Differential acetylcholinesterase activity in rat cerebrum, cerebellum and hypothalamus

Acetylcholinesterase (AChE) has been purified from three different regions of rat brain using Sephadex G 200 column. SDS PAGE (6%) showed single band for the purified AChE fractions. Purified and lyophilized AChE from different (NH4)2SO4 precipitated fractions of three brain parts were utilized for in vitro enzyme kinetics using Dimethoate (Dmt) as inhibitor. K(m) values for cerebellum and hypothalamus were almost similar whereas cerebrum showed a different K(m) value compared to other two regions. With the drug Rivastigmine it was found that % G1 and G4 forms of AChE in three different parts of brain are different.     

A novel mechanism of FSH regulation of DNA synthesis in the granulosa cells of hamster preantral follicles: involvement of a protein kinase C-mediated MAP kinase 3/1 self-activation loop

The objective was to reveal whether a protein kinase C (PKC [all isozymes])-mediated self-sustaining MAPK3/1 (3/1 extracellular signal regulated kinase 2/1, also known as ERK2/1) activation loop was necessary for FSH- or epidermal growth factor (EGF)-induced DNA synthesis in the granulosa cells of intact preantral follicles. For this purpose, hamster preantral follicles were cultured with FSH or EGF in the presence of selective kinase inhibitors FSH or EGF phosphorylated RAF1, MAP2K1, and MAPK3/1. However, a relatively higher dose of EGF was necessary to sustain the MAPK3/1 activity, which was essential for cyclin-dependent kinase 4 (CDK4) activation and DNA synthesis. In intact preantral follicles, FSH or EGF stimulated DNA synthesis only in the granulosa cells. Sustained activation of MAPK3/1 beyond 3 h was independent of EGFR kinase activity but dependent on PKC activity, which appeared to form a self-sustaining MAPK3/1 activation loop by activating RAF1, MAP2K1, and PLA2G4 (phospholipase A2 [all cytosolic isozymes]). Inhibition of PKC activity as late as 4 h after the administration of FSH or EGF arrested DNA synthesis, which corresponded with attenuated phosphorylation of RAF1 and MAPK3/1, thus suggesting an essential role of PKC in MAPK3/1 activation. Collectively, these data present a novel self-sustaining mechanism comprised of MAPK3/1, PLA2G4, PKC, and RAF1 for CDK4 activation leading to DNA synthesis in granulosa cells. Either FSH or EGF can activate the loop to activate CDK4 and initiate DNA synthesis; however, consistent with our previous findings, FSH effect seems to be mediated by EGF, which initiates the event by stimulating EGFR kinase.     

Infrared surface plasmon resonance: a novel tool for real time sensing of variations in living cells

We developed a novel surface plasmon resonance (SPR) method, based on Fourier transform infrared (FTIR) spectroscopy, as a label-free technique for studying dynamic processes occurring within living cells in real time. With this method, the long (micrometer) infrared wavelength produced by the FTIR generates an evanescent wave that penetrates deep into the sample. In this way, it enables increased depth of sensing changes, covering significant portions of the cell-height volumes. HeLa cells cultivated on a gold-coated prism were subjected to acute cholesterol enrichment or depletion using cyclodextrins. Cholesterol insertion into the cell plasma membrane resulted in an exponential shift of the SPR signal toward longer wavelengths over time, whereas cholesterol depletion caused a shift in the opposite direction. Upon application of the inactive analog alpha-cyclodextrin (alpha-CD), the effects were minimal. A similar trend in the SPR signal shifts was observed on a model membrane system. Our data suggest that FTIR-SPR can be implemented as a sensitive technique for monitoring in real time dynamic changes taking place in living cells.     

Reasons for the apparent variation in the probiotic response

The results obtained by probiotic supplementation are a function of the action of live microorganisms on the very complex population of microorganisms in the gut. The outcome of this extremely complicated interaction is bound to be subject to variation and can be influenced by several factors relating to the product, the administrator and the consumer. If we can define the reasons for these inconsistencies we will be better able to develop improved preparations which when given at the right time and in the right way will give the expected response. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia     

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of Celosia cristata

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled pBlueScript SK+ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower protein concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.     

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.     

In vivo tracing of canonical Wnt signaling in Xenopus tadpoles by means of an inducible transgenic reporter tool

The canonical Wnt pathway is recurrently used during embryogenesis and adult life. To track the cellular output of Wnt signaling in a living organism, we designed a hormone-inducible Wnt responsive system, capable to dynamically and specifically report Wnt pathway activities through eGFP expression. In contrast to previous in vivo reporters, our system essentially avoids interference of consecutive signals by remaining dormant until addition of hormone, which makes it a valuable tool to map canonical Wnt signaling in post-embryonic stages. Transgenic Xenopus laevis embryos were analyzed revealing at tadpole stage in specific tissues and organs cell populations with high Wnt pathway activity.