Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Discovery and characterization of endogenous cannabinoids.

The characterization of cannabinoid receptors and signal transduction mechanisms provided the impetus for the searching for endogenous ligands for this system. The result was a family of fatty acid derivatives that interact with cannabinoid receptors to varying degrees. The two ligands that have received the most attention are anandamide (AN) and 2-arachidonolyl-glycerol (Ara-Gl). They are both present in central as well as peripheral tissues. Mechanisms for the synthesis and metabolism of AN have been described. Presently, the physiological stimuli for production and release of AN are unknown. As a result, elucidation of its physiological role remains elusive. However, it seems reasonable to conclude that both AN and 2-Ara-Gl interact with cannabinoid receptors in both peripheral and central tissue to produce a wide range of effects. Administration of these ligands to laboratory animals produce effects that are quite similar to those elicited by delta9-tetrahydrocannabinol (THC), the psychoactive constituent in marijuana. Nevertheless, there are some pharmacological differences between the plant-derived THC and the endogenous cannabinoids that could be due to either pharmadynamic or pharmacokinetics dissimilarities. Extensive structure-activity relationship studies have provided some vital insights into the actions of the endogenous ligands. First and foremost, systematic structural alterations in AN have additional support that it is acting at the cannabinoid receptors in a fashion similar to that of THC. Development of metabolically stable analogs of AN, as well as those with greater receptor affinity, have helped substantiate AN and THC similarities. Nevertheless, pharmacological differences remain between the endogenous and exogenous ligands. Whether these differences are due to the nature of their interaction with the cannabinoid receptors, activation of unique signaling pathways, interactions with non-cannabinoid receptors, or pharmacokinetic considerations remain to be resolved.     

Crinum alkaloids: their chemistry and biology

The current staus of research of free and glycosylated alkaloids occuring in members of the genus Crinum is reviewed. The distribution, isolation, spectral properties, structural properties, inter conventions and biological acitivities of these alkaloids are presented.     

Endothelial nitric oxide synthase is regulated by ERK phosphorylation at Ser602

eNOS (endothelial nitric oxide synthase) contains a MAPK (mitogen-activated protein kinase)-binding site associated with a major eNOS control element. Purified ERK (extracellular-signal-regulated kinase) phosphorylates eNOS with a stoichiometry of 2–3 phosphates per eNOS monomer. Phosphorylation decreases NO synthesis and cytochrome c reductase activity. Three sites of phosphorylation were detected by MS. All sites matched the SP and TP MAPK (mitogen-activated protein kinase) phosphorylation motif. Ser⁶⁰² lies at the N-terminal edge of the 42-residue eNOS AI (autoinhibitory) element. The pentabasic MAPK-binding site lies at the opposite end of the AI, and other critical regulatory features are between them. Thr⁴⁶ and Ser⁵⁸ are located in a flexible region associated with the N terminus of the oxygenase domain. In contrast with PKC (protein kinase C), phosphorylation by ERK did not significantly interfere with CaM (calmodulin) binding as analysed by optical biosensing. Instead, ERK phosphorylation favours a state in which FMN and FAD are in close association and prevents conformational changes that expose reduced FMN to acceptors. The close associations between control sites in a few regions of the molecule suggest that control of signal generation is modulated by multiple inputs interacting directly on the surface of eNOS via overlapping binding domains and tightly grouped targets.     

