Mitotic factors from mammalian cells: A preliminary characterization

The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat-labile, Ca²⁺-sensitive, nondialyzable protein with a sedimentation value of 4-5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.     

What makes a long tail short? Testing Allen's rule in the toque macaques of Sri Lanka

Allen's rule (1877) predicts ecogeographical anatomical variation in appendage proportions as a function of body temperature regulation. This phenomenon has been tested in a variety of animal species. In macaques, relative tail length (RTL) is one of the most frequently measured appendages to test Allen's rule. These studies have relied on museum specimens or the invasive and time-consuming capturing of free-ranging individuals. To augment sample size and lessen these logistical limitations, we designed and validated a novel noninvasive technique using digitalized photographs processed using LibreCAD, an open-source 2D-computer-aided design (CAD) application. This was used to generate pixelated measurements to calculate an RTL equivalent, the Tail to Trunk Index (TTI) = (tail [tail base to anterior tip] pixel count/trunk [neck to tail base] pixel count). The TTI of 259 adult free-ranging toque macaques (Macaca sinica) from 36 locations between 7 and 2,087 m above sea level (m.a.s.l.) was used in the analysis. Samples were collected from all three putative subspecies (M. s. sinica, aurifrons, and opisthomelas), at locations representing all altitudinal climatic zones where they are naturally distributed. These data were used to test whether toque macaque tail length variation across elevation follows Allen's rule, predicting that RTL decreases with increasing elevation and lower temperature. Our results strongly supported this prediction. There was also a statistically significant, negative correlation between elevation and annual average temperature. The best predictor for the TTI index was elevation. Significant subspecies differences in RTL are linked in part to their ecological and altitudinal niche separation, but overall the variation is seen as the species' adaptation to climate. The method developed for the quick morphometric assessment of relative body proportions, applicable for use on unhabituated free-ranging animals, widens the range of materials available for research studying morphological characteristics and their evolution in primates.     

Synthesis, spectroscopy, structure and photophysical properties of dinaphthylmethylarsine complexes of palladium(II) and platinum(II)

Dinaphthylmethylarsine complexes of palladium(II) and platinum(II) with the formulae [MX₂L₂] (M = Pd, Pt; L = di(1-naphthyl)methylarsine = Nap₂AsMe and X = Cl, Br, I), [M₂Cl₂(μ-Cl)₂L₂], [PdCl(S₂CNEt₂)L], [Pd₂Cl₂(μ-OAc)₂L₂] and [MCl₂(PR₃)L] (PR₃ = PEt₃, PPr₃, PBu₃, PMePh₂) have been prepared. These complexes have been characterized by elemental analyses, IR, Raman, NMR (¹H, ¹³C, ³¹P) and UV-vis spectroscopy. The stereochemistry of the complexes has been deduced from the spectroscopic data. The crystal structures of trans-[PdCl₂(PEt₃)(Nap₂AsMe)] and of [Pd(S₂CNEt₂)₂], a follow-up product, were determined. The UV-vis spectra of [MX₂L₂] complexes show a red shift on going from X = Cl to X = I. The complexes [PdX₂L₂] and [PtX₂L₂] are strongly luminescent in fluid solution and in the solid at ambient temperature.     

E,E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins

A series of E,E,E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC(50) figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50-100 microM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.     

Transforming growth factor-β protein inversely regulates in vivo differentiation of interleukin-17 (IL-17)-producing CD4+ and CD8+ T cells

TGF-β is a pleiotropic cytokine that predominantly exerts inhibitory functions in the immune system. Unexpectedly, the in vitro differentiation of both Th17 and Tc17 cells requires TGF-β. However, animals that are impaired in TGF-β signaling (TGF-βRIIDN mice) display multiorgan autoimmune disorders. Here we show that CD4(+) T cells from TGF-βRIIDN mice are resistant to Th17 cell differentiation and, paradoxically, that CD8(+) T cells from these animals spontaneously acquire an IL-17-producing phenotype. Neutralization of IL-17 or depletion of CD8(+) T cells dramatically inhibited inflammation in TGF-βRIIDN mice. Therefore, the absence of TGF-β triggers spontaneous differentiation of IL-17-producing CD8(+) T cells, suggesting that the in vivo and in vitro conditions that promote the differentiation of IL-17-producing CD8(+) T cells are distinct.     

Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.     

Differences in clinical features according to Boryoung and Karp genotypes of Orientia tsutsugamushi

Background

Scrub typhus is an infectious disease caused by Orientia tsutsugamushi. The differences in virulence of O. tsutsugamushi prototypes in humans are still unknown. We investigated whether there are any differences in the clinical features of the Boryoung and Karp genotypes.

Methodology/Principal Findings

Patients infected with O. tsutsugamushi, as Boryoung and Karp clusters, who had visited 6 different hospitals in southwestern Korea were prospectively compared for clinical features, complications, laboratory parameters, and treatment responses. Infected patients in the Boryoung cluster had significantly more generalized weakness, eschars, skin rashes, conjunctival injection, high albumin levels, and greater ESR and fibrinogen levels compared to the Karp cluster. The treatment response to current antibiotics was significantly slower in the Karp cluster as compared to the Boryoung cluster.

Conclusion

The frequency of occurrence of eschars and rashes may depend on the genotype of O. tsutsugamushi.