Minimal cross-recombination between wild-type and loxP511 sites in vivo facilitates truncating both ends of large DNA inserts in pBACe3.6 and related vectors

Contrary to several earlier reports, we find that cross-recombination between wild-type and the mutant loxP511 sites is <0.5% of that between two wild-type sites if Cre protein is expressed by phage P1 during an infection. The finding enabled us to develop a procedure to truncate DNA progressively from both ends of large genomic inserts flanked by these two loxP sites in pBACe3.6 and related vectors with transposons carrying either a wild-type or a loxP511 sequence. Newly constructed loxP511 transposons contained either a kanamycin resistance gene or no marker. Insert DNA ends in deletions were sequenced with primers unique to each transposon-end remaining after the respective recombination. End-sequencing 223 deletions confirmed that the low level of cross-recombination, observed between those sites during the P1 transductions, does not complicate the procedure: truncations from the unintended end of genomic inserts did not occur. Multiple BACs pooled together could also be processed in a single tube to make end-deletions. This deletion technology, utilizing the very minimal cross-recombination between the mutant and wild-type loxP sites of most BAC clones in the public domain and a heterologous one inserted as a transposon, should facilitate functionally mapping long-range gene regulatory sequences and help to isolate genes with defined functional boundaries in numerous projects including those of therapeutic interest.     

Contractile abilities of normal and "mini" triceps surae muscles from mice (Mus domesticus) selectively bred for high voluntary wheel running.

As reported previously, artificial selection of house mice caused a 2.7-fold increase in voluntary wheel running of four replicate selected lines compared with four random-bred control lines. Two of the selected lines developed a high incidence of a small-muscle phenotype ("mini muscles") in the plantar flexor group of the hindlimb, which apparently results from a simple Mendelian recessive allele. At generations 36-38, we measured wheel running and key contractile characteristics of soleus and medial gastrocnemius muscles from normal and mini muscles in mice from these selected lines. Mice with mini muscles ran faster and a greater distance per day than normal individuals but not longer. As expected, in mini-muscle mice the medial and lateral gastrocnemius muscles were approximately 54 and 45% the mass of normal muscles, respectively, but the plantaris muscles were not different in mass and soleus muscles were actually 30% larger. In spite of the increased mass, contractile characteristics of the soleus were unchanged in any notable way between mini and normal mice. However, medial gastrocnemius muscles in mini mice were changed markedly toward a slower phenotype, having slower twitches; demonstrated a more curved force-velocity relationship; produced about half the mass-specific isotonic power, 20-50% of the mass-specific cyclic work and power (only 10-25% the absolute power if the loss in mass is considered); and fatigued at about half the rate of normal muscles. These changes would promote increased, aerobically supported running activity but may compromise activities that require high power, such as sprinting.     

Developmental regulation of skull morphology II: ontogenetic dynamics of covariance

Canalization may play a critical role in molding patterns of integration when variability is regulated by the balance between processes that generate and remove variation. Under these conditions, the interaction among those processes may produce a dynamic structure of integration even when the level of variability is constant. To determine whether the constancy of variance in skull shape throughout most of postnatal growth results from a balance between processes generating and removing variation, we compare covariance structures from age to age in two rodent species, cotton rats (Sigmodon fulviventer) and house mice (Mus musculus domesticus). We assess the overall similarity of covariance matrices by the matrix correlation, and compare the structures of covariance matrices using common subspace analysis, a method related to common principal components (PCs) analysis but suited to cases in which variation is so nearly spherical that PCs are ambiguous. We find significant differences from age to age in covariance structure and the more effectively canalized ones tend to be least stable in covariance structure. We find no evidence that canalization gradually and preferentially removes deviations arising early in development as we might expect if canalization results from compensatory differential growth. Our results suggest that (co)variation patterns are continually restructured by processes that equilibrate variance, and thus that canalization plays a critical role in molding patterns of integration.     

Statins and Selective Inhibition of Rho Kinase Protect Small Conductance Calcium-Activated Potassium Channel Function (KCa2.3) in Cerebral Arteries

Background

In rat middle cerebral and mesenteric arteries the K_Ca2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, K_Ca2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain K_Ca2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA).

