Identification of a cryptic lethal mutation in the mouse t(w73) haplotype

t haplotypes are naturally occurring, variant forms of the t complex on mouse chromosome 17, characterized by the presence of four inversions with respect to wild-type. They harbour mutations causing male sterility, male transmission ratio distortion (TRD) and embryonic lethality. Mice carrying t haplotypes have been found throughout the world, and genetic studies of the lethal mutations have identified at least 16 complementation groups. The embryonic lethal phenotypes of many t haplotypes have been characterized in detail, and are thought to be the consequence of homozygosity for single gene mutations. However, the existence of additional mutations in genes that function at later stages of development would be obscured. Here we investigated the possibility of multiple mutations in t haplotypes by screening the t(w73) haplotype for the presence of novel mutations. Since genetic analysis of t haplotype mutations is hindered by recombination suppression due to the inversions, deletion complexes covering the proximal two-thirds of the t complex were used to uncover the presence of any new lethal alleles. This analysis revealed a novel mutation between D17Jcs41 and D17Mit100, causing mice carrying both t(w73) and selected deletions to die at birth, prior to feeding. The finding of a new, cryptic lethal mutation in t haplotypes is an indication that these recombinationally isolated chromosomes, which already contain at least one lethal mutation that prevents homozygosity, may serve as sinks for the accumulation of additional recessive mutations.     

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.     

Structural comparison of MTA phosphorylase and MTA/AdoHcy nucleosidase explains substrate preferences and identifies regions exploitable for inhibitor design

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.     

Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human beta-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human alpha-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 x 10(5) PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10-50 micro M), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.     

The matrilineal ancestry of Ashkenazi Jewry: portrait of a recent founder event

Both the extent and location of the maternal ancestral deme from which the Ashkenazi Jewry arose remain obscure. Here, using complete sequences of the maternally inherited mitochondrial DNA (mtDNA), we show that close to one-half of Ashkenazi Jews, estimated at 8,000,000 people, can be traced back to only 4 women carrying distinct mtDNAs that are virtually absent in other populations, with the important exception of low frequencies among non-Ashkenazi Jews. We conclude that four founding mtDNAs, likely of Near Eastern ancestry, underwent major expansion(s) in Europe within the past millennium.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

A review of surface roughness in antifouling coatings illustrating the importance of cutoff length

Surface roughness (SR) can affect the hydrodynamic performance of antifouling (AF) coatings and influence the settlement behaviour of fouling larvae, which makes it an important parameter in the evaluation of novel coatings. This paper reviews the causes and consequences of SR in the shipping industry, the methodology used for measuring it, and the importance of measuring and reporting it correctly. SR is a parameter that originates from marine engineering, but has been used extensively by marine scientists to characterise novel coatings and to investigate microtopographies that might inhibit settlement behaviour. One of the integral components of the SR measurement is the use of a cutoff filter. This is a short-pass filter that lets the high wave-number components through and thus separates the waviness from the roughness. Depending on the length of this filter, roughness at different levels of magnification can be investigated. Much of the published work on SR of AF coatings makes no mention of cutoff length, so that the results cannot be compared. It is suggested that an international standard is needed and that if more researchers were aware of the significance of stating cutoff length when reporting SR, more interdisciplinary work between biologists, engineers and material scientists would be possible in this field.     

Using the composite variables of reproductive morphology, histology and steroid hormones to determine age and size at sexual maturity for the thorny skate Amblyraja radiata in the western Gulf of Maine

Age at size was linked to sexual maturity in 77 male and 93 female thorny skates Amblyraja radiata from the western Gulf of Maine using three criteria: 1) gross reproductive morphology, 2) histology and 3) steroid hormone concentrations. Age-bias plots and the coefficient of variation from vertebral band counts suggested that the ageing method represented a non-biased and precise approach to the age assessment of A. radiata . Maturity ogives for males, based on data gathered for clasper length, circulating testosterone concentrations and proportion of mature spermatocysts within the testes, predicted that 50% maturity occurred at a total length ( L _T) of 865 mm and c. 10·90 years of age. For females, maturity ogives, based on data gathered for ovary mass, shell gland mass, follicle size and circulating oestradiol concentrations, predicted that 50% maturity occurred at 875 mm L _T and c. 11·00 years of age. Collectively, the results suggest that analysis of several contemporaneous reproductive variables offers an accurate determination of sexual maturity in the thorny skate.