On the role of small mammals in mediating climatically driven vegetation change

Biotic and abiotic processes jointly influence natural systems, yet opportunities to integrate studies of both processes are uncommon. For two decades we have excluded different subsets of the small mammal community from a series of plots near a grassland-desert ecotone in the northern Chihuahuan Desert. These studies spanned a period of historically high winter rainfall, allowing us to distinguish the effects of climate and small mammals on the composition and patch structure of vegetation. Removal of only kangaroo rats ( Dipodomys ) or of all small mammals led to increased cover of large herbaceous vegetation. The size of vegetative patches increased in all plots but this increase was three times greater where all rodents were removed. Thus, the activity of small mammals that forage under and near shrub canopies appear to significantly inhibit the expansion of existing vegetative patches, and may have a stronger influence on habitat structure than previously recognized.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

Evolution of the WANCY region in amniote mitochondrial DNA

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.      

Stephanodiscus kusuensis, sp. nov a new Pleistocene diatom from Southern Japan

A new fossil diatom, Stephanodiscus kusuensis sp. nov., is described from a diatomite deposit from the Kusu Basin, Oita Prefecture Japan. Radiometric age estimates of the strata in which the taxon occurs are 400–500 kyr. The new species is distinct from all other known species of Stephanodiscus because it possesses a highly pronounced double concentric undulation and two distinct arrangements of central strutted processes. Cladistic analysis is used to identify the phylogenetic position of this taxon with respect to other Stephanodiscus species.     

Monoclonal antibody that blocks phosphoinositide-dependent activation of mouse tumor DNA polymerase alpha

A monoclonal antibody (MaB) against mouse sarcoma DNA polymerase alpha was isolated from the culture medium of an IgG-secreting hybridoma. The MaB demonstrated reactivity against two murine DNA polymerase alpha preparations and a calf thymus DNA polymerase alpha. Murine sarcoma polymerase was activated in vitro by phosphatidylinositol-4-monophosphate (PIP) showing increased deoxynucleotidyltransferase activity and enhanced binding affinity to activated DNA template. The MaB did not neutralize polymerase activity, but blocked further activation of the enzyme by PIP. Treatment of polymerase with MaB prior to treatment with PIP inhibited both increased enzyme activation and increased binding of the enzyme to DNA template. Treatment of polymerase with MaB subsequent to treatment with PIP did not block enzyme activation or increased DNA template binding. The data suggest that this anti-DNA polymerase alpha IgG is directed against a regulatory subunit of the polymerase rather than the catalytic subunit. The antibody may serve to distinguish between DNA polymerase alpha preparations with distinctly different regulatory subunits.     

Need for open access non-screening mammography in a hospital with a specialist breast clinic service.

OBJECTIVE--Assessment of open access non-screening mammography in a hospital with a breast clinic. DESIGN--Retrospective analysis of patients sent for first mammogram to our open access service by general practitioners and breast clinic in the year April 1989 to March 1990. SETTING--District general hospital serving 200,000 people before the introduction of breast screening. SUBJECTS--361 symptomatic women referred directly by general practitioners and 226 women referred by the breast clinic for first, non-screening mammograms. MAIN OUTCOME MEASURES--Radiographic reports on all patients. Final diagnosis in patients reported as having possible or probable neoplasm. RESULTS--Of the women referred directly by general practitioners one (0.2%) was reported as showing probable malignancy (later histologically confirmed) and 15 (4%) as showing possible malignancy (on follow up none had proved malignancy). Of the women referred by the breast clinic 38 (17%) were reported as showing probable malignancy (all had confirmed carcinomas) and 35 (15%) as showing possible malignancy (19 (54%) had proved malignancy). 18 of the proved malignancies were in women under 50 years old, 26 were in women over 64 years, and 14 were in women of screening age. 54 (93%) of the 58 patients with proved breast cancer and an abnormal mammogram had a discrete breast lump. CONCLUSIONS--General practitioners accurately divided women into low and high risk groups, resulting in few abnormalities being detected in patients referred directly for mammography. This suggests that an open access non-screening mammography service for general practitioners is unnecessary in an area with a specialist breast clinic. The large proportion of cancers in women outside of screening age emphasises the need for such clinics.     

Trifluoromethyl ketones as inhibitors of histone deacetylase

Trifluoromethyl ketones were found to be inhibitors of histone deacetylases (HDACs). Optimization of this series led to the identification of submicromolar inhibitors such as 20 that demonstrated antiproliferative effects against the HT1080 and MDA 435 cell lines.     

