Identification of the MMRN1 binding region within the C2 domain of human factor V

In platelets, coagulation cofactor V is stored in complex with multimerin 1 in alpha-granules for activation-induced release during clot formation. The molecular nature of multimerin 1 factor V binding has not been determined, although multimerin 1 is known to interact with the factor V light chain. We investigated the region in factor V important for multimerin 1 binding using modified enzyme-linked immunoassays and recombinant factor V constructs. Factor V constructs lacking the C2 region or entire light chain had impaired and absent multimerin 1 binding, respectively, whereas the B domain deleted construct had modestly reduced binding. Analyses of point mutated constructs indicated that the multimerin 1 binding site in the C2 domain of factor V partially overlaps the phosphatidylserine binding site and that the factor V B domain enhances multimerin 1 binding. Multimerin 1 did not inhibit factor V phosphatidylserine binding, and it bound to phosphatidylserine independently of factor V. There was a reduction in factor V in complex with multimerin 1 after activation, and thrombin cleavage significantly reduced factor V binding to multimerin 1. In molar excess, multimerin 1 minimally reduced factor V procoagulant activity in prothrombinase assays and only if it was added before factor V activation. The dissociation of factor V-multimerin 1 complexes following factor V activation suggests a role for multimerin 1 in delivering and localizing factor V onto platelets prior to prothrombinase assembly.     

Regulation of adiponectin by adipose tissue-derived cytokines: in vivo and in vitro investigations in humans

Adiponectin is an adipose tissue-specific protein that is abundantly present in the circulation and suggested to be involved in insulin sensitivity and development of atherosclerosis. Because cytokines are suggested to regulate adiponectin, the aim of the present study was to investigate the interaction between adiponectin and three adipose tissue-derived cytokines (IL-6, IL-8, and TNF-alpha). The study was divided into three substudies as follows: 1) plasma adiponectin and mRNA levels in adipose tissue biopsies from obese subjects [mean body mass index (BMI): 39.7 kg/m2, n = 6] before and after weight loss; 2) plasma adiponectin in obese men (mean BMI: 38.7 kg/m2, n = 19) compared with lean men (mean BMI: 23.4 kg/m2, n = 10) before and after weight loss; and 3) in vitro direct effects of IL-6, IL-8, and TNF-alpha on adiponectin mRNA levels in adipose tissue cultures. The results were that 1) weight loss resulted in a 51% (P < 0.05) increase in plasma adiponectin and a 45% (P < 0.05) increase in adipose tissue mRNA levels; 2) plasma adiponectin was 53% (P < 0.01) higher in lean compared with obese men, and plasma adiponectin was inversely correlated with adiposity, insulin sensitivity, and IL-6; and 3) TNF-alpha (P < 0.01) and IL-6 plus its soluble receptor (P < 0.05) decreased adiponectin mRNA levels in vitro. The inverse relationship between plasma adiponectin and cytokines in vivo and the cytokine-induced reduction in adiponectin mRNA in vitro suggests that endogenous cytokines may inhibit adiponectin. This could be of importance for the association between cytokines (e.g., IL-6) and insulin resistance and atherosclerosis.     

Identification of Rev-erbalpha as a physiological repressor of apoC-III gene transcription

Elevated serum levels of triglyceride-rich remnant lipoproteins (TRL) are a major risk factor predisposing a subject to atherosclerosis. Apolipoprotein C-III (apoC-III) is a major constituent of TRL that impedes triglyceride hydrolysis and remnant clearance and, as such, may exert pro-atherogenic activities. In the present study, transient cotransfection experiments in rat hepatocytes in primary culture and rabbit kidney RK13 cells demonstrated that overexpression of Rev-erbalpha specifically decreases basal and HNF-4 stimulated human apoC-III promoter activity. A Rev-erbalpha response element was mapped by promoter deletion, mutation analysis, and gel-shift experiments to a AGGTCA half-site located at position -23/-18 (downstream of the TATA box) in the apoC-III promoter. Finally, Rev-erbalpha-deficient mice displayed elevated serum and liver mRNA levels of apoC-III together with increased serum VLDL triglycerides. Taken together, our data identify Rev-erbalpha as a regulator of apoC-III gene expression, providing a novel, physiological role for this nuclear receptor in the regulation of lipid metabolism.     

