Phosphorylation of 4E-BP1 in the Mammalian Brain Is Not Altered by LRRK2 Expression or Pathogenic Mutations

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of autosomal dominant familial Parkinson''s disease (PD). LRRK2 encodes a multi-domain protein containing GTPase and kinase enzymatic domains. Disease-associated mutations in LRRK2 variably influence enzymatic activity with the common G2019S variant leading to enhanced kinase activity. Mutant LRRK2 induces neuronal toxicity through a kinase-dependent mechanism suggesting that kinase activity is important for mediating the pathogenic effects of LRRK2 mutations. A number of LRRK2 kinase substrates have been identified in vitro but whether they represent authentic physiological substrates in mammalian cells or tissues is not yet clear. The eukaryotic initiation factor 4E (eIF4E)-binding protein, 4E-BP1, was recently identified as a potential substrate of LRRK2 kinase activity in vitro and in Drosophila with phosphorylation occurring at Thr37 and Thr46. Here, we explore a potential interaction of LRRK2 and 4E-BP1 in mammalian cells and brain. We find that LRRK2 can weakly phosphorylate 4E-BP1 in vitro but LRRK2 overexpression is not able to alter endogenous 4E-BP1 phosphorylation in mammalian cells. In mammalian neurons LRRK2 and 4E-BP1 display minimal co-localization, whereas the subcellular distribution, protein complex formation and covalent post-translational modification of endogenous 4E-BP1 are not altered in the brains of LRRK2 knockout or mutant LRRK2 transgenic mice. In the brain, the phosphorylation of 4E-BP1 at Thr37 and Thr46 does not change in LRRK2 knockout or mutant LRRK2 transgenic mice, nor is 4E-BP1 phosphorylation altered in idiopathic or G2019S mutant PD brains. Collectively, our results suggest that 4E-BP1 is neither a major nor robust physiological substrate of LRRK2 in mammalian cells or brain.     

Respiratory changes due to extreme cold in the Arctic environment

Effects of acute exposure and acclimatisation to cold stress on respiratory functions were investigated in healthy tropical Indian men (n=10). Initial baseline recordings were carried out at Delhi and thereafter serially thrice at the arctic region and once on return to Delhi. For comparison the respiratory functions were also evaluated on Russian migrants (RM;n=7) and Russian natives (RN;n=6). The respiratory functions were evaluated using standard methodology on a Vitalograph: In Indians, there was an initial decrease in lung vital capacity (VC), forced vital capacity (FVC), forced expiratory volume 1st s (FEV1), peak expiratory flow rate (PEFR) and maximum voluntary ventilation (MVV) on acute exposure to cold stress, followed by gradual recovery during acclimatisation for 4 weeks and a further significant improvement after 9 weeks of stay at the arctic region. On return to India all the parameters reached near baseline values except for MVV which remained slightly elevated. RM and RN showed similar respiratory functions at the beginning of acute cold exposure at the arctic zone. RN showed an improvement after 10 weeks of stay whereas RM did not show much change. The respiratory responses during acute cold exposure are similar to those of initial altitude responses.     

Cooperative Use of VCD and XRD for the Determination of Tetrahydrobenzoisoquinolines Absolute Configuration: A Reliable Proof of Memory of Chirality and Retention of Configuration in Enediyne Rearrangements

The absolute configurations (AC) of azaheterocylic compounds resulting from the cascade rearrangement of enediynes involving only light atoms were unambiguously assigned by the joint use of vibrational circular dichroism (VCD) and copper radiation single crystal X‐ray diffraction (XRD). These AC determinations proved that the rearrangements of enediynes proceeded with memory of chirality and retention of configuration. Chirality 25:832–839, 2013. © 2013 Wiley Periodicals, Inc.     

A Simple Novel Agar Diffusion Method for Isolation of Indigenous Microalgae Chlamydomonas sp. CRP7 and Chlorella sp. CB4 from Operational Swampy Top Soil

A simple agar diffusion method is developed where pure colony of Chlamydomonas sp. CRP7 was isolated from Chlorella sp. CB4 mixtures by passing through agar migration with a light exposure of 6,000 lux for 7 h. The main concept behind it is that Chlamydomonas has flagella and the rhodopsin pigment is attracted towards light. Thus the above two microalgae species can be separated from the mixtures as eye spot serves as a navigator and flagella serves as a propeller for Chlamydomonas spp. Further the genomic DNA was isolated and purified from the above mentioned two species after the separation from the mixtures. PCR amplification was carried out for ITS1, 5.8S and ITS2 regions. The amplified products were sequenced and the sequence analysis confirmed that they belong to Chlamydomonas sp. and Chlorella sp. This is an important augmentation for isolation and separation of microalgae.     

