Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.     

A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus

Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.     

Accuracy of diagnostic registers and management of chronic obstructive pulmonary disease: the Devon primary care audit

Background

Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.

Methods

Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.

Results

Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.

Conclusion

Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.     

You can't always get what you want: size assortative mating by mutual mate choice as a resolution of sexual conflict

Background  

Assortative mating patterns for mate quality traits like body size are often observed in nature. However, the underlying mechanisms that cause assortative mating patterns are less well known. Sexual selection is one important explanation for assortment, suggesting that i) one (usually the female) or both sexes could show preferences for mates of similar size or ii) mutual mate choice could resolve sexual conflict over quality traits into assortment. We tested these hypotheses experimentally in the socially monogamous cichlid fish Pelvicachromis taeniatus, in which mate choice is mutual.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

A universal molecular method for identifying underground plant parts to species

As part of a large project to determine rooting depth and resource uptake on the Edwards Plateau of central Texas, we developed a DNA-based technique that allows the below-ground parts of all plants to be identified to the level of genus and usually to species. Identification is achieved by comparing DNA sequences of the internal transcribed spacer (ITS) region of the 18S-26S nuclear ribosomal DNA repeat, derived from below-ground plant material, with a reference ITS region database for plants at a site. The method works throughout plants because the plant ITS region can be PCR amplified using a set of universal primers. Congeneric species can usually be identified because the ITS region evolves relatively rapidly. In our study, all roots were easily identified to the level of genus; most congeneric species were identified solely by ITS sequence differences but some required a combination of ITS sequence data and above-ground surveys of species at a site. In addition to showing the feasibility and efficacy of our technique, we compare it with another DNA-based technique used to identify below-ground plant parts. Finally, we also describe a DNA extraction and purification technique that reliably provides high-quality DNA of sufficient quantity from roots so that PCR can be readily accomplished. Our technique should allow the below-ground parts of plants in any system to be identified and thereby open new possibilities for the study of below-ground plant communities.