Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.     

Seroprevalence of Strongyloides stercoralis infection in a South Indian adult population

BackgroundThe prevalence of Strongyloides stercoralis infection is estimated to be 30–100 million worldwide, although this an underestimate. Most cases remain undiagnosed due to the asymptomatic nature of the infection. We wanted to estimate the seroprevalence of S. stercoralis infection in a South Indian adult population.MethodsTo this end, we performed community-based screening of 2351 individuals (aged 18–65) in Kanchipuram District of Tamil Nadu between 2013 and 2020. Serological testing for S. stercoralis was performed using the NIE ELISA.ResultsOur data shows a seroprevalence of 33% (768/2351) for S. stercoralis infection which had a higher prevalence among males 36% (386/1069) than among females 29.8% (382/1282). Adults aged ≥55 (aOR = 1.65, 95% CI: 1.25–2.18) showed higher adjusted odds of association compared with other age groups. Eosinophil levels (39%) (aOR = 1.43, 95% CI: 1.19–1.74) and hemoglobin levels (24%) (aOR = 1.25, 95% CI: 1.11–1.53) were significantly associated with S. stercoralis infection. In contrast, low BMI (aOR = 1.15, 95% CI: 0.82–1.61) or the presence of diabetes mellitus (OR = 1.18, 95% CI: 0.83–1.69) was not associated with S. stercoralis seropositivity.ConclusionsOur study provides evidence for a very high baseline prevalence of S. stercoralis infection in South Indian communities and this information could provide realistic and concrete planning of control measures.     

A Highlights from MBoC Selection: Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions, including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer’s disease. Here we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show selective inhibition of Cdk5/p25 ­hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson’s disease.     

Specific inhibition of cyclin-dependent kinase 5 activity induces motor neuron development in vivo

Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in motor neuron development remains unexplored. Here, using gain and loss-of-function analyses in developing zebrafish embryos, we report that cdk5 plays a critical role in spinal and cranial motor neuron development. Cdk5 knockdown results in supernumerary spinal and cranial motor neurons. While a dominant negative, kinase-dead cdk5 promotes the generation of supernumerary motor neurons; over-expression of cdk5 suppresses motor neuron development. Thus, modulating cdk5 activity seems promising in inducing motor neuron development in vivo.     

Development of a process to manufacture PEGylated orally bioavailable insulin

To make insulin orally bioavailable, insulin was modified by covalent attachment (conjugation) of a short-chain methoxy polyethylene glycol (mPEG) derivative to the ε-amino group of a specific amino acid residue (LysB(29)). During the conjugation process, activated PEG can react with any of the free amino groups, the N-terminal of the B chain (PheB(1)), the N-terminal of the A chain (GlyA(1)), and the ε-amino group of amino acid (LysB(29)), resulting in a heterogeneous mixture of conjugated products. The abundance of the desired product (Methoxy-PEG(3)-propionyl--insulin at LysB(29):IN-105) in the conjugation reaction can be controlled by changing the conjugation reaction conditions. Reaction conditions were optimized for maximal yield by varying the proportions of protein to mPEG molecule at various values of pH and different salt and solvent concentrations. The desired conjugated molecule (IN-105) was purified to homogeneity using RP-HPLC. The purified product, IN-105, was crystallized and lyophilized into powder form. The purified product was characterized using multiple analytical methods including ESI-TOF and peptide mapping to verify its chemical structure. In this work, we report the process development of new modified insulin prepared by covalent conjugation of short chain mPEG to the insulin molecule. The attachment of PEG to insulin resulted in a conjugated insulin derivative that was biologically active, orally bioavailable and that showed a dose-dependent glucose lowering effect in Type 2 diabetes patients.     

Green Revolution Trees: Semidwarfism Transgenes Modify Gibberellins, Promote Root Growth, Enhance Morphological Diversity, and Reduce Competitiveness in Hybrid Poplar