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

Cell differentiation in microsporangia of Pinus sylvestris. V. Diakinesis to tetrad formation

Following the diffuse stage, the progression of meiosis in Pinus sylvestris unlike that earlier in meiosis, was conspicuously asynchronous. During the diffuse stage of meiosis tapetal cells dedifferentiated. Plasmodesmata were formed, the cells developed a uniform, meristematic appearance and the nuclei underwent mitosis. Throughout the stages covered by this report tapetal cells redifferentiated, again becoming hypersecretory cells, and Ubisch bodies (orbicules) formed. In angio-sperms Ubisch bodies apparently form only once whereas in Pinus they are produced several or many times with a different and characteristic form each time. The future Ubisch bodies are filled from connections with cisternae of the endoplasmic reticu-lum, then coated by plasma membrane and its glycocalyx. The plasma membrane and glycocalyx coating are likely to be responsible for the specific exine form of Ubisch bodies. Cytokinesis after meiosis was typical of plant cells, but no cell wall formed. Thus deep invasions of callose between microspores give an appearance of furrowing, as was often suggested in classical literature.     

Morphology and citric acid production of Aspergillus niger PM 1

Summary Aspergillus niger PM 1 was grown in a tubular loop and a stirred tank bioreactor. Batch fermentations were performed under various agitation conditions and pH. Citric acid, oxalic acid, extracellular polysaccharides and proteins were assayed. The following morphological parameters were measured: mean perimeter of clumps, mean perimeter of the central core of clumps, mean length of filaments and mean diameter of filaments. Citric acid production and morphology in both reactors were dependent on agitation intensity and pH. The length of the filaments was shown to be the only parameter that could be related to citric acid production in both reactors: the shorter the filaments the more citric acid was produced. However, for the same amount of citric acid produced the morphology of the organism grown in the stirred tank differed considerably from that grown in the loop reactor.     

Metabolism of carbohydrate derivatives by Pseudomonas acidovorans.

Wild-type Pseudomonas acidovorans strain A1 was unable to grow on glycerol or glucose as sole source of carbon and energy although it grew well on gluconate. Spontaneous glycerol-positive mutants, which apparently had become permeable to glycerol, were readily isolated, but glucose-positive mutants did not occur. P. acidovorans lacked glucose dehydrogenase and glucokinase, which were sufficient to account for its inability to grow on glucose. Gluconate was degraded exclusively via a noncoordinately induced Entner-Doudoroff pathway. Phosphogluconate dehydrogenase was undetectable. In contrast to P. aeruginosa, P. acidovorans possessed a single glyceraldehyde-phosphate dehydrogenase activity, which was NAD+ specific and constitutive, and an inducible pyruvate kinase. Moreover, growth of glycerol-positive strain K2 on glycerol did not induce any of the enzymes related to metabolism of hexosephosphate derivatives as occurs in fluorescent pseudomonads.     

Purification and partial characterisation of a 1.57 kDa thermostable esterase from Bacillus stearothermophilus

The molecular mass of esterases usually falls in the range of 20–160 kDa, although an esterase of 5.7 kDa from Candida lipolytica has been described. Three other enzymes smaller than 10 kDa have been reported, all of which were more thermostable than their higher molecular mass counterparts. This paper describes the purification of an extracellular esterase hydrolysing fluorescein dibutyrate from Bacillus stearothermophilus NCIMB 13335. The esterase had a molecular mass of 1.57 kDa when analysed by SDS-PAGE, gel filtration and MALDI-TOF spectrometry. This enzyme retained more than 90% of its activity after incubation at 90°C for 2 h.     

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.