Plant cytokinin analogues with inhibitory activity on cyclin-dependent kinases exert their antiproliferative effect through induction of apoptosis initiated by the mitochondrial pathway: determination by a multiparametric flow cytometric analysis

OBJECTIVE: Regulation of the cell cycle by cyclin-dependent kinase (CDK) activity occurs at multiple levels and is often altered in human cancers. Therefore, CDK activity has been targeted for drug discovery, and a number of small molecules have now been identified as CDK inhibitors. Plant cytokinin analogues with CDK inhibitory activity and antiproliferative effects were studied to characterize the cellular basis of the cytotoxic effect. METHODS: The IC(50) value (concentration at which 50% of the cell proliferation is inhibited) and AC(50) value (concentration at which 50% of the cell population is apoptotic) were determined by flow cytometry and microscopy, respectively. A new multiparametric flow cytometric analysis was used to study the sequence of different apoptotic events. In this assay, analysis of phosphatidylserine exposure, mitochondrial membrane depolarization, activation of caspases and DNA condensation were combined. RESULTS: Treatment of Jurkat and KG1 cells with the CDK inhibitors results in a decrease of viable cells and a parallel increase in percentage of apoptotic cells. Apoptosis was accompanied by a rapid decrease of mitochondrial membrane potential, which precedes DNA condensation, exposure of phosphatidylserine and activation of caspases. CONCLUSIONS: The main cellular mechanism of the antiproliferative effect of plant cytokinin analogues with CDK inhibitory activity is the induction of apoptosis. The multiparametric flow cytometric technique allowed to follow the kinetics of various aspects of apoptotic cell changes and demonstrated that cytokinin analogue-induced apoptosis starts through the mitochondrial pathway. This technique could also become of value for the rapid screening of pro-apoptotic properties of chemotherapeutic compounds.     

Intrapituitary adenoviral administration of 7B2 can extend life span and reverse endocrinological deficiencies in 7B2 null mice

The prohormone convertase PC2 requires the aid of a helper protein, known as 7B2, for production of active enzyme. Deletion of 7B2 results in a lethal phenotype resembling Cushing's disease. In this study, we have investigated the effect of a single low dose of recombinant adenovirus vector encoding 7B2 and delivered directly to the pituitary of 7B2 nulls on pituitary ACTH, plasma ACTH, corticosterone, alpha MSH and glucose, and survival time. We show that after injection of recombinant adenovirus encoding 27-kDa 7B2 into 7B2 nulls, transgene expression, as measured by RIA for 7B2, exhibits a transient elevation in the pituitary and blood, with a slight but significant elevation of PC2 activity in pituitaries of 7B2 nulls and a drop in the level of circulating ACTH concomitant with a small increase in circulating alpha MSH. The level of circulating blood glucose was increased, and that of corticosterone was decreased. Lastly, slight but significantly prolonged survival times were observed. These data showing partial rescue of 7B2 nulls support the idea that adenoviral administration of 7B2 will represent an effective means to study the role of this interesting neuroendocrine protein on endocrine function in vivo.     

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.

When exposed to cadmium a highly resistant strain P. aeruginosa san ai responds by an increased metalloprotein expression (particularly denitrification proteins), an enhanced respiration, and a pronounced thiol-disulfide protein modifications.     

Osteoinductive 3D scaffolds prepared by blend centrifugal spinning for long-term delivery of osteogenic supplements

Bone regeneration is a long-term process requiring proper scaffolding and drug delivery systems. The current study delivers a three-dimensional (3D) scaffold prepared by blend centrifugal spinning loaded with the osteogenic supplements (OS) β-glycerol phosphate, ascorbate-2-phosphate and dexamethasone. The OS were successfully encapsulated into a fibrous scaffold and showed sustained release for 30 days. Furthermore, biological testing showed the osteoinductive properties of the scaffolds on a model of human mesenchymal stem cells and stimulatory effect on a model of osteoblasts. The osteoinductive properties were further proved in vivo in critical size defects of rabbits. The amount of bone trabecules was bigger compared to control fibers without OS. The results indicate that due to its long-term drug releasing properties, single step fabrication process and 3D structure, the system shows ideal properties for use as a cell-free bone implant in tissue-engineering.

Bone regeneration is a long-term process requiring proper scaffolding and drug delivery systems.     

