Cell cycle inhibitory effects of leaf extract from Curcuma vamana M. Sabu & Mangaly on mitotically synchronous cultures of Physarum polycephalum Schw.

Leaf extracts of C. vamana, endemic to Kerala state in India, were found to inhibit cell cycle progression in synchronous cultures of P. polycephalum in a concentration and phase-specific manner. Crude alkaloid extract (CAE) elicited maximum cell cycle delays in comparison to soxhletted chloroform, acetone and aqueous extracts. Total alkaloid content of CAE was found to be 64.9 mg/g. CAE showed lowest DPPH radical scavenging activity. Other extracts with higher free radical scavenging activity exhibited lesser cell cycle inhibiting potential. Upto 21% decrease in nuclear DNA was observed in CAE treated samples. However, genotoxicity as evidenced by comet assay was not observed. The extracts were also found to be non-toxic to human RBCs at the highest concentration tested (750 microg/mL). CAE treatment completely suppressed a 63 kDa polypeptide with a concomitant, but weak induction of a 60 kDa polypeptide suggesting that these may be cell cycle related. CAE was found to possess potent antiproliferative activity against PBLs. The study clearly demonstrates the cell cycle inhibitory activity of C. vamana leaf extracts, with CAE being the most potent of them.

An alternative splicing variant in Clcn7-/- mice prevents osteopetrosis but not neural and retinal degeneration.

The ubiquitously expressed chloride channel 7 (CLCN7) is present within the ruffled border of osteoclasts. Mutations in the CLCN7 gene in humans (homologous to murine Clcn7) are responsible for several types of osteopetrosis in humans, and deficiencies in CLCN7 can present with retinal degeneration and a neuronal storage disease. A previously reported Clcn7(-/-) mouse showed diffuse osteopetrosis accompanied by severe retinal and neuronal degeneration. In contrast, the authors produced a novel Clcn7(-/-) mutant where mice did not develop osteopetrosis but still developed lethal neural and retinal degeneration. In these mice, there was a rapid progressive loss of the outer nuclear layer and photoreceptor layers of the retina. Laminar degeneration and necrosis of neurons in layers IV and V of the cerebral cortex and in the CA2/CA3 regions of the hippocampus were associated with intraneuronal accumulations of autofluorescent granules (periodic acid-Schiff positive). The extensive reactive gliosis was always associated with the accumulation of intraneuronal cytoplasmic material. The authors found, through quantitative real time polymerase chain reaction analyses, that an alternate Clcn7 transcript (previously identified only in bone marrow) showed minimal expression in the brain and eye but moderate expression in bone, which correlates with rescue of the osteopetrotic phenotype in the face of continued retinal and neuronal degeneration. Findings in this knockout mouse model prove that osteopetrotic compression of the brain is not responsible for neuronal and retinal degeneration in CLCN7-deficient mice; rather, they suggest that neurotoxicity is most likely due to lysosomal dysfunction as a result of the functional lack of this chloride channel in the central nervous system and eye.

Netrin-G2 and netrin-G2 ligand are both required for normal auditory responsiveness.

Mice in which netrin-G2 has been genetically inhibited do not startle to an acoustic stimulus, but otherwise perform normally through a behavioral test battery. Light microscopic examination of the inner ear showed no obvious structural abnormalities. Brainstem responses to acoustic stimuli (auditory brainstem responses, ABR) were also present, confirming the lack of any overarching defects in the inner ear or auditory nerve. Genetic inhibition of netrin-G2 ligand produced a nearly identical phenotype, that is, no startle with ABR present, and otherwise normal. This similarity confirms that these two proteins act in the same biological pathway. We have also determined that the affinity between the two proteins is strong, around 2.5 nM, similar to that observed between netrin-G1 and netrin-G1 ligand - 2.3 nM in our hands. The combination of equivalent phenotypes when genetically inhibited coupled with evidence of a strong biochemical interaction supports the notion of a receptor-ligand interaction between these two proteins in vivo. This interaction is critical for auditory synaptic responsiveness in the brain.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo.

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein-protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein-protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

RET(Men2B)-transgene produces sympathoadrenal tumors but does not prevent intestinal aganglionosis in gdnf-/- or gfr alpha-1(-/-) mice.

