The molecular basis of differential subcellular localization of C2 domains of protein kinase C-alpha and group IVa cytosolic phospholipase A2

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.     

Association of ischemic stroke onset time with presenting severity,acute progression,and long-term outcome: A cohort study

BackgroundPreclinical data suggest circadian variation in ischemic stroke progression, with more active cell death and infarct growth in rodent models with inactive phase (daytime) than active phase (nighttime) stroke onset. We aimed to examine the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in human ischemic stroke.Methods and findingsIn a Korean nationwide multicenter observational cohort study from May 2011 to July 2020, we assessed circadian effects on initial stroke severity (National Institutes of Health Stroke Scale [NIHSS] score at admission), END, and favorable functional outcome (3-month modified Rankin Scale [mRS] score 0 to 2 versus 3 to 6). We included 17,461 consecutive patients with witnessed ischemic stroke within 6 hours of onset. Stroke onset time was divided into 2 groups (day-onset [06:00 to 18:00] versus night-onset [18:00 to 06:00]) and into 6 groups by 4-hour intervals. We used mixed-effects ordered or logistic regression models while accounting for clustering by hospitals. Mean age was 66.9 (SD 13.4) years, and 6,900 (39.5%) were women. END occurred in 2,219 (12.7%) patients. After adjusting for covariates including age, sex, previous stroke, prestroke mRS score, admission NIHSS score, hypertension, diabetes, hyperlipidemia, smoking, atrial fibrillation, prestroke antiplatelet use, prestroke statin use, revascularization, season of stroke onset, and time from onset to hospital arrival, night-onset stroke was more prone to END (adjusted incidence 14.4% versus 12.8%, p = 0.006) and had a lower likelihood of favorable outcome (adjusted odds ratio, 0.88 [95% CI, 0.79 to 0.98]; p = 0.03) compared with day-onset stroke. When stroke onset times were grouped by 4-hour intervals, a monotonic gradient in presenting NIHSS score was noted, rising from a nadir in 06:00 to 10:00 to a peak in 02:00 to 06:00. The 18:00 to 22:00 and 22:00 to 02:00 onset stroke patients were more likely to experience END than the 06:00 to 10:00 onset stroke patients. At 3 months, there was a monotonic gradient in the rate of favorable functional outcome, falling from a peak at 06:00 to 10:00 to a nadir at 22:00 to 02:00. Study limitations include the lack of information on sleep disorders and patient work/activity schedules.ConclusionsNight-onset strokes, compared with day-onset strokes, are associated with higher presenting neurologic severity, more frequent END, and worse 3-month functional outcome. These findings suggest that circadian time of onset is an important additional variable for inclusion in epidemiologic natural history studies and in treatment trials of neuroprotective and reperfusion agents for acute ischemic stroke.

Wi-Sun Ryu and colleagues investigate the association of stroke onset time with presenting severity, early neurological deterioration (END), and long-term functional outcome in ischemic stroke.     

Role of Protein Tyrosine Phosphatase Non-Receptor Type 7 in the Regulation of TNF-α Production in RAW 264.7 Macrophages

Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.     

CAPS markers using mitochondrial consensus primers for molecular identification of Panax species and Korean ginseng cultivars (Panax ginseng C. A. Meyer)

Cleaved amplified polymorphic sequence (CAPS) marker system using mitochondrial consensus primers was applied for molecular identification of Korean ginseng cultivars (Panax ginseng). Initially, a total of 34 primers were tested to six Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, four primers (mt7, mt11, mt13, and mt18) generated co-dominant polymorphic banding patterns discriminating the Korean ginseng cultivars from P. quinquefolius and P. notoginseng. In the CAPS analysis results, the majority of the cleaved PCR products also yielded additional latent polymorphisms between the Korean ginseng cultivars and two foreign Panax species. Specific latent CAPS polymorphisms for cultivar Gopoong and Chunpoong were detected from internal region amplified with mt9 primer by treating HinfI and Tsp509I endonucleases, respectively. Sequencing analysis revealed that the length of amplified region of Korean ginseng cultivars was 2,179 bp, and those of P. quinquefolius and P. notoginseng were 2,178 and 2,185 bp, respectively. Blast search revealed that the amplified region was a mitochondrial cytochrome oxidase subunit 2 (cox2) gene intron II region. Nineteen single nucleotide polymorphisms (SNP) including each specific SNP for Gopoong and Chunpoong, and three insertion and deletion (InDel) polymorphisms were detected by sequence alignment. The CAPS markers developed in this study, which are specific to Gopoong and Chunpoong, and between the Korean ginseng cultivars and two foreign Panax species, will serve as a practical and reliable tool for their identification, purity maintenance, and selection of candidate lines and cultivars.     

