Metabolism

Dysregulation of sleep and metabolism has enormous health consequences. Sleep
loss is linked to increased appetite and insulin insensitivity, and epidemiological studies
link chronic sleep deprivation to obesity-related disorders. Interactions between sleep and
metabolism involve the integration of signalling from brain regions regulating sleep,
feeding, and metabolism, as well as communication between the brain and peripheral
organs. In this series of studies, using the fruit fly as a model organism, we investigated
how feeding information is processed to regulate sleep, and how peripheral tissues
regulate sleep through the modulation of energy stores.
In order to address these questions, we performed a large RNAi screen to identify
novel genetic regulators of sleep and metabolism. We found that, the mRNA/DNA
binding protein, Translin (trsn), is necessary for the acute modulation of sleep in
accordance with feeding state. Flies mutant for trsn or selective knockdown of trsn in
Leucokinin (Lk) neurons abolishes starvation-induced sleep suppression. In addition, genetic silencing of Lk neurons or a mutation in the Lk locus also disrupts the integration
between sleep and metabolism, suggesting that Lk neurons are active during starvation.
We confirmed this hypothesis by measuring baseline activity during fed and starved
states. We found that LHLK neurons, which have axonal projections to sleep and
metabolic centers of the brain, are more active during starvation. These findings suggest
that LHLK neurons are modulated in accordance with feeding state to regulate sleep.
Finally, to address how peripheral tissues regulate sleep, we performed an RNAi
screen, selectively knocking down genes in the fat body. We found that knockdown of
Phosphoribosylformylglycinamidine synthase (Ade2), a highly conserved gene involved
the biosynthesis of purines, regulates sleep and energy stores. Flies heterozygous for two
Ade2 mutations are short sleepers and this effect is partially rescued by restoring Ade2 to
the fly fat body. These findings suggest Ade2 functions within the fat body to promote
both sleep and energy storage, providing a functional link between these processes.
Together, the experimental evidence presented here provides an initial model for how the
peripheral tissues communicate to the brain to modulate sleep in accordance with
metabolic state.

The pathology of Alzheimer's disease (AD) remains elusive. Competing evidence links amylois \U+fffd\-peptide (A\U+fffd\) amyloid formation to the phenotype of AD (1). The mechanism of amyloid fibril formation has been an ongoing investigation for many years. A\U+fffd\10-23 peptide, a fragment of A\U+fffd\1-42 peptide, contained crucial hydrophobic core residues (2). In this study, an investigation was launched to study the aggreagation process of A\U+fffd\1023 peptide and its ability to form amyloid fibrils. Furthermore, the presence of its hydrophobic core showed importance for its ability to aggregate and form amyloid fibrils. Thereafter, the inhibition of A\U+fffd\1-42 peptide aggregation was studied by using pyrimidine-based compounds. A\U+fffd\1-42 peptides, known to be neurotoxic, aggregate to form amyloid fibrils (3). This investigation may provide insight into the development of novel small molecular candidates to treat AD.

We have studies oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation... Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal rold in controlling oxidized RNA and protecting cells under oxidative stress.

p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway.

We employed three genotypes of GAD 65, wildtype (GAD 65 +/+), heterozygous (GAD 65 +/-) and knockout (GAD 65 -/-) to investigate the role of GAD 65 in survival of pancreatic islets. We analyzed the mRNA expression of pro-survival proteins including Bcl2 and Bax in pancreas of wildtype, heterozygous and knockout using Reverse Transcriptase Polymerase Chain Reaction (RTPCR). The level of expression of Bcl2 mRNA was down regulated in knockout mice pancreas and Bax to Bcl2 ratio was found higher in knockout mice pancreas suggesting higher cell death rate. However, further studies are required to recognize and understand the specific connections between apoptotic pathways and GAD 65 in pancreatic islets.