Multiple endocrine neoplasia type 2B (MEN2B) syndrome is caused by a missense mutation in the RET gene, which replaces Met918 by Thr in the intracellular kinase domain of the protein. This single amino acid substitution transforms the receptor into a constitutively active monomeric kinase (RET(Men2B)) and produces an autosomal dominant syndrome characterized by medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies, and mucosal ganglioneuromas. The ligand, GDNF, stimulates RET activity through a co-receptor, GFR alpha-1. In vitro studies have shown that the kinase and mitogenic properties of RET(Men2B) are enhanced by GDNF/GFR alpha-1 stimulation. A relevant clinical question is whether ablation of either GDNF or GFR alpha-1 could alter penetrance or severity of the MEN2B syndrome. We report that ganglioneuromatous tumors caused by a RET(Men2B) transgene in mice are not affected grossly or microscopically by the absence of gdnf or gfr alpha-1. Loss-of-function mutations in ret, gdnf, or gfr alpha-1 cause pan-intestinal aganglionosis in mice. We find that expression of the RET(Men2B) transgene in enteric neural progenitors, after they colonize the gut, does not prevent intestinal aganglionosis associated with gdnf or gfr alpha-1 deficiency.

Dendritic dynamics in vivo change during neuronal maturation.

In vivo imaging of optic tectal neurons in the intact Xenopus tadpole permits direct observation of the structural dynamics that occur during dendritic arbor formation. Based on images of single DiI-labeled neurons collected at daily intervals over a period of 6 d, we divided tectal cell development into three phases according to the total length of the dendritic arbor. During phase 1, the cell differentiates from a neuroepithelial cell type and extends an axon out of the tectum. The total dendritic branch length (TDBL) is <100 micrometers. During phase 2, when TDBL is 100-400 micrometers, the dendritic arbor grows rapidly. During phase 3, when TDBL is >400 micrometers, the dendritic arbor grows slowly and appears stable. Neurons at different positions along the rostrocaudal developmental axis of the tectum were imaged at 2 hr intervals over 6 hr and at 24 hr intervals over several days. Images collected at 2 hr intervals were analyzed to determine rates of branch additions and retractions. Morphologically complex, phase 3 neurons show half the rate of branch additions and retractions as phase 2 neurons. Therefore, rapidly growing neurons have dynamic dendritic arbors, and slower-growing neurons are structurally stable. The change in growth rate and dendritic arbor dynamics from phase 2 to phase 3 correlates with the developmental increase in synaptic strength in neurons located along the rostrocaudal tectal axis. The data are consistent with the idea that strong synaptic inputs stabilize dendritic arbor structures and that weaker synaptic inputs are permissive for a greater degree of dynamic rearrangements and a faster growth rate in the dendritic arbor.

NMDA receptor activity stabilizes presynaptic retinotectal axons and postsynaptic optic tectal cell dendrites in vivo.

To investigate the role of N-methyl-D-aspartate (NMDA) receptor activity in the stability of the presynaptic axon arbor and postsynaptic dendritic arbors in vivo, we took time-lapse confocal images of single DiI-labeled Xenopus retinotectal axons and optic tectal neurons in the presence and absence of the NMDA receptor antagonist, APV. Retinotectal axons or tectal neurons were imaged at 30-min intervals over 2 h, or twice over a 24-h period. Retinal axons in animals exposed to DL-APV (100 microM) showed an increase in rates of branch additions and a decrease in branch lifetimes over 2 h compared to untreated axons. Under the same experimental conditions, tectal neurons showed a decreased rate of branch tip additions and retractions. APV treatment over 24 h had no apparent effect on axon arbor morphology, but did decrease tectal cell dendritic arbor elaboration. These observations demonstrate that NMDA receptor activity in postsynaptic neurons stabilizes pre- and postsynaptic neuronal morphology in vivo.. However, when NMDA receptor activity is blocked, presynaptic retinal axons respond with increased arbor dynamics while postsynaptic tectal cell dendrites decrease arbor dynamics. Such differential responses of pre- and postsynaptic partners might increase the probability of coactive afferents converging onto a common target under conditions of lower NMDA receptor activity.

Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo.