An approach for immunodiagnosis of clinical cases of filariasis

In this communication an immunodiagnostic approach has been adopted for detection of antigen and antibody in amicrofilaeamic Mf(-) patients by countercurrent immuno electrophoresis (CCIE) and immunodiffusion (ID). Using Setaria cervi and Immune Complex (IC) antigens, out of fifteen clinical cases the number of positive patients in CCIE were twelve and ten respectively. Sixty percent of the Mf(-) cases were positive in antigen detection against both the homologous and heterologous antibody. In ID nine Mf(-) cases gave precipitin bands against S. cervi antigen while with IC antigens ten patients were positive. In similar experiments, it was found that out of fifteen Mf(-) cases nine and eleven patients were positive in antigen detection against microfilaraemic Mf(+) sera and S. cervi antibody respectively. All the Mf(+) cases were positive in both antibody and antigen detection. From the standpoint of immunodiagnosis the data were analysed by two-way analysis of variance study and a newly developed system using Binomial distribution. The sera from the control group were negative in all the immunodiagnostic tests.     

An operator-induced conformational change in the C-terminal domain of the lambda repressor.

4,4'-bis(1-anilino-8-naphthalenesulfonic acid (Bis-ANS), an environment-sensitive fluorescent probe for hydrophobic region of proteins, binds specifically to the C-terminal domain of lambda repressor. The binding is characterized by positive cooperativity, the magnitude of which is dependent on protein concentration in the concentration range where dimeric repressor aggregates to a tetramer. In this range, positive cooperativity becomes more pronounced at higher protein concentrations. This suggests a preferential binding of Bis-ANS to the dimeric form of the repressor. Binding of single operator OR1 to the N-terminal domain of the repressor causes enhancement of fluorescence of the C-terminal domain bound Bis-ANS. The binding of single operator OR1 also leads to quenching of fluorescence of tryptophan residues, all of which are located in the hinge or the C-terminal domain. Thus two different fluorescent probes indicate an operator-induced conformational change which affects the C-terminal domain. The significance of this conformational change with respect to the function of lambda repressor has been discussed.     

Structural and Functional Properties of Exopolysaccharide Excreted by a Novel <Emphasis Type="Italic">Bacillus anthracis</Emphasis> (Strain PFAB2) of Hot Spring Origin

Exopolysaccharide produced by a unique avirulent Bacillus anthracis strain PFAB2 of hot spring origin has been characterized and its functional properties are investigated which is a first report. Maximum yield of EPS is 7.66 g/l with 2% glucose and 1% peptone as optimum carbon and nitrogen source respectively. The EPS is found to be a homopolymer consisting of only glucose as principle monosaccharide component. Through ¹H NMR study, different dextran-like proton peaks are observed. Molecular weight of the EPS resembles low molecular weight bacterial origin polysaccharides. Melting transition of the EPS has started after 276 °C which indicates good thermal stability. The EPS also shows potent antioxidant activity in terms of DPPH and ABTS mediated free radical scavenging property compared to standard ascorbic acid. Emulsifying property of the EPS is also observed and has shown good emulsification of vegetable oils. The polysaccharide forms a thermo resistant gel during the heating phase, with G′ higher than G″ indicating excellent shear-thinning behaviour and viscoelastic nature of the EPS.     

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains

Alpha-synuclein (α-syn) protein is abundantly expressed mainly within neurons, and exists in a number of different forms - monomers, tetramers, oligomers and fibrils. During disease, α-syn undergoes conformational changes to form oligomers and high molecular weight aggregates that tend to make the protein more insoluble. Abnormally aggregated α-syn is a neuropathological feature of Parkinson''s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Biochemical characterization and analysis of insoluble α-syn using buffers with increasing detergent strength and high-speed ultracentrifugation provides a powerful tool to determine the development of α-syn pathology associated with disease progression. This protocol describes the isolation of increasingly insoluble/aggregated α-syn from post-mortem human brain tissue. This methodology can be adapted with modifications to studies of normal and abnormal α-syn biology in transgenic animal models harbouring different α-syn mutations as well as in other neurodegenerative diseases that feature aberrant fibrillar deposits of proteins related to their respective pathologies.     

Demonstration of an endogenous activator for the Na+, K+-ATPase system

A heat-labile, non-dialysable and protease-sensitive endogenous activator (NaAF) capable of stimulating the Na⁺, K⁺-ATPase system has been demonstrated. The activator (NaAF) activity was partially enriched (about 10 fold) by dialysis (30 kDa cutoff) under negative pressure and pH 4.8 precipitation. The NaAF has been found to occur in the cytosolic fractions of tissues such as the kidney and brain from two different species (rabbit and pig) tested so far. Also, the factor from one tissue stimulates with equal efficacy the Na⁺, K⁺-ATPase systems of other tissues regardless of the species; thus demonstrating universal nature of the activator. Some degree of cross-reactivity was noted between the activating effects of this activator (for the Na⁺,K⁺-ATPase) and that for the H⁺,K⁺-ATPase recently described (J. Biol. Chem. 262:5664–5670, 1987). The purified NaAF obtained from sephacryl S-300 column chromatography activates the pure renal medullary Na⁺,K⁺-ATPase in a dose dependent manner.A preliminary account of this work was published in Fed. Proc. 46(4): 4466, 1987