T1alpha/podoplanin is essential for capillary morphogenesis in lymphatic endothelial cells

The lymphatic vasculature functions to maintain tissue perfusion homeostasis. Defects in its formation or disruption of the vessels result in lymphedema, the effective treatment of which is hampered by limited understanding of factors regulating lymph vessel formation. Mice lacking T1alpha/podoplanin, a lymphatic endothelial cell transmembrane protein, have malformed lymphatic vasculature with lymphedema at birth, but the molecular mechanism for this phenotype is unknown. Here, we show, using primary human lung microvascular lymphatic endothelial cells (HMVEC-LLy), that small interfering RNA-mediated silence of podoplanin gene expression has the dramatic effect of blocking capillary tube formation in Matrigel. In addition, localization of phosphorylated ezrin/radixin/moesin proteins to plasma membrane extensions, an early event in the capillary morphogenic program in lymphatic endothelial cells, is impaired. We find that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA early (by 30 min) after plating on Matrigel, and Rac1 shows a delay in its activation. Further indication that podoplanin action is linked to RhoA activation is that use of a cell-permeable inhibitor of Rho inhibited lymphatic endothelial capillary tube formation in the same manner as did podoplanin gene silencing, which was not mimicked by treatment with a Rac1 inhibitor. These data clearly demonstrate that early activation of RhoA in the lymphangiogenic process, which is required for the successful establishment of the capillary network, is dependent on podoplanin expression. To our knowledge, this is the first time that a mechanism has been suggested to explain the role of podoplanin in lymphangiogenesis.     

Phosphorylation- and Nucleotide-Binding-Induced Changes to the Stability and Hydrogen Exchange Patterns of JNK1β1 Provide Insight into Its Mechanisms of Activation

Many studies have characterized how changes to the stability and internal motions of a protein during activation can contribute to their catalytic function, even when structural changes cannot be observed. Here, unfolding studies and hydrogen–deuterium exchange (HX) mass spectrometry were used to investigate the changes to the stability and conformation/conformational dynamics of JNK1β1 induced by phosphorylative activation. Equivalent studies were also employed to determine the effects of nucleotide binding on both inactive and active JNK1β1 using the ATP analogue, 5?-adenylyl-imidodiphosphate (AMP-PNP). JNK1β1 phosphorylation alters HX in regions involved in catalysis and substrate binding, changes that can be ascribed to functional modifications in either structure and/or backbone flexibility. Increased HX in the hinge between the N- and C-terminal domains implied that it acquires enhanced flexibility upon phosphorylation that may be a prerequisite for interdomain closure. In combination with the finding that nucleotide binding destabilizes the kinase, the patterns of solvent protection by AMP-PNP were consistent with a novel mode of nucleotide binding to the C-terminal domain of a destabilized and open domain conformation of inactive JNK1β1. Solvent protection by AMP-PNP of both N- and C-terminal domains in active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, concomitantly stabilizing the kinase. This suggests that phosphorylation activates JNK1β1 in part by increasing hinge flexibility to facilitate interdomain closure and the creation of a functional active site. By uncovering the complex interplay that occurs between nucleotide binding and phosphorylation, we present new insight into the unique mechanisms by which JNK1β1 is regulated.     

Nine years of climber community dynamics in a Nigerian lowland rain forest 31 years after a ground fire

Successional studies in tropical forests have generally emphasized the tree component, ignoring the community dynamics of non-tree life-forms, and so there is a limited understanding of how the plant community as a whole is changing during succession within forests. Thus, this study examined the changes in climber community composition and structure in a regenerating secondary lowland rain forest at Ile-Ife that was ravaged by a ground fire 31 years ago using six sample plots. All individual climbers in each sample plot were identified, enumerated and their girths at breast height were measured. The girths were measured at 1.3 m height or just before the point of branching. Our data were compared with those of previous studies in the forest to determine the changes in floristics and structure of climber community over the years using Sorenson’s similarity index. The mortality and recruitment rates in the forest during the course of succession were determined. Climber species increased from 49 (2005) to 61 (2014). Climber density increased astronomically from 448–1152 ha^?1 (2005) to 1712–4492 ha^?1 but basal area only increased slightly from 0.37–1.10 m² ha^?1 (2005) to 0.40–1.14 m² ha^?1. The recruitment rate (8%) was higher than the mortality rate (5.8%). The similarity of the climber species composition calculated using the Sorenson similarity index showed that the similarity between the two periods of study was 0.53 (53%). This study concluded that during the study period, the climber community changed, and climber species abundance and structure increased.     

The lung as a route for systemic delivery of therapeutic proteins and peptides

The large surface area, good vascularization, immense capacity for solute exchange and ultra-thinness of the alveolar epithelium are unique features of the lung that can facilitate systemic delivery via pulmonary administration of peptides and proteins. Physical and biochemical barriers, lack of optimal dosage forms and delivery devices limit the systemic delivery of biotherapeutic agents by inhalation. Current efforts to overcome these difficulties in order to deliver metabolic hormones (insulin, calcitonin, thyroid-stimulating hormone [TSH], follicle-stimulating hormone [FSH] and growth hormones) systemically, to induce systemic responses (immunoglobulins, cyclosporin A [CsA], recombinant-methionyl human granulocyte colony-stimulating factor [r-huG-CSF], pancreatic islet autoantigen) and to modulate other biological processes via the lung are reviewed. Safety aspects of pulmonary peptide and protein administration are also discussed.     

