Neuroprotective Action of Hypothalamic Peptide PRP-1 at Various Time Survivals Following Spinal Cord Hemisection

The purpose of the present study was to evaluate the neuroprotective action of proline-rich peptide-1 (PRP-1) produced by hypothalamic nuclei cells (nuclei paraventricularis and supraopticus) following lateral hemisection of spinal cord (SC). The dynamics of rehabilitative shifts were investigated at various periods of postoperative survival (1–2, 3, and 4 weeks), both with administration of PRP-1 and without it (control). We registered evoked spike flow activity in both interneurons and motoneurons of the same segment of transected and symmetric intact sides of SC and below it on the stimulation of mixed (n. ischiadicus), flexor (n. gastrocnemius) and extensor (n. peroneus communis) nerves. In the control group (administration of 0.9% saline as placebo), no significant decrease of post-stimulus activity of neurons was observed on the transected side by the 2nd week. This activity strongly decreased by week 3 postaxotomy, with some increase on the intact side, possibly of compensatory origin. No shifts occurred by the 4th week. Regardless of the period of administration, PRP-1 increased neuronal activity on the transected side, with the same activation levels on both SC sides. These data were confirmed by histochemical investigation. PRP-1 administration, both daily and every other day, for a period of 2–3 weeks led to prevention of scar formation and promotion of the re-growth of white matter nerve fibers in the damaged area. It also resulted in prevention of neuroglial elements degeneration and reduction in gliosis expression in the lesion supporting neuronal survival. Thus, PRP-1 achieved protection against “tissue stress”, which was also confirmed by the registration of activity on the level of transection and restoration of the motor activity on the injured side. The obtained data propose the possibility of PRP-1 application in clinical practice for prevention of neurodegeneration of traumatic origin.     

On methods of isolation of active, tightly coupled mitochondria of wheat seedlings

Tightly coupled mitochondria can be isolated from wheat seedlings 1) if young seedlings are used, 2) if the grinding medium is buffered and contains bovine serum albumin (BSA), 3) if the isolation procedure, including centrifugation is 10 to 11 minutes long. Mitochondria isolated in this manner oxidize α-ketoglutarate, malate and NADH. Respiratory control (R.C.) values are around 6, 3, and 10, respectively. ADP:O values with those substrates are 4.0, 2.9, and 1.7, respectively. Mitochondria are stable for 3 hours. It is suggested that the technique may be used for preparation of mitochondria for comparative studies since activities of these mitochondria are highly reproducible.     

Combining bleach and mild predigestion improves ancient DNA recovery from bones

The feasibility of genome‐scale studies from archaeological material remains critically dependent on the ability to access endogenous, authentic DNA. In the majority of cases, this represents a few per cent of the DNA extract, at most. A number of specific pre‐extraction protocols for bone powder aimed to improve ancient DNA recovery before library amplification have recently been developed. Here, we test the effects of combining two of such protocols, a bleach wash and a predigestion step, on 12 bone samples of Atlantic cod and domestic horse aged 750–1350 cal. years before present. Using high‐throughput sequencing, we show that combined together, bleach wash and predigestion consistently yield DNA libraries with higher endogenous content than either of these methods alone. Additionally, the molecular complexity of these libraries is improved and endogenous DNA templates show larger size distributions. Other library characteristics, such as DNA damage profiles or the composition of microbial communities, are little affected by the pre‐extraction protocols. Application of the combined protocol presented in this study will facilitate the genetic analysis of an increasing number of ancient remains and will reduce the cost of whole‐genome sequencing.     

Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.     

Alternatively activated macrophages in intestinal helminth infection: effects on concurrent bacterial colitis

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host's response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-alpha response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-alpha in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.     

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.     

PRP-1 Protective Effect against Central and Peripheral Neurodegeneration following n. ischiadicus Transection

We investigated the action of the new hypothalamic proline-rich peptide (PRP-1), normally produced by neurosecretory cells of hypothalamic nuclei (NPV and NSO), 3 and 4 weeks following rat sciatic nerve transection. The impulse activity flow of interneurons (IN) and motoneurons (MN) on stimulation of mixed (n. ischiadicus), flexor (n. gastrocnemius – G) and extensor (n. peroneus communis – P) nerves of both injured and symmetric intact sides of spinal cord (SC) was recorded in rats with daily administration of PRP-1 (for a period of 3 weeks) and without it (control). On the injured side of SC in control, there were no responses of IN and MN on ipsilateral G and P stimulation, while responses were elicited on contralateral nerve stimulation. The neuron responses on the intact side of SC were revealed in a reverse ratio. Thus, there were no effects upon stimulation of the injured nerve distal stump in the control because of the absence of fusion between transected nerve stumps. This was also testified by the atrophy of the distal stump and the absence of motor activity of the affected limb. In PRP-1-treated animals, the responses of SC IN and MN in postaxotomy 3 weeks on the injured side of SC at ipsilateral nerve stimulation and on the intact side at contralateral nerve stimulation were recorded because of the obvious fusion of the severed nerve stumps. The histochemical data confirmed the electrophysiological findings. Complete coalescence of transected fibers together with restoration of the motor activity of the affected limb provided evidence for reinnervation on the injured side. Thus, it may be concluded that PRP-1 promotes nerve regeneration and may be used clinically to improve the outcome of peripheral nerve primary repair.