Brain glutamic acid decarboxylase 65 (GAD65) catalyzes the rate-limiting step in the biosynthesis of the major inhibitory neurotransmitter-amino butyric acid (GABA) from the substrate L-glutamic acid. Severe lapse in GABA neurotransmission is one of the etiologies documented in the manifestation of certain neurodegenerative diseases such as epilepsy, Parkinson's disease, Huntington's disease etc. Because GAD65 synthesizes GABA, any modulation of GAD65, therefore, has direct implications on the quanta of GABA released at the synapse. Hence, the major objective of this study was to focus on the regulation of GAD65, with special emphasis on investigating the proteolytic cleavage of fGAD65. Previously, we have shown in vitro that GAD65 was cleaved to form its truncated form (tGAD65), which was more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiologica l stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this study, we examined the cleavage of fGAD65 under a range of conditions encompassing both physiological and pathological aspects, including rats under ischemia/reperfusion insult, rat brain synaptosomes or primary neuronal cultures subjected to excitotoxic stimulation with KCl. It was observed that the formation of tGAD65 progressively increased with increasing stimulus concentration. More importantly, cleavage of synaptic vesicle (SV) - associated fGAD65 by calpain was demonstrated, and the resulting tGAD65 harboring the active site of the enzyme was detached from the SVs. Vesicular uptake of the newly synthesized GABA into the SVs was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate indica

The purpose of this study was to determine the acute effects of caffeine supplementation on strength and muscular endurance in resistance-trained women. In a randomized manner, 15 women consumed caffeine (6 mg/kg) or placebo (PL) seven days apart. Sixty minutes following supplementation, participants performed a one repetition maximum (1RM) barbell bench press test and repetitions to failure at 60% of 1RM. Heart rate and blood pressure were assessed at rest, 60 minutes post-consumption, and immediately following completion of repetitions to failure. Repeated measures ANOVA indicated a significantly greater bench press maximum with caffeine (p<0.05) (52.9 « 11.1 kg vs. 52.1 « 11.7 kg) with no significant differences between conditions in 60% 1RM repetitions (p=0.81). Systolic blood pressure was significantly greater post-exercise, with caffeine (p<0.05) (116.8 « 5.3 mmHg vs. 112.9 « 4.9 mmHg). Our findings indicate a moderate dose of caffeine may be sufficient for enhancing strength performance in resistance-trained women.

RNA damage occurring under oxidative stress has been shown to cause RNA dysfunction and must be detrimental to cells and organisms. We propose that damaged RNA can be removed by specific RNA surveillance activities. In this work, we investigated the role of polynucleotide phosphorylase (PNPase), a 3'->5' exoribonuclease, in protecting the cells against oxidative stress and eliminating oxidatively-damaged RNA. Previously, it was reported that E. coli PNPase has a higher affinity to poly(8-oxoG:A). We further confirmed that E. coli PNPase can specifically bind to an oxidized RNA with a high affinity. An E. coli strain deficient in PNPase (pnp) is hypersensitive to hydrogen peroxide (H2O2). Importantly, the level of H2O2-induced RNA damage, measured by the content of 8-hydroxyguanosine, increases significantly in the pnp mutant cells. Consistent with the notion that PNPase plays a direct role in these processes, introduction of the pnp gene encoding E. coli PNPase can restore the viability and RNA oxidation level of the pnp mutant cells in response to H2O2 treatment. Interestingly, degradosome-association is not required for PNPase to protect cell against oxidative stress. PNPase is evolutionary conserved in most of organisms of all domains of life. The human polynucleotide phosphorylase (hPNPase) localizes mainly in mitochondria and plays pleiotropic roles in cell differentiation and has been previously shown to bind 8- oxoG-RNA with a high affinity. Here we show that similar to E. coli PNPase, hPNPase plays an indispensable role in protecting HeLa cells against oxidative stress. The viability in HeLa cell and 8-oxoG levels in RNA are inversely correlated in response to H2O2- treatment. After removal of oxidative challenge, the elevated level of 8-oxoG in RNA decreases, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA.