Rapid engineering of the geldanamycin biosynthesis pathway by Red/ET recombination and gene complementation

Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.     

PIV measurements of flow in a centrifugal blood pump: time-varying flow

Measurements of the time-varying flow in a centrifugal blood pump operating as a left ventricular assist device (LVAD) are presented. This includes changes in both the pump flow rate as a function of the left ventricle contraction and the interaction of the rotating impeller and fixed exit volute. When operating with a pulsing ventricle, the flow rate through the LVAD varies from 0-11 L/min during each cycle of the heartbeat. Phase-averaged measurements of mean velocity and some turbulence statistics within several regions of the pump, including the inlet, blade passage, exit volute, and diffuser, are reported at 20 phases of the cardiac cycle. The transient flow fields are compared to the constant flow rate condition that was reported previously in order to investigate the transient effects within the pump. It is shown that the quasi-steady assumption is a fair treatment of the time varying flow field in all regions of this representative pump, which greatly simplifies the comprehension and modeling of this flow field. The measurements are further interpreted to identify the effects that the transient nature of the flow field will have on blood damage. Although regions of recirculation and stagnant flow exist at some phases of the cardiac cycle, there is no location where flow is stagnant during the entire heartbeat.     

Genetic correlations do not constrain the evolution of sexual dimorphism in the moss Ceratodon purpureus

The trajectory of phenotypic evolution is constrained in the short term by genetic correlations among traits. However, the extent to which genetic correlations impose a lasting constraint is generally unknown. Here, I examine the genetic architecture of life-history variation in male and female gametophytes from two populations of the moss Ceratodon purpureus, focusing on genetic correlations within and between the sexes. A significant negative correlation between allocation to vegetative and reproductive tissue was evident in males of both populations, but not females. All traits showed between-sex correlations of significantly less than one, indicating additive genetic variance for sexual dimorphism. The degree of dimorphism for traits was significantly negatively associated with the strength of the between-sex correlation. The structure of genetic correlations among life-history traits was more divergent between the two populations in females than in males. Collectively, these results suggest that genetic correlations do not impose a lasting constraint on the evolution of life-history variation in the species.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.     

Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation

In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.     

Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion

Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.     

A threefold genetic allee effect: population size affects cross-compatibility, inbreeding depression and drift load in the self-incompatible Ranunculus reptans

A decline in population size can lead to the loss of allelic variation, increased inbreeding, and the accumulation of genetic load through drift. We estimated the fitness consequences of these processes in offspring of controlled within-population crosses from 13 populations of the self-incompatible, clonal plant Ranunculus reptans. We used allozyme allelic richness as a proxy for long-term population size, which was positively correlated with current population size. Crosses between plants of smaller populations were less likely to be compatible. Inbreeding load, assessed as the slope of the relationship between offspring performance and parental kinship coefficients, was not related to population size, suggesting that deleterious mutations had not been purged from small populations. Offspring from smaller populations were on average more inbred, so inbreeding depression in clonal fitness was higher in small populations. We estimated variation in drift load from the mean fitness of outbred offspring and found enhanced drift load affecting female fertility within small populations. We conclude that self-incompatibility systems do not necessarily prevent small populations from suffering from inbreeding depression and drift load and may exacerbate the challenge of finding suitable mates.     

Bryophyte dispersal inferred from colonization of an introduced substratum on Whiteface Mountain, New York

A long-standing debate in bryophyte biogeography concerns the frequency of long-distance spore dispersal. The diversity of bryophytes on mortared rock walls along the Veterans Memorial Highway on Whiteface Mountain, New York, USA, was studied to document the recruitment of species over the 65 years since the highway was constructed. The highway is situated in the Adirondack Mountains, a relatively unpopulated region with a largely acidic flora. The introduction of mortar has increased the bryophyte diversity by 50% above that of native lithic substrata on the mountain. The composition of the native and mortar floras differed greatly, suggesting that the walls were not colonized by locally abundant ruderal species. Many of the species sampled on the walls are typically found only in lower elevation forested sites, distant (～5 km or more) from the highway, and not on anthropogenic calcium carbonate. These results suggest that a bryophyte community consisting of common and uncommon species assembled from distant sites at the rate of at least one species per year in the last 65 years. These data provide the ecological context for experimental and phylogeographic studies and suggest that some bryophytes may be capable of routine dispersal over distances of at least 5 km.