Resistance to mutagenesis of cells biochemically transformed by herpesvirus DNA fragments

LM(TK-) mouse fibroblast cells that were biochemically transformed to the dThd kinase-positive phenotype by restriction nuclease fragments of herpes simplex virus or marmoset herpesvirus DNA, all of which contained the virus dThd kinase coding region, or by HeLa S3 DNA were more resistant to mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine or 5-bromodeoxyuridine than were dThd kinase-positive LM and LM(TK-) cells. Measurements of dNTP pool sizes did not reveal relative imbalances for representative cell lines under several conditions of growth.     

POSITIONAL AND SEASONAL VARIATION IN OAK (QUERCUS; FAGACEAE) LEAF MORPHOLOGY

Within-tree and seasonal variation in quantitative characters of oak leaves were evaluated by factorial analysis of variance. All linear and areal measurements illustrate marked within-tree and seasonal variation. Numbers of lobes and bristle tips and primary vein angle appear relatively stable within trees and among seasons. In many instances, size-correction reduces both within-tree and seasonal variance to nonsignificant levels. However, all characters do not illustrate the same trends either within trees or across the seasons. The results have important implications for those attempting to evaluate among-tree and among-population variation.     

Strabismus regulates asymmetric cell divisions and cell fate determination in the mouse brain

The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn–enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.     

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.     

The taxonomic value of fruit wing types in the order Apiales

This study shows that structural data, when carefully examined, can provide valuable characters for delimiting monophyletic groups and can complement DNA with observable features to recognize and circumscribe taxa. In the angiosperm order Apiales, traditional classification has relied heavily (often exclusively) on fruit characters. Recent molecular systematic studies, however, provided a radically different picture of relationships, calling into question the utility of fruit characters. We have studied fruit anatomy from 18 genera (Annesorhiza, Asteriscium, Astrotricha, Choritaenia, Dasispermum, Elaeoselinum, Heptaptera, Hermas, Heteromorpha, Laretia, Molopospermum, Myodocarpus, Pachypleurum, Peucedanum, Polemanniopsis, Polylophium, Rouya, and Tordylium) that represent all major taxonomic groups of Apiales characterized by winged fruits and the full range of wing types. Fruit anatomy closely corresponded with the phylogenetic position of these genera, as suggested by molecular studies. Fruit features of taxonomic importance include developmental origin of the wings, carpel shape, presence of vittae, woodiness of the endocarp, position of crystals, and type of carpophores. Despite the long history of recognizing umbellifers as a "natural group," few studies have been able to provide structural characters to help circumscribe the clades identified by molecular data. The interpretations presented are an important step toward erecting a stable system of classification for this difficult family.     

The bacterial metallome: composition and stability with specific reference to the anaerobic bacterium Desulfovibrio desulfuricans

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe₁Mn_0.3Zn_0.26Cu_0.03Co_0.03Mo_0.03. Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c ₃ and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.     

Islet Autoimmunity Identifies a Unique Pattern of Impaired Pancreatic Beta-Cell Function,Markedly Reduced Pancreatic Beta Cell Mass and Insulin Resistance in Clinically Diagnosed Type 2 Diabetes

There is a paucity of literature describing metabolic and histological data in adult-onset autoimmune diabetes. This subgroup of diabetes mellitus affects at least 5% of clinically diagnosed type 2 diabetic patients (T2DM) and it is termed Latent Autoimmune Diabetes in Adults (LADA). We evaluated indexes of insulin secretion, metabolic assessment, and pancreatic pathology in clinically diagnosed T2DM patients with and without the presence of humoral islet autoimmunity (Ab). A total of 18 patients with at least 5-year duration of clinically diagnosed T2DM were evaluated in this study. In those subjects we assessed acute insulin responses to arginine, a glucose clamp study, whole-body fat mass and fat-free mass. We have also analyzed the pancreatic pathology of 15 T2DM and 43 control cadaveric donors, using pancreatic tissue obtained from all the T2DM organ donors available from the nPOD network through December 31, 2013. The presence of islet Ab correlated with severely impaired β-cell function as demonstrated by remarkably low acute insulin response to arginine (AIR) when compared to that of the Ab negative group. Glucose clamp studies indicated that both Ab positive and Ab negative patients exhibited peripheral insulin resistance in a similar fashion. Pathology data from T2DM donors with Ab or the autoimmune diabetes associated DR3/DR4 allelic class II combination showed reduction in beta cell mass as well as presence of autoimmune-associated pattern A pathology in subjects with either islet autoantibodies or the DR3/DR4 genotype. In conclusion, we provide compelling evidence indicating that islet Ab positive long-term T2DM patients exhibit profound impairment of insulin secretion as well as reduced beta cell mass seemingly determined by an immune-mediated injury of pancreatic β-cells. Deciphering the mechanisms underlying beta cell destruction in this subset of diabetic patients may lead to the development of novel immunologic therapies aimed at halting the disease progression in its early stage.     

Isolation of a protein kinase induced by herpes simplex virus type 1

We have isolated a new cyclic AMP-independent protein kinase activity induced in HeLa cells by infection with herpes simplex virus type 1. Induction of the enzyme does not occur in cells treated with cycloheximide at the time of infection, or in cells infected with UV-inactivated herpes simplex virus type 1. The amount of enzyme induced in infected cells is dependent upon the multiplicity of infection. An enzyme with identical properties to the appearing in infected HeLa cells is also induced by herpes simplex virus type 1 in BHK cells.