Methods

MCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence.

Results

Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of K_Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. K_Ca2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation.

Conclusions

Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K_Ca2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of K_Ca2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.     

Fine mapping of "mini-muscle," a recessive mutation causing reduced hindlimb muscle mass in mice

Prolonged selective breeding of Hsd:ICR mice for high levels of voluntary wheel running has favored an unusual phenotype (mini-muscle [MM]), apparently caused by a single Mendelian recessive allele, in which hindlimb muscle mass is reduced by almost 50%. We recently described the creation and phenotypic characterization of a population suitable for mapping the genomic location of the MM gene. Specifically, we crossed females from a high-runner line fixed for the MM allele with male C57BL/6J. F1 males were then backcrossed to the MM parent females. Backcross (BC) mice exhibited a 50:50 ratio of normal to MM phenotypes. Here, we report on linkage mapping of MM in this BC population to a 2.6335-Mb interval on MMU11. This region harbors approximately 100 expressed or predicted genes, many of which have known roles in muscle development and/or function. Identification of the genetic variation that underlies MM could potentially be very important in understanding both normal muscle function and disregulation of muscle physiology leading to disease.     

The biology and future prospects of antivirulence therapies

The emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics demand the discovery of new therapeutic approaches. Targeting bacterial virulence is an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium. Certain virulence factors have been shown to be potential targets for drug design and therapeutic intervention, whereas new insights are crucial for exploiting others. Targeting virulence represents a new paradigm to empower the clinician to prevent and treat infectious diseases.     

Biodegradation of polyalcohol ethoxylate by a wastewater microbial consortium

Polyalcohol ethoxylate (PAE), an anionic surfactant, is the primary component in most laundry and dish wash detergents and is therefore highly loaded in domestic wastewater. Its biodegradation results in the formation of several metabolites and the fate of these metabolites through wastewater treatment plants, graywater recycling processes, and in the environment must be clearly understood. Biodegradation pathways for PAE were investigated in this project with a municipal wastewater microbial consortium. A microtiter-based oxygen sensor system was utilized to determine the preferential use of potential biodegradation products. Results show that while polyethylene glycols (PEGs) were readily degraded by PAE acclimated microorganisms, most of the carboxylic acids tested were not degraded. Biodegradation of PEGs suggests that hydrophobe–hydrophile scission was the dominant pathway for PAE biodegradation in this wastewater community. Ethylene glycol (EG) and diethylene glycol (DEG) were not utilized by microbial populations capable of degrading higher molecular weight EGs. It is possible that EG and DEG may accumulate. The microtiter-based oxygen sensor system was successfully utilized to elucidate information on PAE biodegradation pathways and could be applied to study biodegradation pathways for other important contaminants.     

The early years--across the bench from Bruce (1963-1966)

This personal reflection on the author's experience as Bruce Merrifield's first graduate student has been adapted from a talk given at the Merrifield Memorial Symposium at the Rockefeller University on November 13, 2006.     

Back to the future: ribonuclease A

Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best-studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and synthesized in the laboratory of Bruce Merrifield, all at the Rockefeller Institute/University. It has proven to be an excellent model system for many different types of experiments, both as an enzyme and as a well-characterized protein for biophysical studies. Of major significance was the demonstration by Chris Anfinsen at NIH that the primary sequence of RNase encoded the three-dimensional structure of the enzyme. Many other prominent protein chemists/enzymologists have utilized RNase as a dominant theme in their research. In this review, the history of RNase and its offspring, RNase S (S-protein/S-peptide), will be considered, especially the work in the Merrifield group, as a preface to preliminary data and proposed experiments addressing topics of current interest. These include entropy-enthalpy compensation, entropy of ligand binding, the impact of protein modification on thermal stability, and the role of protein dynamics in enzyme action. In continuing to use RNase as a prototypical enzyme, we stand on the shoulders of the giants of protein chemistry to survey the future.