Glutamatergic retinotectal inputs mediated principally by NMDA receptors can be recorded from optic tectal neurons early during their morphological development in Xenopus tadpoles. As tectal cell dendrites elaborate, retinotectal synaptic responses acquire an AMPA receptor-mediated synaptic component, in addition to the NMDA component. Here, we tested whether glutamatergic activity was required for the elaboration of dendritic arbors in Xenopus optic tectal neurons. In vivo time-lapse imaging of single DiI-labeled neurons shows that the NMDA receptor antagonist APV (100 microM) blocked the early development of the tectal cell dendritic arbor, whereas the AMPA receptor antagonist CNQX (20 microM) or the sodium channel blocker TTX (1 microM) did not. The decreased dendritic development is attributable to failure to add new branches and extend preexisting branches. These observations indicate that NMDA-type glutamatergic activity promotes the initial development of the dendritic arbor. At later stages of tectal neuron development when AMPA receptor-mediated synaptic transmission is strong, both APV and CNQX decrease dendritic arbor branch length, consistent with a role for glutamatergic synaptic transmission in maintaining dendritic arbor structure. These results indicate that AMPA and NMDA receptors can differentially influence dendritic growth at different stages of neuronal development, in correlation with changes in the relative contribution of the receptor subtype to synaptic transmission.

Heart rate variability as an index of cue reactivity in alcoholics.

BACKGROUND: Autonomic responses follow exposure to conditioned stimuli such as contextual factors associated with alcohol ingestion. Heart rate variability is under autonomic control and may be a measure of such response. METHODS: Twenty alcoholics and 23 matched social drinkers (all male) were exposed to a neutral cue and then an alcohol cue in identical settings, during which the electrocardiogram of these subjects was recorded. Time and frequency domain parameters of heart rate variability (HRV) were computed by a blind rater. RESULTS: Coefficient of variation of R-R intervals and absolute powers of HRV spectrum (in frequency bands 0.05-0.15 Hz and 0.01-0.05 Hz) following alcohol cue were significantly higher in alcoholics than social drinkers. The mean heart rate (MHR) failed to reflect this difference. CONCLUSIONS: HRV paradigm appears more sensitive than MHR to measure cue reactivity.

Mesodermal guidance of pioneer axon growth.

Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs the in situ path of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.

Phenomenological study of late-onset schizophrenia.

The phenomenological presentation of late-onset schizophrenia is a topic of considerable debate. This study aims to look at the clinical presentation of late-onset schizophrenia. Charts of all subjects who received a diagnosis of schizophrenia between January 1990 and December 1993 with age of onset being 45 or more were systematically analysed using the OPCRIT checklist. Of the 89 subjects chosen for analysis, 59 satisfied the ICD-10 DCR criteria for schizophrenia and formed the sample of the study. Of them, 20 were males and 39 were females. The mean age of onset for males was 51.6±5.7 years and for females, 53.2±16.7 years. The commonest phenomenon was persecutory delusion (83%) followed by delusion of influence and hallucinations in any modality (66%each). 53% of the subjects had third person auditory hallucinations, while 42% showed negative symptoms. Delusional perception and thought echo were not found in any subject. The implications of these phenomenological findings are discussed.

Error correction during guidance of pioneer axons in the leg of the cockroach embryo.

Axons of the Til and Fe2 pioneer neurons in the legs of insect embryos possess separate and highly stereotyped proximal projections towards the CNS. However, quantitative analyses of deviations from the standard paths during the period of axon growth indicate that transient errors occur unexpectedly often. The distribution of legs with axons following deviant paths among the embryos analyzed is used to determine whether these errors are caused by random developmental noise or by non-random genetic or environmental factors. During the formation of the Til pathway all the errors are characterized by defasciculation of the 2 axons, occur with an average incidence of 7% and are statistically shown to be randomly caused. In comparison, during the formation of the Fe2 pathway the errors are characterized by both defasciculation and elongation in an inappropriate distal direction, occur with an incidence of 16%, and as revealed by statistical analyses, are caused by a non-random factor. Therefore, during pathfinding by these 2 pairs of axons there is a need for error-correcting mechanisms to insure the stereotypy of the final projections. These error-correcting mechanisms are suggested to have properties similar to those producing canalization as proposed by Waddington.