Optimal Induction of Hepatitis C Virus Envelope-Specific Immunity by Bicistronic Plasmid DNA Inoculation with the Granulocyte-Macrophage Colony-Stimulating Factor Gene

In this study, we have constructed various DNA vaccine vectors that carried hepatitis C virus (HCV) envelope genes without and with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in several different ways. In Buffalo rats that received plasmids carrying the HCV envelope genes, which encode envelope proteins E1 and E2, both antibody and lymphoproliferative responses against these proteins were induced. These responses were greatly enhanced by the codelivery of the GM-CSF gene. In particular, inoculation with a bicistronic plasmid that independently expressed the GM-CSF gene and the envelope genes in the same construct generated the highest antibody titers and significantly increased lymphoproliferative responses against these proteins. Moreover, strong antibody responses to homologous and heterologous hypervariable region 1 peptides were elicited in the immunized rats.     

Establishment of a Captive All‐male Group of Proboscis Monkey (Nasalis larvatus) at the Singapore Zoo

Surplus male proboscis monkeys at the Singapore Zoo pose a considerable problem for maintenance and maximizing of exhibition potential. In 2008, a new exhibit was constructed to house and display a group of six proboscis monkey males born in Singapore Zoo. To document and monitor the all‐male group establishment in the new exhibit, we conducted observations on intragroup interactions between the monkeys, spatial use of their new exhibit, and visitor effects on their behavior. We found contact aggressive interactions between the monkeys to be consistently lower than noncontact aggressive interactions and by week six of introduction to the new exhibit, contact aggression was almost nonevident. Affiliative interactions also developed between individuals in the group, with an interface of aggressive and socioreconcilatory behavior influenced by food competition and a dominance hierarchy. This was evident from significantly higher overall aggression and affiliation during feeding times compared to nonfeeding times, and this was reduced when food competition was mitigated by modifying the feeding regime. We measured the groups’ spatial use of the exhibit and the relation to behavior, crowd size, and density. Our results showed that the proboscis monkeys utilized the available exhibit space, were largely unaffected by visitor crowd size and density, and were able to exhibit a variety of natural behaviors, including swimming. Our accomplishment in maintaining and displaying an all‐male group of proboscis monkeys in captivity provides viable options for more comprehensive captive management and breeding programs for this endangered species. Zoo Biol. 32:281‐290, 2013. © 2012 Wiley Periodicals, Inc.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

Genetic Analysis of Genes Related to Tight Junction Function in the Korean Population with Non-Syndromic Hearing Loss

Tight junctions (TJs) are essential components of eukaryotic cells, and serve as paracellular barriers and zippers between adjacent tissues. TJs are critical for normal functioning of the organ of Corti, a part of the inner ear that causes loss of sensorineural hearing when damaged. To investigate the relation between genes involved in TJ function and hereditary loss of sensorineural hearing in the Korean population, we selected the TJP2 and CLDN14 genes as candidates for gene screening of 135 Korean individuals. The TJP2 gene, mutation of which causes autosomal dominant non-syndromic hearing loss (ADNSHL), lies at the DFNA51 locus on chromosome 9. The CLDN14 gene, mutation of which causes autosomal recessive non-syndromic hearing loss (ARNSHL), lies at the DFNB29 locus on chromosome 21. In the present study, we conducted genetic analyses of the TJP2 and CLDN14 genes in 87 unrelated patients with ADNSHL and 48 unrelated patients with either ARNSHL or potentially sporadic hearing loss. We identified two pathogenic variations, c.334G>A (p.A112T) and c.3562A>G (p.T1188A), and ten single nucleotide polymorphisms (SNPs) in the TJP2 gene. We found eight non-pathogenic variations in the CLDN14 gene. These findings indicate that, whereas mutation of the TJP2 gene might cause ADNSHL, CLDN14 is not a major causative gene for ARNSHL in the Korean population studied. Our findings may improve the understanding of the genetic cause of non-syndromic hearing loss in the Korean population.