Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation.     

Reduction of Visceral Adipose Tissue and Improvement of Metabolic Indices: Effect of Dexfenfluramine in NIDDM

Increased visceral adipose tissue is thought to contribute to impaired glucose tolerance. We studied 10 men with non-insulin dependent diabetes (NIDDM) before and after a 12-week intervention study using dexfenfluramine. Subjects had a mean body mass index (BMI) of 26.4 ± 1.7 kg\m² and had an abdominal distribution of body fatness (waist-to hip ratio >0.9). Anthropometric indices, biochemistry, macronutrient intake from 7-day food records as well as a euglycaemic glucose clamp and magnetic resonance imaging (MRI) were performed at week 0 and week 12. Abdominal adipose tissue area measured by MRI was reduced from 854 ± 270 cm² to 666 ± 231 cm² (p=0.003) due mainly to a selective 32% reduction in visceral fat area from 484 ± 230 cm² to 333 ± 72 cm² (p=0.002). Insulin sensitivity improved from 0.29 ± 0.13 [min^?1 (mU/L)] to 0.54 ± 0.21 [min^?1 (mU/L)] (p=0.01) and C-peptide levels reduced from 0.77 ± 0.24 μmol/L to 0.58 ± 0.15 μmol/L (p=0.002). The reductions in fasting glucose and glycated haemoglobin failed to achieve significance. Fasting total cholesterol and triglyceride levels significantly reduced (p=<0.001 and p=0.021 respectively). There was a reduction in total energy intake (p=0.005) due to a significant reduction in calories obtained from fat (p<0.001). Thus dexfenfluramine was shown to be a useful adjunct therapy for the reduction of visceral fat in abdominally-obese men with NIDDM with an associated improvement in insulin sensitivity.     

Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis.

In the absence of E1B, the 289- and 243-residue E1A products of human adenovirus type 5 induce p53-dependent apoptosis. However, our group has shown recently that the 289-residue E1A protein is also able to induce apoptosis by a p53-independent mechanism (J. G. Teodoro, G. C. Shore, and P. E. Branton, Oncogene 11:467-474, 1995). Preliminary results suggested that p53-independent cell death required expression of one or more additional adenovirus early gene products. Here we show that both the E1B 19-kDa protein and cellular Bcl-2 inhibit or significantly delay p53-independent apoptosis. Neither early region E2 or E3 appeared to be necessary for such cell death. Analysis of a series of E1A mutants indicated that mutations in the transactivation domain and other regions of E1A correlated with E1A-mediated transactivation of E4 gene expression. Furthermore, p53-deficient human SAOS-2 cells infected with a mutant which expresses E1B but none of the E4 gene products remained viable for considerably longer times than those infected with wild-type adenovirus type 5. In addition, an adenovirus vector lacking both E1 and E4 was unable to induce DNA degradation and cell killing in E1A-expressing cell lines. These data showed that an E4 product is essential for E1A-induced p53-independent apoptosis.     

The phytogeography of European and Mediterranean heath species (Ericoideae, Ericaceae): a quantitative analysis

The geographic ranges of heath species in Europe and the Mediterranean and their relationships with environmental (climatic and ecogeographic) variables and biological features are analysed by means of multivariate methods. In particular, twinspan classifications into floristic elements and floristic regions, DCA floristic ordinations, CCA environment-constrained ordinations and CCA biology-constrained ordinations are carried out. Results of the analyses show a correspondence with conventional regionalization analyses based on broader criteria, and less correspondence with numerical analyses of other taxonomic groups at a similar scale. This lack of fit depends on the particular history and ecology of the taxonomic groups under study. A number of climatic (temperature and water stress) and geographic (coast length) variables are associated with different types of heaths according to their geographical ranges (continental, Mediterranean, Atlantic). Biological features of heaths account for a small part of the variation in range, but the association of temperate heaths with a preference for acid soils, of Mediterranean heaths with pubescence, and of Atlantic heaths with plant height is of interest. Heath species richness throughout Europe and the Mediterranean is analysed by multiple regression analyses and, apart from a strong influence of area size, a significant effect of water conditions, temperature and proximity to sea is detected. The area with the highest heath species richness is Western Mediterranean. Additional classification, ordination and multiple regression analyses of heaths in the Iberian Peninsula (Spain and Portugal, the areas with the highest heath diversity) revealed similar patterns to those found in Europe and the Mediterranean. The effect of heterogeneity of the studied units at this latter scale is removed in the Iberian analysis because of the relative homogeneity of the units considered at this scale.     

Lethal and sub-lethal selectivity of fenbutatin oxide and sulfur to the predator Iphiseiodes zuluagai (Acari: Phytoseiidae) and its prey, Oligonychus ilicis (Acari: Tetranychidae), in Brazilian coffee plantations

Lethal concentration (LC) has been widely used to estimate pesticide toxicity. However, it does not consider the sub-lethal effects. Therefore we included the instantaneous rate of increase in association with LC to estimate population-level effects of the acaricides fenbutatin oxide and sulfur on the predator Iphiseiodes zuluagai and its prey, the phytophagous southern red mite, Oligonychus ilicis. The predator was 32.84x and 17.20x more tolerant to fenbutatin oxide and sulfur, respectively, than its prey, based on LC₅₀ estimates obtained from acute concentration–mortality bioassays. The instantaneous rate of population growth in both mite species decreased with increasing acaricide concentration. Both acaricides provided effective control of O. ilicis at their recommended concentrations, but sulfur drastically compromised the predator populations quickly leading them to extinction due to the low reproductive potential of this species compared with its prey.     

The natural compound benzoxazolin-2(3H)-one selectively retards cell cycle in lettuce root meristems

Benzoxazolin-2(3H)-one (BOA) is a natural plant product that is phytotoxic to target plant species, inhibiting germination and growth and causing oxidative damage. We investigated its effects on the root meristems of seedlings of lettuce (Lactuca sativa) by means of light and transmission electron microscopy, flow cytometry, and conventional determination of mitotic index. Flow cytometry analyses and mitotic index showed a retard of cell cycle in BOA-treated meristems with selective activity at G2/M checkpoint.     

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N=10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N=10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69+/-80.31; 24.46+/-2.58; 70.51+/-7.66, respectively) in immunized rabbits when compared with animals from control group (164.91+/-15.67; 12.89+/-1.05; 32+/-3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.