Semidwarfism has been used extensively in row crops and horticulture to promote yield, reduce lodging, and improve harvest index, and it might have similar benefits for trees for short-rotation forestry or energy plantations, reclamation, phytoremediation, or other applications. We studied the effects of the dominant semidwarfism transgenes GA Insensitive (GAI) and Repressor of GAI-Like, which affect gibberellin (GA) action, and the GA catabolic gene, GA 2-oxidase, in nursery beds and in 2-year-old high-density stands of hybrid poplar (Populus tremula × Populus alba). Twenty-nine traits were analyzed, including measures of growth, morphology, and physiology. Endogenous GA levels were modified in most transgenic events; GA₂₀ and GA₈, in particular, had strong inverse associations with tree height. Nearly all measured traits varied significantly among genotypes, and several traits interacted with planting density, including aboveground biomass, root-shoot ratio, root fraction, branch angle, and crown depth. Semidwarfism promoted biomass allocation to roots over shoots and substantially increased rooting efficiency with most genes tested. The increased root proportion and increased leaf chlorophyll levels were associated with changes in leaf carbon isotope discrimination, indicating altered water use efficiency. Semidwarf trees had dramatically reduced growth when in direct competition with wild-type trees, supporting the hypothesis that semidwarfism genes could be effective tools to mitigate the spread of exotic, hybrid, and transgenic plants in wild and feral populations.Semidwarfism is a valuable trait in many crop species and agricultural environments. In cereal crops, it can result in decreased lodging, increased yield, and improved harvest index (Dalrymple, 1985; Hedden, 2003). Therefore, it was a critical foundation of the “Green Revolution” that resulted in large improvements of yield in wheat (Triticum aestivum) and rice (Oryza sativa; Hargrove and Cabanilla, 1979; Perovic et al., 2008). Semidwarfism has had substantial benefits for fruit tree production, where it enables earlier fruit bearing, higher yields, and easier harvests in orchards (Battisini and Battisini, 2005). Semidwarf woody species are also extensively used in ornamental horticulture, where they allow more compact forms to be fit into small areas around homes and on streets and reduce the need for pruning to avoid interference with structures and transmission lines (Busov et al., 2003).Although against the current orthodoxy of forest tree breeding, where height growth is emphasized, semidwarfism might also have benefits for wood and biomass production (Bradshaw and Strauss, 2001). Such trees could be useful if they were less prone to wind throw due to their shorter, stockier forms and expected greater allocation to roots. Reduced stature could also result in less bending and slanting of trunks in the face of wind and gravity on hillslopes and thus reduce the extent of reaction wood formation, which degrades the performance and value of solid wood and pulp products. Reduced height and increased allocation of growth to roots might enhance stress tolerance, soil nutrient uptake, bioremediation, and carbon sequestration.Semidwarfism can be achieved by the modification of several types of genes and physiological mechanisms, but the most prevalent and advanced forms in agriculture affect GAs or their signaling (for review, see Busov et al., 2008). GAs are endogenous plant hormones that influence several aspects of plant growth and development, including seed germination, leaf expansion, shoot growth, cell division, flower induction, and fruit development (Sun and Gubler, 2004; Fleet and Sun, 2005; Swain and Singh, 2005). With respect to shoot growth, the most obvious effect of GA is its promotion of stem elongation by stimulating both cell elongation and division (Marth et al., 1956). GA modification also has significant effects on plant biochemistry, changing the amounts and distribution of a wide variety of metabolites in shoots and roots (Rossetto et al., 2003; Chen et al., 2004; Busov et al., 2006).Little is known about how semidwarfism affects belowground growth. GA has been shown to play a controlling role in lateral root development (Gou et al., 2010), and GA and ethylene synergistically promote both the initiation and growth of adventitious roots (Osmont et al., 2007). In tomato (Solanum lycopersicum), isogenic GA-deficient mutants (gib) allocate more biomass to roots compared with shoots (Nagel et al., 2001). In poplar (Populus spp.), semidwarf transgenic plants grown in vitro had a lower shoot-to-root ratio, which was at least partly due to proliferation of lateral roots (Busov et al., 2006; Gou et al., 2010).As a domestication trait, semidwarfism has been proposed as a means for mitigating the spread of transgenic plants within and outside of crop environments (Al-Ahmad et al., 2005). The genetic dominance of most semidwarfism transgenes would cause reduced height growth in transgene-containing progeny, reducing their ability to compete for light. Moreover, because of the close linkage of the semidwarfism genes to other genes that were cointroduced on the same plasmid, they would also powerfully retard their spread or introgression, even in cases where the linked transgene would, on their own, impart a selective advantage. However, there have been very few plant species where this concept has been explicitly tested (Al-Ahmad and Gressel, 2006; Gressel and Valverde, 2009), and we know of no examples in woody or perennial plants.To study the effects of semidwarfism genes in a woody plant grown under field conditions, we inserted a number of dominant GA-modifying transgenes into hybrid poplar (Populus tremula × Populus alba), the widely recognized model woody plant for genomics and biotechnology (Herschbach and Kopriva, 2002; Brunner et al., 2004a; Tuskan et al., 2004). Most of the genes studied were overexpressed forms of GA 2-oxidase, GA-Insensitive (GAI), or Repressor of GAI-Like (RGL), all known to cause semidwarfism in other plant species. GA 2-oxidase is a major GA catabolic enzyme in plants, and GAI and RGL are negative regulators of the GA signal transduction pathway (Appleford et al., 2007; Busov et al., 2008). The transgenic trees were first analyzed in the greenhouse (Busov et al., 2006) and then assayed for their effect on height growth in a 2-year field trial (Zawaski et al., 2011), from which we selected 10 transgenic events that grew at approximately three-quarters the rate of wild-type trees. The goal was to select semidwarf trees whose phenotype was not so severe as to be irrelevant to possible crop uses but strong enough to give a clear phenotype in a field study. In this study, we analyzed changes in a number of morphological, physiological, and growth traits and investigated the prospect for semidwarfism to be used as a mitigation tool to reduce the frequency of spread of transgenic and exotic species.     

Conservation genetics of endangered medicinal plant Commiphora wightii in Indian Thar Desert

To ascertain the conservation priorities and strategies for Commiphora wightii, an endangered medicinal plant of Indian Thar Desert, genetic diversity was estimated within and among different populations. The total of 155 amplification products were scored using ten each of RAPD and ISSR primers, exhibiting an overall 86.72% polymorphism across 45 individuals representing eight populations. The cumulative data of two markers were used to compute pair-wise distances. The Neighbor-Joining tree revealed high genetic differentiation among populations except Kiradu population. Nei's gene diversity (h) ranged between 0.082 and 0.193 with total diversity at species level is 0.294. Shannon's information index (I) ranged between 0.118 and 0.275 with an overall diversity of 0.439. Analysis of molecular variance showed more diversity among population level (56.65%) than at within population level (43.35%). The low gene flow value (Nm = 0.349) and high coefficient of genetic differentiation (G_ST = 0.589) and high fixation index (F_ST = 0.566) demonstrated elevated genetic differentiation among the population and can be predicted that these populations are not in Hardy–Weinberg proportions. Principal Co-ordinate Analysis confirms that Akal population has become phylogenetically more distinct and less diverse than the rest of the samples. Mantel's test revealed no correlation between genetic and geographical distances of populations (R² = 0.122). Overall highest diversity was observed in the population of Machiya Safari Park and Kiradu, while lowest in Akal population, later may constitute an evolutionary significant unit, having merit for special management.     

Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases

Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.