Functionalities and input methods for recording food intake: A systematic review

BackgroundIncreasing healthcare costs related to lifestyle-related chronic diseases require new solutions. Research on self-management tools is expanding and many new tools are emerging. Recording food intake is a key functionality in many of these tools. Nutrition monitoring is a relevant method to gain an overview of factors influencing health. However, keeping a food diary often constitutes a challenge for a patient, and developing a user-friendly and useful electronic food diary is not straightforward.PurposeTo gain insight into the existing approaches to recording food intake, and to analyze current functionalities and input methods.MethodsWe searched digital libraries, vendor markets and social networks focusing on nutrition. Selection criteria were publications written in English, and patient-oriented tools that offered recording of food intake or nutrition. The system properties that we searched for were types of data, types of terminal, target population, and types of reports and sharing functionalities. We summarized the properties based on their frequency in the reviewed sample.Results31 publications met the selection criteria. The majority of the identified food recording systems (67%) facilitated entry of food type and the consumed quantity of food; 16% of the systems were able to record more than one type of data. The three most frequent target populations were people with obesity, diabetes and overweight. Mobile phones were used as terminals in 35% of the cases, personal computers (PCs) in 29%, and personal digital assistants in 23%. Only 10% supported both PCs and mobile phones. Data sharing was provided by 71% and reports by 51% of the systems. We searched for apps in Google Play and the Apple Store and tested 45 mobile applications that stored food intake data, of which 62% supported recording of types of food, 24% recording of carbohydrate intake and 15% recording of calorie intake. The majority of the mobile applications offered some kind of reports and data sharing, mainly via All of the tested social-network-enabled applications supported access from a personal computer and a mobile phone, search in a food database, reports, graphical presentation, listing of favorite foods, overview of own meals, and entering of consumed food type and quantity.ConclusionThe analyzed apps reflected a variety of approaches to recording food intake and nutrition using different terminals – mostly mobile phones (35%), followed by PCs (29%) and PDAs (23%) for older studies, designed mainly for users with obesity (45%), diabetes mellitus (42%) and overweight (32%), or people who want to stay healthy (10%). The majority of the reviewed applications (67%) offered only input of food type and quantity. All approaches (n = 31), except for two, relied on manual input of data, either by typing or by selecting a food type from a database. The exceptions (n = 2) used a barcode scanning function. Users of mobile phone applications were not limited to data recording, but could view their data on the screen and send it via email. The tested web applications offered similar functionalities for recording food intake. The systems studied provided some degree of personalization: users can access some systems via PCs or mobile phones and they can choose among various types of data input content for recording food intake. Many functions, such as search in a food database, reports, graphical presentation, listing of favorite foods, and overview of the user's own meals, are optimized to simplify the recording process and save time. Data sharing and reports are common features of the reviewed systems. However, none use the user's recorded food history to make suggestions on new nutritional intake, during the food recording process. This may be an area for future research.     

Clinical picture of cardiogenic shock as primary manifestation of sepsis originating in the knee joint

This is a case report of a male with infection in the right knee joint progressing to sepsis. However, the patient initially complained mainly of dysarthria and breathlessness. He rapidly developed respiratory insufficiency with the loss of consciousness. Echocardiography revealed severe dysfunction of the left ventricle, suggesting acute failure of the chronically failing heart. The patient was referred to a coronary care unit. Only the further course of the disease, particularly progression of the local finding on the right leg and development of fever, together with significantly elevated inflammatory parameters in laboratory findings, resulted in the diagnosis of sepsis which also included myocardial dysfunction and brain hypoperfusion manifested as dysarthria.     

Survivin gene promoter polymorphism -31G/C as a risk factor for keratocystic odontogenic tumor development

Several single nucleotide polymorphisms in survivin gene promoters, notably -31G/C, have been shown to modulate the expression and activity of the survivin protein. Consequently, the -31G/C polymorphism has been identified as a risk factor for the development of several types of tumors. The aim of this study was to investigate a possible association between the -31G/C polymorphism and the risk for keratocystic odontogenic tumor (KCOT) development. DNA from 52 biopsy specimens of KCOTs and from 82 buccal swabs of healthy individuals was subjected to PCR restriction fragment length polymorphism analysis to identify individual genotypes. The distribution of genotypes in KCOT and control groups, respectively, was: GG: 30 (57.7%) vs. 26 (31.7%); CG: 17 (32.7%) vs. 45 (54.9%); and CC: 5 (9.6%) vs. 11 (13.4%), respectively. These differences were statistically significant. The G allele was more common in the KCOT group than in the control group: 76 (74%) vs. 96 (59%), respectively. Logistic regression analysis showed that GC heterozygotes had a considerably decreased susceptibility for KCOTs compared with GG homozygotes. The same was true for GC+CC vs. GG. The GG genotype of the -31G/C polymorphism might be a risk factor for KCOT development.     

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-x(L) is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-x(L) pathway "resets," allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using "knock-in" mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-x(L) response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-x(L) induction provides a "stop-gap" in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-x(L) may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms.