Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

Antifertility effect of Andrographis paniculata (Nees) in male albino rat

Dry leaf powder of A. paniculata, when fed orally to male albino rats, at a dose level of 20 mg powder per day for 60 days, resulted in cessation of spermatogenesis, degenerative changes in the seminiferous tubules, regression of Leydig cells and regressive and/or degenerative changes in the epididymis, seminal vesicle, ventral prostate and coagulating gland. There was reduction in the weight and fluid content of the accessory glands. The treatment also resulted in accumulation of glycogen and cholesterol in the testis, and increased activities of lactate dehydrogenase in testis and alkaline phosphatase in testis and ventral prostate. The results suggest antispermatogenic and/or antiandrogenic effect of the plant.     

Molecular nature of β-Galactosidase from different tissues in two strains of the house mouse

One inbred mouse strain, C57BL/Kl, has high galactosidase activities in all tissues while another strain, DBA/2/Kl, has low activities determined by the Bgs locus. Beta-Galactosidase from these two strains was partly purified by a five-step procedure: acidification, ammonium sulfate precipitation, gel filtration at two pHs, and isoelectric focusing. No qualitative differences were found between the enzyme preparations from the two strains. They had identical heat inactivation curves, pH optima, molecular weight, and isoelectric points, and the Km values were very similar. It thus seems that this genetic difference in enzyme activity probably cannot be explained by a variation of the galactosidase-specific activity but rather reflects a difference in number of enzyme molecules. Eight different isoenzymes were separated from liver, kidney, and spleen. Each isoenzyme has a different electrophoretic mobility and there is a stepwise increase in molecular weight from 143,000 to 380,000 beginning with the protein having the lowest isoelectric point. A likely interpretation is that the isoenzymes bind a smaller polypeptide in varying numbers in addition to the enzymatic polypeptide per se.     

A novel ABCB11 mutation in an Iranian girl with progressive familial intrahepatic cholestasis

Progressive familial intrahepatic cholestasis is an autosomal recessive liver disorder caused by (biallelic) mutations in the ATP8B1 of ABCB11 gene. A nine-year-old girl with cholestasis was referred for genetic counseling. She had a family history of cholestasis in two previous expired siblings. Genetic analysis of the ABCB11 gene led to the identification of a novel homozygous mutation in exon 25. The mutation 3593- A > G lead to a missense mutation at the amino acid level (His1198Arg). This mutation caused PFIC2 due to abnormal function in the bile salt export pump protein (BSEP).     

Aging of mesenchymal stem cell in vitro

Background  

A hot new topic in medical treatment is the use of mesenchymal stem cells (MSC) in therapy. The low frequency of this subpopulation of stem cells in bone marrow (BM) necessitates their in vitro expansion prior to clinical use. We evaluated the effect of long term culture on the senescence of these cells.