Production of triploid plants from endosperm cultures of Phlox drummondii

Triploid plants of ornamental Phlox drummondii Hook. were raised from cultures of endosperm excised from immature fruits having zygotic embryo at early dicotyledonous stage. Endosperm tissue was firstly cultured with the embryo on the Murashige and Skoog’s (MS) medium supplemented with 5 μM 6-benzylaminopurine (BAP) + 10 μM α-napthaleneacetic acid (NAA) for 7 d and recultured after the embryo was removed. A friable callus appeared two weeks after removal of the embryo and it became compact callus mass in another three weeks. Upon transfer of this 5-week-old callus to the MS medium with 10 μM BAP + 2.5 μM indole-3-acetic acid (IAA), maximum percentage of green nodular shoot buds appeared from which regenerated dwarf shoots. Elongation of the dwarf shoots, however, required transfer of the individual dwarf shoots excised from the callus on the fresh medium and best results achieved on medium with low concentration of IAA (0.5 μM) in presence of 10 μM BAP. The shoots were then rooted in vitro and plants subsequently established in pots containing soil. Over 70 % of plants were triploid with a chromosome number of 2n=3x=21. Size of stem, leaves, flowers, pollen, and stomata of these triploid plants were higher and the plants were more vigorous as compared to naturally occurring diploid plants. In particular, flowers showed bright colour with enlarged central eye adding to their ornamental value.     

Impact of Postovulatory Food Deprivation on the Ova Transport, Hormonal Profiles and Metabolic Changes in Sows

The effect of food deprivation on ova transport, hormonal profiles and metabolic changes was studied in 20 crossbred multiparous sows during their second oestrus after weaning. To determine the time of ovulation, transrectal ultrasonographic examination was performed. The sows were divided into 2 groups, one control group (C-group), which was fed according to Swedish standards, and one experimental group (E-group). The E-group sows were deprived of food from the first morning meal after ovulation until slaughter. Blood samples were collected every second hour from about 12 h before expected ovulation in the second oestrus after weaning until slaughter and were analysed for progesterone, prostaglandin F_2α-metabolite, insulin, glucose, free fatty acids and triglycerides. All sows were slaughtered approximately 48 h after ovulation and the genital tract was recovered. The isthmic part of the oviduct was divided into 3 equally long segments and flushed separately with phosphate buffered saline (PBS). Uterine horns were also flushed with PBS. A significantly greater number of ova were found in the first and second part of the isthmus in the E-group (p = 0.05) while in the C-group most of the ova were found in the third part of the isthmus or the uterus (p = 0.01). The level of prostaglandin F_2α-metabolite was significantly higher in the E-group compared with the C-group. The concentration of progesterone increased in both groups after ovulation but there were no significant differences between the groups. The other blood parameters showed that the food-deprived sows were in a catabolic state. The 48 h period of fasting results, directly or indirectly in an delayed ova transport, which may be due to a delayed relaxation in the smooth circular muscle layer of the isthmus.     

Appraisal of Transdermal Water-in-Oil Nanoemulgel of Selegiline HCl for the Effective Management of Parkinson’s Disease: Pharmacodynamic,Pharmacokinetic, and Biochemical Investigations

In the present study, the potential of transdermal nanoemulsion gel of selegiline hydrochloride for the treatment of Parkinson’s disease was investigated. Water-in-oil nanoemulsions were developed by comparing low- and high-energy methods and were subjected to thermodynamic stability tests, in vitro permeation, and characterization studies. In vitro studies indicated that components of nanoemulsion acted as permeation enhancers with highest flux of 3.531 ± 1.94 μg/cm²/h from nanoemulsion SB6 containing 0.5 mg selegiline hydrochloride, 3% distilled water, 21% S _mix (Span 85, Tween 80, PEG 400), and 76% isopropyl myristate by weight. SB6 with the least droplet size of 183.4 ± 0.35 nm, polydispersity index of 0.42 ± 0.06 with pH of 5.9 ± 0.32 and viscosity of 22.42 ± 0.14 cps was converted to nanoemulsion gel NEGS4 (viscosity = 22,200 ± 400 cps) by addition of Viscup160® for ease of application and evaluated for permeation, safety, and pharmacokinetic profile in Wistar rats. It provided enhancement ratio 3.69 times greater than conventional gel. NEGS4 showed 6.56 and 5.53 times increase in bioavailability in comparison to tablet and conventional gel, respectively, along with sustained effect. Therefore, the developed water-in-oil nanoemulsion gel promises to be an effective vehicle for transdermal delivery of selegiline hydrochloride.