A Novel Bicistronic High-Capacity Gutless Adenovirus Vector That Drives Constitutive Expression of Herpes Simplex Virus Type 1 Thymidine Kinase and Tet-Inducible Expression of Flt3L for Glioma Therapeutics

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, affecting ∼18,000 new patients every year; its prognosis remains poor despite standard treatment with surgery, radiotherapy, and chemotherapy (temozolomide) (36-38, 44; J. C. Buckner, presented at the ASCO Annual Meeting, 2009). Complete resection is mostly impossible due to the highly infiltrative nature of this disease. Residual GBM cells remaining within the nonneoplastic brain parenchyma eventually lead to tumor recurrence that is resistant to conventional chemotherapy and radiotherapy, ultimately leading to the patient''s death (44). Several dendritic cell vaccination strategies aiming to stimulate the patient''s immune system to seek out and destroy residual brain tumor cells are currently under preclinical and clinical development and constitute a promising adjuvant treatment for GBM (18, 24, 42, 43, 45).We have developed a novel immunotherapeutic approach for GBM using first-generation adenoviral vectors (Ads) to deliver a combination of therapeutic transgenes into the tumor mass (2, 7, 9, 12, 14, 17, 48) which is slated to begin phase I clinical testing this year. In our strategy, rather than vaccinating against tumor antigens, we aim to reconstruct an immune circuit that is absent from the normal brain. Our gene therapy strategy consists of the conditionally cytotoxic herpes simplex virus type 1 thymidine kinase (TK) (2, 12), which kills proliferating tumor cells in the presence of the prodrug ganciclovir (GCV), used in combination with human soluble Fms-like tyrosine kinase 3 ligand (Flt3L), which recruits bone marrow-derived dendritic cells (DCs) to the normal brain or brain tumor microenvironment in mice (11, 12) and rats (1, 13). We previously showed that codelivery of Ad-Flt3L and Ad-TK into the brain tumor milieu induces an anti-GBM-specific immune response (9, 12, 17, 27, 48), leading to long-term survival of rats bearing intracranial CNS1, 9L, and F98 tumors (2, 17, 27) and mice bearing intracranial GL26, GL261, and B16-F10 tumors (12, 48). In addition, the combination of Ad-Flt3L plus Ad-TK induces GBM-specific immunological memory that improves survival in intracranial multifocal and recurrent models of GBM in both rats and mice (9, 12, 17, 20, 22, 27).First-generation adenoviral vectors have been used in clinical trials to deliver HSV1-TK (31). Ad-TK delivered into the margins of the tumor cavity after surgical resection of GBM was well tolerated in over 70 patients in six early clinical trials (19, 34). Publication of final results from a large, multicenter phase III trial involving 251 patients is awaited; preliminary analysis indicates small but statistically significant benefits from adding gene therapy to standard care. Final analysis will have to wait for the detailed publications (3, 16).To provide stable, long-term therapeutic transgene expression, “gutless,” high-capacity adenoviral (HC-Ad) vectors have been engineered in which all viral encoding genes have been eliminated and replaced with inert, noncoding stuffer DNA sequences (26, 30). In the presence of an anti-adenoviral immune response, transgene expression in the brain from first-generation Ads is reduced, while expression from HC-Ad vectors remains stable for at least 1 year (4, 25, 28). Therefore, we engineered two HC-Ad vectors to either constitutively express TK (HC-Ad-TK) (21, 27) or express Flt3L under the control of the tightly regulatable mCMV-TetOn expression system (HC-Ad-TetOn-Flt3L) (6, 8, 27, 47). Intratumoral administration of HC-Ad-TK and HC-Ad-TetOn-Flt3L led to high levels of therapeutic efficacy, and the therapy was well tolerated, with no overt toxicity and no spread of vector genomes outside the injected brain hemisphere in treated Lewis rats bearing syngeneic intracranial brain tumors (27).To reduce vector dose and facilitate GMP manufacturing of the clinical product, we engineered a novel, bicistronic HC-Ad vector that encodes, for the first time, both constitutively expressed HSV1-TK and inducible Flt3L from a single HC-Ad vector genome. Herein we demonstrate therapeutic efficacy of the bicistronic HC-Ad in naïve rats bearing intracranial tumors and also in tumor-bearing rats which had been exposed to adenoviruses and therefore exhibited anti-Ad immunity. The safety profile of this approach was assessed by evaluating the biodistribution of HC-Ad vector genomes, systemic and neurological toxicity, and behavioral abnormalities over the course of 1 year posttreatment. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.