Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Evaluation of the RF heating of a generic deep brain stimulator exposed in 1.5 T magnetic resonance scanners

The radio frequency (RF) electromagnetic field of magnetic resonance (MR) scanners can result in significant tissue heating due to the RF coupling with the conducting parts of medical implants. The objective of this article is to evaluate the advantages and shortcomings of a new four‐tier approach based on a combined numerical and experimental procedure, designed to demonstrate safety of implants during MR scans. To the authors' best knowledge, this is the first study analyzing this technique. The evaluation is performed for 1.5 T MR scanners using a generic model of a deep brain stimulator (DBS) with a straight lead and a helical lead. The results show that the approach is technically feasible and provides sound and conservative information about the potential heating of implants. We demonstrate that (1) applying optimized tools results in reasonable uncertainties for the overall evaluation; (2) each tier reduces the overestimation by several dB at the cost of more demanding evaluation steps; (3) the implant with the straight lead would cause local temperature increases larger than 18 °C at the RF exposure limit for the normal operating mode; (4) Tier 3 is not sufficient for the helical implant; and (5) Tier 4 might be too demanding to be performed for complex implants. We conclude with a suggestion for a procedure that follows the same concept but is between Tier 3 and 4. In addition, the evaluation of Tier 3 has shown consistency with current scan practice, namely, the resulting heat at the lead tip is less than 3.5 °C for the straight lead and 0.7 °C for the helix lead for scans at the current applied MR scan restrictions for deep brain stimulation at a head average SAR of 0.1 W/kg. Bioelectromagnetics 34:104–113, 2013. © 2012 Wiley Periodicals, Inc.     

Vasopressin-induced polyphosphoinositide and phosphatidylcholine degradation in fibroblasts. Temporal relationship for formation of phospholipase C and phospholipase D hydrolysis products

Cultured fibroblasts (REF52 cells) were employed to investigate phospholipid degradation in response to vasopressin (VP) treatment. There have been few studies in fibroblasts which characterize the pattern and relationship of phosphatidylinositol 4,5-bisphosphate (PIP2) and non-phosphoinositide hydrolysis elicited by VP. Here we demonstrate that VP-induced PIP2 hydrolysis is closely accompanied by phosphatidylcholine (PC) degradation by phospholipase D. Cells prelabeled with [3H]arachidonic acid showed rapid formation and diminution of [3H]diacylglycerol (DG) (5-15s) when treated with VP; this was accompanied by a reduction in polyphosphoinositide radioactivity. Radiolabeled inositol trisphosphate was generated with a similar time frame. In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into cellular PC, VP elicited the generation of [3H]myristoyl phosphatidate (PA) as early as 15 s, in the absence of an increase in labeled DG. In the presence of ethanol the pattern of [3H]myristoyl phosphatidylethanol (PEt) formation coincided with [3H]myristoyl-PA formation in the absence of ethanol. PEt was similarly formed, in response to VP treatment, in cells prelabeled with 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine. The formation of PC-derived [3H]myristoyl-DG was characterized by a lag period of approximately 1 min, after which DG increased steadily over a 10-min period. Biphasic formation of DG was observed in cells prelabeled with [3H]arachidonic acid, and the formation of [3H]PA occurred in an uninterrupted fashion. Two protein kinase C agonists, phorbol diester and dioctanoylglycerol, elicited the formation of [3H]myristoyl-PEt. The inclusion of staurosporine, a protein kinase C inhibitor, blocked VP-induced [3H]myristoyl-PEt formation by 88%. These data demonstrate that VP elicits the coordinated hydrolysis of PIP2 by phospholipase C and PC hydrolysis by phospholipase D. This event results in the prolonged generation of PA and biphasic formation of DG. From the time courses shown, we hypothesize that the early generation of PA, heretofore ascribed to products of the polyphosphoinositide cycle, are in part derived from PC by phospholipase D.     

The Course of Serum Inflammatory Biomarkers Following Whiplash Injury and Their Relationship to Sensory and Muscle Measures: a Longitudinal Cohort Study

Tissue damage or pathological alterations are not detectable in the majority of people with whiplash associated disorders (WAD). Widespread hyperalgisa, morphological muscle changes and psychological distress are common features of WAD. However little is known about the presence of inflammation and its association with symptom persistence or the clinical presentation of WAD. This study aimed to prospectively investigate changes in serum inflammatory biomarker levels from the acute (<3 weeks) to chronic (>3 months) stages of whiplash injury. It also aimed to determine relationships between biomarker levels and hyperalgesia, fatty muscle infiltrates of the cervical extensors identified on MRI and psychological factors. 40 volunteers with acute WAD and 18 healthy controls participated. Participants with WAD were classified at 3 months as recovered/mild disability or having moderate/severe disability using the Neck Disability Index. At baseline both WAD groups showed elevated serum levels of CRP but by 3 months levels remained elevated only in the moderate/severe group. The recovered/mild disability WAD group had higher levels of TNF-α at both time points than both the moderate/severe WAD group and healthy controls. There were no differences found in serum IL-1β. Moderate relationships were found between hyperalgesia and CRP at both time points and between hyperalgesia and IL-1β 3 months post injury. There was a moderate negative correlation between TNF-α and amount of fatty muscle infiltrate and pain intensity at 3 months. Only a weak relationship was found between CRP and pain catastrophising and no relationship between biomarker levels and posttraumatic stress symptoms. The results of the study indicate that inflammatory biomarkers may play a role in outcomes following whiplash injury as well as being associated with hyperalgesia and fatty muscle infiltrate in the cervical extensors.     

Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

Background

There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort.

Methods

We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test.

Results

Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05).

Conclusions

Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae. However, discordance between inflammation and health status was observed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0114-1) contains supplementary material, which is available to authorized users.     

Dietary use and conservation concern of edible wetland plants at indo-burma hotspot: a case study from northeast India

Background

The wetlands of the North East India fall among the global hotspots of biodiversity. However, they have received very little attention with relation to their intrinsic values to human kind; therefore their conservation is hardly addressed. These wetlands are critical for the sustenance of the tribal communities.

Methods

Field research was conducted during 2003 to 2006 in seven major wetlands of four districts of Manipur state, Northeast India (viz. Imphal-East, Imphal-West, Thoubal, and Bishnupur). A total of 224 wetland-plant-collectors were interviewed for the use and economics of species using semi-structured questionnaires and interview schedules. Imphal, Bishenpur and Thoubal markets were investigated in detail for influx and consumption pattern of these plants. The collectors were also inquired for medicinal use of wetland species. Nutritive values of 21 species were analyzed in laboratory. The vouchers were collected for all the species and deposited in the CSIR-NEIST (Formerly Regional Research Laboratory), Substation, Lamphelpat, Imphal, Manipur, India.

Results

We recorded 51 edible wetland species used by indigenous people for food and medicinal purposes. Thirty eight species had high medicinal values and used in the traditional system to treat over 22 diseases. At least 27 species were traded in three markets studied (i.e. Imphal, Thoubal and Bishenpur), involving an annual turnover of 113 tons of wetland edible plants and a gross revenue of Rs. 907, 770/- (US$1 = Rs. 45/-). The Imphal market alone supplies 60% of the total business. Eighty per cent of the above mentioned species are very often used by the community. The community has a general opinion that the availability of 45% species has depleted in recent times, 15 species need consideration for conservation while another 7 species deserved immediate protection measures. The nutrient analysis showed that these species contribute to the dietary balance of tribal communities.

Conclusions

Considering the importance of wild wetland plants in local sustenance, it is suggested to protect their habitats, develop domestication protocols of selected species, and build programs for the long-term management of wetland areas by involving local people. Some medicinal plants may also be used to develop into modern medicines.

     

Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D₃₇ values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.A survey of bacteria and archaea identifies 11 phyla that contain species with unusually high resistance to the lethal effects of ionizing radiation (IR). These phyla are not closely related to each other and do not share a common lineage, and all include genera that are considered radiosensitive (9). The existence of so many unrelated and isolated radioresistant species in the phylogenetic tree argues that the molecular mechanisms that protect against IR-induced damage evolved independently in these organisms, suggesting that at least some species have the capacity to acquire radioresistance through evolutionary processes if they are subjected to appropriate selective pressure.The first of these species to be discovered, and the best studied to date, is the bacterium Deinococcus radiodurans. The molecular basis of the extraordinary radioresistance of Deinococcus has not been elucidated, but well-constructed proposals abound. Radioresistance has variously been attributed to the condensed structure of the nucleoid (29, 40, 56), the elevated levels of Mn ion present in the cytosol as a mechanism to control protein oxidation (11, 12), a specialized RecA-independent DNA repair process (54), and other species attributes (9). Radioresistance in Deinococcus is probably mechanistically related to desiccation resistance derived from evolution in arid environments (37, 45), although this may not be the origin of the phenotype in all relevant species (9).An understanding of the genetic underpinnings of bacterial radiation resistance holds promise for yielding insights into the mechanistic basis of radiation toxicity, along with the potential for new approaches to facilitate recovery from radiation injury in other organisms, including humans. To better define the genetic, biochemical, and physiological characteristics most important for radioresistance, we employed a strategy to allow the cells to inform us. In brief, we generated radioresistant variants of radiosensitive bacteria and defined the genetic changes underlying the new phenotype.In 1946, Evelyn Witkin established that it was possible to increase the resistance of Escherichia coli B to DNA damage (50). She exposed cultures to high doses of UV light, killing most of the population and selecting for variants better able to tolerate UV. In the 6 decades since the Witkin report, additional investigators have repeated this result, demonstrating that iterative cycles of high-dose exposure to a DNA damaging agent can heritably enhance a culture''s ability to tolerate that DNA damaging agent. Increases in IR resistance have been reported for E. coli (17), Salmonella enterica serovar Typhimurium (14), and Bacillus pumulis (44), organisms that are otherwise considered radiosensitive. Davies and Sinskey (14) showed that for S. enterica serovar Typhimurium LT2, the number of cycles of exposure and recovery correlates with the level of radioresistance achieved. After 84 cycles, they generated a strain displaying inactivation kinetics similar to that of Deinococcus radiodurans, with a D₁₀ value (the dose needed to inactivate 90% of the population) 200-fold higher than that of the parental strain.For this study, we expanded on these earlier studies by independently generating four IR-resistant populations of Escherichia coli K-12 MG1655 (4). Our effort included an important innovation relative to the earlier studies—we characterized the evolved populations with an experimental program that included the complete genomic resequencing of multiple strains purified from three of the populations, taking advantage of new sequencing technologies. The result is an increasingly detailed data set—based on a single robust model system—that allows us to (i) explore the molecular basis of radiation resistance in bacteria and (ii) test current hypotheses and search for novel mechanisms of radiation resistance.