Application of simple fed-batch technique to high-level secretory production of insulin precursor using <Emphasis Type="Italic">Pichia pastoris</Emphasis> with subsequent purification and conversion to human insulin

Background  

The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.     

Evaluation of intra-amniotic surfactant administration for lung maturation in preterm sheep

We aimed to evaluate the effects of intra-amniotic surfactant administration on alveolar lecithin/sphingomyelin ratio, density of type II pneumocytes, and fetal lung function in preterm merino sheep. Pregnant ewes at 119 days gestation either received 200 mg intra-amniotic surfactant (n=4) or saline solution (n=4). After 24 h, the lambs were delivered by hysterotomy and mechanically ventilated. Lecithin/sphingomyelin ratios in alveolar fluid, inflating pressure–volume relationships, and type II pneumocyte counts in histological specimens were compared among the groups. All of the lambs completed the protocol. Mean lecithin/sphingomyelin ratio increased significantly in amniotic (p=0.03) and alveolar fluid (p=0.03) samples of surfactant-treated animals. Lung function in terms of pressure–volume curves did not differ between two groups. Type II pneumocyte density tended to be higher (p=0.057) after intra-amniotic surfactant administration. Single-dose treatment with intra-amniotic surfactant seems to improve amniotic and alveolar lecithin/sphingomyelin ratio questionably by increasing alveolar type II cells. Pressure–volume relationships from inflation of the lungs might be unaltered with intra-amniotic surfactant treatment.     

Synthesis and Biological Activity of Some Bromophenols and Their Derivatives Including Natural Products

In addition to the first synthesis of the natural bromophenol butyl 2-(3,5-dibromo-4-hydroxyphenyl)acetate ( 1 ), indene derivatives 34 and 35 were synthesized from 3-phenylpropenal derivatives in BBr₃ medium. Five known natural bromophenols and some derivatives were synthesized by known methods. Cholinesterase (ChEs) inhibitors reduce the breakdown of acetylcholine and are used in the treatment of Alzheimer's disease (AD) and dementia symptoms. The inhibition effects of all obtained compounds were examined towards acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glycosidase enzymes. All synthesized compounds demonstrated the strong inhibition effects against both cholinergic enzymes. For determination of Ki values of novel bromophenols Lineweaver-Burk graphs were obtained. Ki values were found in the ranging of 0.13–14.74 nM for AChE, 5.11–23.95 nM for BChE, and 63.96–206.78 nM for α-glycosidase, respectively. All bromophenols and their derivatives exhibit effective inhibition profile when compared to positive controls.     

Urinary selenium in healthy and diabetic Saudi Arabians

We examined Se in urine of 170 Saudi Arabian diabetics (19 insulin-dependent [type 1] and 151 insulin-independent [type 2]) and in an equal number of control subjects of the same origin by measuring the ratio of the concentration of this metal (C_Se) to that of creatinine in urine (C_creat) for each subject. The mean (and SEM) of C_Se/C_creat for the control subjects was 56 (2.9) μmol/mol creat, whereas, the value for the diabetics combined or separated into type 1 and type 2 was 56.7 (3.2), 51.5 (6.3), and 57.4 (3.5) μmol/mol creat, respectively. With the exception of type 2 diabetics who were treated with insulin in addition to oral hypoglycemic and diet (35 patients) (mean [SEM]=43 (4.3) μmol/mol creat), there was no significant difference in C_Se/C_creat between the diabetics and control subjects. Also, there was no significant correlation between C_Se/C_creat and age, sex, or weight of diabetics, whereas, the correlation with the degree of diabetic control was significant (p≤0.0136). Of all diabetes-associated disorders (cardiovascular diseases, neuropathy, ophthalmologic diseases, infections, and hepatic disease), only ophthalmologic diseases appears to cause a significant (p≤0.05) reduction in C_Se/C_creat, but only among type 2 diabetics. Inasmuch as Se status is reflected by urinary Se, healthy Saudi Arabians appear to have Se status that is comparable or higher than those reported for other populations.     

Chromogranin A (CGA) in the gastro-entero-pancreatic (GEP) endocrine system

Summary Chromogranin A (CGA), a protein at first detected in the adrenal medulla, has recently been found also in other organs, e.g. the endocrine pancreas. However, immunohistochemical findings concerning the cellular source of pancreatic CGA were controversial. Therefore, the endocrine pancreas of 10 mammalian species (man, tupaia, mole, cat, dog, pig, guinea pig, rabbit, rat) was investigated immunohistochemically for CGA-like immunoreactivities on serial semithin plastic sections using a high-titer polyclonal antiserum against bovine CGA. The results show that basically all pancreatic endocrine cell types are CGA-immunoreactive; however, every species has its own pattern of CGA-immunoreactive cell types. Other findings of the present studies indicate that the physiological function of CGA in pancreatic endocrine cells is related to the storage mechanisms of peptide hormones. Finally, a methodological approach is given to obtain not only qualitative but also semiquantitative data during immunohistochemical investigations.     

Identification of factors involved in target RNA-directed microRNA degradation

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.     

Prognostic value of circulating levels of stem cell growth factor beta (SCGF beta) in patients with Chagas’ disease and idiopathic dilated cardiomyopathy

Chagas’ disease (CD) often leads to dilated cardiomyopathy (DCM), and during its chronic stage hematopoietic stem or progenitor cells are involved in its pathological process. However, it is not clear whether stem cell growth factor (SCGF) beta can be regulated in patients with CD and idiopathic DCM. In present study, we aim to investigate the plasma SCGF beta concentration and its correlation with echocardiographic parameters and clinical outcome.In this prospective cohort study, SCGF beta levels were quantified in patients with CD (n = 94), DCM (n = 48), and control healthy subjects (n = 25). In comparison with healthy subjects, no statistical difference can be detected in NYHA classes I–II patients. However, SCGF beta was significantly increased in advanced heart failure patients (NYHA III–IV), compared to CD patients without heart failure. There was no group difference between CD and DCM. However, despite this significant increase in advanced heart failure patients, SCGF beta had no significant correlation with echocardiographic parameters, and it cannot be used as a prognostic marker for mortality and heart transplantation.To our best knowledge, this is the first report of SCGF beta in heart failure patients. Although it is significantly increased in advanced heart failure patients caused by CD or DCM, its prognostic value for end points is minor.     

VEB4: Early zygotic mRNA expressed asymmetrically along the animal-vegetal axis of the sea urchin embryo

We have analyzed a gene, designated VEB4 , that is expressed transiently in very early blastulae of the sea urchin, Strongylocentrotus purpuratus . Sequence analysis of the complete open reading frame shows that VEB4 encodes an unusual, highly charged protein with a pl of 9.55. We show here that VEB4 mRNA accumulate in a spatial pattern that is indistinguishable from that of two other recently described genes encoding metallo-endoproteases, SpAN , related to astacin and SpHE , the hatching enzyme (Reynolds et al . 1992). VEB4 and other members of this gene set encode the earliest strictly zygotic gene products that have been identified. The asymmetric accumulation of VEB4 mRNA in non-vegetal blastomeres of the 16 cell embryo and their descendants reflects the animal-vegetal maternal developmental axis.