Genetic divergence and evolutionary instability in ospE-related members of the upstream homology box gene family in Borrelia burgdorferi sensu lato complex isolates

A series of related genes that are flanked at their 5' ends by a conserved upstream sequence element called the upstream homology box (UHB) have been identified in Borrelia burgdorferi. These genes have been referred to as the UHB or erp gene family. We previously demonstrated that among a limited number of B. burgdorferi isolates, the UHB gene family is variable in composition and organization. Prior to this report the UHB gene family in other species of the B. burgdorferi sensu lato complex had not been studied, and if this family is important in the pathogenesis or biology of the Lyme disease spirochetes, then a wide distribution among species and isolates of the B. burgdorferi sensu lato complex would be expected. To assess this, we screened for the UHB element by Southern hybridization and determined its restriction fragment length polymorphism (RFLP) patterns. The UHB element was found to be carried by all B. burgdorferi sensu lato complex species tested (B. burgdorferi, B. garinii, B. afzelii, B. japonica, B. valaisiana sp. nov., and B. andersonii), but the RFLP patterns varied widely at both the inter- and intraspecies levels. Variation in both the number and size of the hybridizing restriction fragments was evident. PCR analyses also revealed the presence of polymorphic, ospE-related alleles in many isolates. Sequence analyses identified the molecular basis of the polymorphisms as being primarily insertions and deletions. Sequence variation and the insertions and deletions were found to be clustered in two distinct domains (variable domains 1 and 2). In many isolates variable domain 1 is flanked by direct repeat elements, some as long as 38 bp. Computer analyses of the deduced amino acid sequences encoded within variable domain 1 predict them to be hydrophilic, surface exposed, and antigenic. The analyses conducted here suggest that the UHB gene family, as evidenced by the variable UHB RFLP patterns, is not evolutionarily stable and that the polymorphic ospE alleles are derived from a common ancestral gene which has been modified through mutation or recombination events. The characterization of ospE-related genes of the UHB gene family among B. burgdorferi sensu lato species will prove important in attempts to construct a model for UHB gene family organization and in deciphering the role of the UHB gene family in the biology and pathogenesis of the Lyme disease spirochetes.     

Comparison of different oxygen exchange models

A functional distribution of coronary volume can be estimated from the response of arterio-venous O2 content difference (AVO2) to a flow step. However, the results depend on the assumed O2 exchange model. The previously used model consisted of a single mixed compartment with O2 exchange (reference model). The purpose of this study is to provide an estimate of the errors made in the volume estimations by not taking into account factors as flow heterogeneity, different mixing sites or Krogh-like O2 exchange. The approach is indirect: the response of the AVO2 to a flow step has been calculated with alternative O2 exchange models in which factors mentioned are incorporated. These transients are fitted with the reference model. The resulting estimated volumes are different from the volumes assumed in the alternative models. Large differences are obtained with some of the alternative models, e.g. the model with Krogh characteristics. However, these models seem unrealistic because capillary pO2 is higher than venous pO2. Only small differences in volume are obtained with the more realistic models. Therefore, these results indicate that the coronary volumes are approximated well by the estimations obtained with the reference model. These volume estimations were 9.9 and 3.8 ml 100 g-1 for the O2 exchange vessels and the distal venous volume, respectively.     

Temporal and spatial expression of erbB4 in ectodermal and mesenchymal cells during primary palatogenesis in noncleft and cleft strains of mice

Primary palatogenesis in mice is similar to that in humans, and spontaneous cleft lip appears to be multifactorially determined in both. Binding of a ligand to erbB4 has been shown to stimulate the receptor's protein kinase activity, which subsequently stimulates a signal-transduction cascade leading to cell growth and differentiation, and to morphogenesis during development. In this study, an immunohistochemical technique was used to investigate the temporal and spatial expression of erbB4 in the primary palate of cleft (A/WySn) and noncleft strains of mice (BALB/cBy). Positive staining of erbB4 was found in ectodermal and mesenchymal cells of facial prominences before the primary palate formation stage (day 10, hour 20) in both strains. During the primary palate formation stage (day 11, hour 20), positive staining of erbB4 was found in the ectodermal and mesenchymal cells of the facial prominences of the noncleft strain, but not in those of the cleft strain. These results suggest erbB4 expression may be associated with normal primary palatogenesis of mice and, conversely, cleft lip may be associated with a deficiency of erbB4 expression during primary palate formation in mice.     

Intracellular maturation of the mouse metalloprotease disintegrin MDC15

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.     

Basilar membrane vibration in the gerbil hemicochlea

Excised gerbil cochleae were cut along the mid-modiolar plane (hemicochlea). Along one-half turn of this preparation, fluorescent microbeads were placed on the basilar membrane (BM). The BM was vibrated with click stimuli (50 micros) produced mechanically by a piezo pusher. The stimulus delivery probe could be positioned either more apical or more basal from the beads. Vibration patterns were measured with a wide bandwidth photomultiplier from the movements of the beads. When the probe was positioned more basal, the responses to click stimuli were brief, damped sinusoids. According to the fast Fourier transforms (FFTs) of the averaged time wave forms, the best frequency between successive beads decreased toward the apex (0.8 octave/mm). Sharpness of tuning of the normalized FFT spectra (NQ10dB) on average was 1.5. Response amplitude at a fixed input level, measured at different beads away from the stimulation site, dropped exponentially (58 dB/mm). In addition, for each individual bead, amplitude dropped linearly with decreasing stimulus intensity. In experiments where the stimulating probe was placed more apical, two major properties were observed: first, beads revealed only the spectral components present in the motion of the probe. Second, magnitude reduction of the displacement of the cochlear partition was greater, on average 155 dB/mm, indicating a lack of significant propagation in the reverse direction.     