Cation exchange as a single polishing step for conjugated peptides

Purification of peptides typically includes expensive reverse phase (RP) processes, which utilize high pressure and large volumes of solvent. For two conjugated peptides, chromatography process development targeted a low-pressure aqueous process that could achieve target product purities of ≥95%, comparable to purities seen with traditional RP. A high throughput screening approach of different modalities was used to identify binding and elution conditions on a cation exchange resin and small-scale columns were used in order to assess impurity removal and process yield. The parameters for load and gradient elution were optimized to increase product purity and process productivity with a wide operating window identified where high purity and productivity are achieved. Computational modeling was then used to validate experimental chromatography results and to gain insight on the effect of the chemical modifications on the surface properties of the two peptides. Both modeling and experimental data showed that with optimization, cation exchange could be utilized as a single polishing step for conjugated peptides. Similar purities were achieved as those seen with RP with up to double the productivity.     

Distribution of endosulfan in water, sediment and fish from Warri river, Niger delta, Nigeria

This article presents the first attempt to quantify the levels and distribution pattern of endosulfan, an organochlorine pesticide in surface water, sediment and fish ( Chrysichthys furcatus and Tilapia zilli ). The samples were collected from three stations (Ovwian, Ekakpamre and Ovu) of Warri River in the western Niger Delta of Nigeria in 2006 during the dry and wet seasons (January–August). A total of 96 samples made up of 24 samples each for water, sediment and fish were analysed in this study. The pesticide levels were analysed using high performance liquid chromatography (HPLC model CECIL 1010) to elucidate its distribution in various environmental compartments. The ranges of concentrations of the pesticide in the matrices were: 0.01–9.23 μg/l (water), 0.06–11.98 μg/gdw (sediment), 0.01–15.06 μg/gdw ( Chrysichthys furcatus) and 0.01–1.80 μg/gdw ( Tilapia zilli ). From this result, decreasing order of occurrence of the pesticide is as follows: fish > sediment > water. The concentrations observed in fish ( Chrysichthys furcatus ) were higher than the levels observed in sediment and water suggesting bioaccumulation of the pesticide by the fish. Spatial variations occurred with downstream stations having statistically higher concentrations in all matrices at P  < 0.05. Seasonal variations occurred with higher concentrations in dry season for water and sediment only, while the fish species had higher concentrations in the wet season. The observed values of endosulfan were above the ecological bench marks (0.02 μg/l) recommended by Nigeria Environmental Protection Agency and European Union. They were also relatively higher than those in previous studies on the Nigerian environment, an observation that calls for regular monitoring of the Niger Delta water bodies.     

Interception, pH and solid content of rainfall in a Nigerian lowland rain forest

Rainfall interception by forest trees, pH, alkalinity, total suspended and dissolved solids of incident rainfall, stemflow and throughfall and the effect of tree species in these parameters were investigated in three open sites and a secondary rain forest at Ile-Ife, Nigeria, in 1994 and 1995. The tree species Blighia sapida , Holarrhena floribunda and Piptadeniastrum africanum intercepted different proportions of incident rainfall, 1.6, 39.4, and 7.4%, respectively. The pH of stemflow was higher than that of throughfall and the pH of both was always higher than that of incident rainfall. The total suspended solids were higher at the beginning and towards the end of the rainy season. The total dissolved solids concentration in all rainfall fractions was highest at the early rains and during the heavy rain months, especially in throughfall and stemflow. The total suspended and dissolved solids concentration of stemflow was higher than that of throughfall and those of both were higher than that of the incident rain. Total alkalinity of incident rainfall and throughfall decreased, while that of stemflow increased with increase in rainfall. There were significant differences in the alkalinity of stemflow and throughfall under the crowns of the tree species.     

Expression,Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase

The Protein Journal - Patients in health-care settings develop nosocomial infections due to prolonged hospital stay. The Gram negative Klebsiella pneumoniae (K. pneumoniae), is a bacterial pathogen...     

De novo achondroplasia causing four consecutive unsuccessful pregnancies: a case report

ABSTRACT: INTRODUCTION: The incidence of achondroplasia is very low, and the birth of two or more consecutive babies with achondroplasia to unaffected parents is a rarity. We report a rare case of recurrent achondroplasia in babies of unaffected parents. CASE PRESENTATION: A 29-year-old Nigerian Igbo woman who has had three consecutive dead achondroplastic babies presented at a gestational age of 31 weeks with a two-hour history of drainage of liquor and vaginal bleeding. Neither she nor her husband had features of achondroplasia. Fundal height was compatible with the gestational age. Fetal heart activity was present. An abdominal ultrasound examination showed a viable fetus with short long bones, oligohydramnios, and a fundal placenta with a small retroplacental blood clot. Our patient was stabilized and had an emergency Cesarean section for grade 1 abruptio placentae. A live male baby with Apgar scores of 4 at one minute and 5 at 10 minutes was delivered. The baby had classic features of achondroplasia and died shortly after birth. CONCLUSIONS: To the best of our knowledge, this is the first reported case of recurrent achondroplasia in siblings of unaffected parents in Nigeria. Management is challenging, and the outcomes of future pregnancies appear bleak. However, proper counseling and follow-up are needed. There is also a need to establish preconception clinics and facilities for prenatal genetic diagnosis and gene therapy in developing countries.