conditions that promote reliable signalling within an age class. Because males have opportunities for future reproduction, they will reduce their levels of advertising compared with a semelparous species. In addition, because higher-quality males have more future reproduction, they will reduce their advertising more than low-quality males. Thus, the conditions for reliable signalling in a semelparous organism are generally not sufficient to produce reliable signalling in species with multiple reproductive bouts. This result is due to the possibility of future reproduction so that, as individuals age and the opportunities for future reproduction fade, signalling becomes more reliable. This provides a novel rationale for female preference for older mates; older males reveal more information in their sexual displays.     

The evolution of virulence in vector-borne and directly transmitted parasites

Ewald (1994) has suggested that vector-borne parasites are expected to evolve a higher level of host exploitation than directly transmitted parasites, and this should thereby result in them being more virulent. Indeed, some data do conform to this general pattern. Nevertheless, his hypothesis has generated some debate about the extent to which it is valid. I explore this issue quantitatively within the framework of mathematical epidemiology. In particular, I present a dynamic optimization model for the evolution of parasite replication strategies that explicitly explores the validity of this hypothesis. A few different model assumptions are explored and it is found that Ewald's hypothesis has only qualified support as a general explanation for why vector-borne parasites are more virulent than those that are directly transmitted. I conclude by suggesting that an alternative explanation might lie in differences in inoculum size between these two types of transmission.     

Normal conjunctival flora in the North American opossum (Didelphis virginiana) and raccoon (Procyon lotor)

We documented the normal conjunctival bacterial flora from 17 opossums (Didelphis virginiana) and 10 raccoons (Procyon lotor) trapped in Manhattan, Kansas (USA) from November 1999 to January 2000. Both raccoons and opossums were free of apparent ocular disease. The inferior conjunctival sacs of each animal were swabbed for aerobic bacterial and Mycoplasma culture and polymerase chain reaction (PCR) for Mycoplasma and Chlamydia detection. All conjunctival samples were positive for one or more species of aerobic bacteria. The most common isolate from opossums was Staphylococcus spp. Other isolates included Streptococcus spp., Bacillus spp., Corynebacterium spp., and Enterococcus faecalis. The most common isolates in raccoons was Bacillus spp. Other isolates included Streptococcus spp., Staphylococcus spp., non-hemolytic Escherichia coli, and Enterococcus faecalis. Mycoplasma culture was negative in samples from opossums and raccoons. Evidence of Mycoplasma and Chlamydia presence was detected by PCR.     

Free tissue coverage of chronic traumatic wounds of the lower leg

Thirty-eight consecutive patients who underwent 42 free flaps for chronic wounds of the lower leg were identified over an 11-year period. All wounds were open for a minimum of 1 month (mean, 40 months; median, 8 months; range, 1 month to 30 years). The average age was 37 years (range, 7 to 68 years), there were 31 male patients and seven female patients, and the average follow-up time was 30 months (range, 12 to 72 months). The original injury was an open fracture in 28 patients, wound dehiscence after open reduction and internal fixation of a closed fracture in nine patients, and a shrapnel wound in one patient. A total of 23 patients had osteomyelitis, which was classified as local (involving less than 50 percent of the bone diameter) in 15 patients and as diffuse (involving greater than 50 percent of the bone diameter or infected nonunion) in eight patients. The wounds were treated with sequential debridement, antibiotics, and flap coverage. Ancillary procedures included antibiotic beads in 18 patients, saucerization in 16, Ilizarov bone transport in three, calcanectomy in two, and fibular resection and ankle fusion in one. Thirty-four of 42 flaps survived, four having undergone a repeat free flap. There were three failures out of 25 flaps (12 percent) among those with a normal angiogram and five failures out of 15 flaps (33 percent) among those with an abnormal angiogram (p > 0.05). The failure rate of those with osteomyelitis was six of 26 (23 percent) versus two of 26 (13 percent) for those without osteomyelitis (p > 0.05). Successful reconstruction (bone healed, patient ambulatory and infection-free) was achieved in 33 of 38 patients (87 percent). The failure of reconstruction for those patients with osteomyelitis was four of 23 (22 percent) versus one of 15 (7 percent) for others (p > 0.05). The failure rate of flaps in patients with diffuse osteomyelitis was three of eight (38 percent) versus two of 30 for others (7 percent, p = 0.053). The presence of diffuse osteomyelitis was associated with a lower rate of successful limb reconstruction. An abnormal angiogram and the presence of osteomyelitis both were associated with a lower rate of successful limb reconstruction, but this was not significant, probably because of the small size of the cohort.     

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.     

Germinal vesicle material is essential for nucleus remodeling after nuclear transfer

Successful cloning by nuclear transfer has been reported with somatic or embryonic stem (ES) cell nucleus injection into enucleated mouse metaphase II oocytes. In this study, we enucleated mouse oocytes at the germinal vesicle (GV) or pro-metaphase I (pro-MI) stage and cultured the cytoplasm to the MII stage. Nuclei from cells of the R1 ES cell line were injected into both types of cytoplasm to evaluate developmental potential of resulting embryos compared to MII cytoplasmic injection. Immunocytochemical staining revealed that a spindle started to organize 30 min after nucleus injection into all three types of cytoplasm. A well-organized bipolar spindle resembling an MII spindle was present in both pro-MI and MII cytoplasm 1 h after injection with ES cells. However, in the mature GV cytoplasm, chromosomes were distributed throughout the cytoplasm and a much bigger spindle was formed. Pseudopronucleus formation was observed in pro-MI and MII cytoplasm after activation treatment. Although no pronucleus formation was found in GV cytoplasm, chromosomes segregated into two groups in response to activation. Only 8.1% of reconstructed embryos with pro-MI cytoplasm developed to the morula stage after culture in CZB medium. In contrast, 53.5% of embryos reconstructed with MII cytoplasm developed to the morula/blastocyst stage, and 5.3% of transferred embryos developed to term. These results indicate that GV material is essential for nucleus remodeling after nuclear transfer.