A Ca2+-activated whole-cell Cl- conductance in human placental cytotrophoblast cells activated via a G protein

Evidence now suggests that some people can exert some degree of control over seizure initiation and inhibition. In order to explore this further, 79 young people with epilepsy, attending specialist residential schools, were interviewed regarding awareness of seizure precipitants; recognition of seizure warnings; attempts at seizure inhibition, and ways of self-inducing seizures. Questionnaires with the same content were completed by residential care staff. Results show that many subjects claimed to identify seizure precipitants (63.3%), experienced warnings (70.9%), and had developed means of trying to inhibit seizure occurrence (50.6%). However, in each instance, staff reports were much lower (56.9%, 47.2%, and 22.2%, respectively), and one-to-one concordance was poor. A larger than expected percentage of self-induction was reported for both subjects (8.9%) and staff (9.7%). The implication of these results for both the investigation and treatment of epilepsy are discussed further.     

Crystal structure of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme Rad6 at 2.6 A resolution

The Saccharomyces cerevisiae ubiquitin-conjugating enzyme (UBC) Rad6 is required for several functions, including the repair of UV damaged DNA, damage-induced mutagenesis, sporulation, and the degradation of cellular proteins that possess destabilizing N-terminal residues. Rad6 mediates its role in N-end rule-dependent protein degradation via interaction with the ubiquitin-protein ligase Ubr1 and in DNA repair via interactions with the DNA binding protein Rad18. We report here the crystal structure of Rad6 refined at 2.6 A resolution to an R factor of 21.3%. The protein adopts an alpha/beta fold that is very similar to other UBC structures. An apparent difference at the functionally important first helix, however, has prompted a reassessment of previously reported structures. The active site cysteine lies in a cleft formed by a coil region that includes the 310 helix and a loop that is in different conformations for the three molecules in the asymmetric unit. Residues important for Rad6 interaction with Ubr1 and Rad18 are on the opposite side of the structure from the active site, indicating that this part of the UBC surface participates in protein-protein interactions that define Rad6 substrate specificity.     

Hypertensive cardiac hypertrophy--is genetic variance the missing link?

1. Hypertensive cardiac hypertrophy is a major independent predictor of adverse cardiovascular events. In man the cardiac response to increased afterload is very variable, even when ambulatory blood pressure monitoring is used. Analysis of breeding experiments using normotensive and hypertensive rat strains, human twin studies and other data indicate that genetic factors play a significant role in regulating cardiac mass; in other words, a large component of total variability is accounted for by genetic variance. 2. The observation that some patients with only mild-to-moderate hypertension exhibit gross left ventricular hypertrophy (LVH) similar to the inherited hypertrophic cardiomyopathies such as familial hypertrophic cardiomyopathy (FHC) and Friedreich's ataxia (FA) has prompted us to investigate the hypothesis that genetic factors associated with excessive myocardial hypertrophy, viz. mutations in FHC and FA genes alter the hypertrophic response of the heart to pressure overload. Here we review briefly three lines of study: (i) association analysis to test whether the allele frequencies differ in hypertensive patients with or without left ventricular hypertrophy; (ii) characterization of the cardiac manifestations of FA to understand the mechanism by which the heart is affected in a disease associated with pathology in a subgroup of neurons, and (iii) creation of transgenic models to facilitate the investigation of the interaction between hypertrophic stimuli and underlying genetic predisposition. 3. Information on the nature of the cardiac-mass-modifying genes involved may be useful not only for selecting high risk patients in strategies aimed at preventing the development of LVH, but also in opening new avenues of research on the reprogramming of cardiac myocytes to encourage them to hypertrophy in situations where cardiac muscle has been damaged or is hypoplastic.     

Specification of anteroposterior cell fates in Caenorhabditis elegans by Drosophila Hox proteins

Antennapedia class homeobox (Hox) genes specify cell fates in successive anteroposterior body domains in vertebrates, insects and nematodes. The DNA-binding homeodomain sequences are very similar between vertebrate and Drosophila Hox proteins, and this similarity allows vertebrate Hox proteins to function in Drosophila. In contrast, the Caenorhabditis elegans homeodomains are substantially divergent. Further, C. elegans differs from both insects and vertebrates in having a non-segmented body as well as a distinctive mode of development that involves asymmetric early cleavages and invariant cell lineages. Here we report that, despite these differences, Drosophila Hox proteins expressed in C. elegans can substitute for C. elegans Hox proteins in the control of three different cell-fate decisions: the regulation of cell migration, the specification of serotonergic neurons, and the specification of a sensory structure. We also show that the specificity of one C. elegans Hox protein is partly determined by two amino acids that have been implicated in sequence-specific DNA binding. Together these findings suggest that factors important for target recognition by specific Hox proteins have been conserved throughout much of the animal kingdom.