Role of asparagine-linked oligosaccharides in rhodopsin maturation and association with its molecular chaperone, NinaA

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn(20) is the sole site for glycosylation. The Rh1(N20I) protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1(N20I); therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1(N20I)/NinaA interaction. We further show that Rh1(N20I) interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.     

Studies on the chemical modification of potato (Solanum tuberosum) lectin and its effect on haemagglutinating activity

1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups, activated with carbodi-imide, with α-aminobutyric acid methyl ester led to the incorporation of 5.3 residues of the ester per molecule of lectin. Presence of inhibitor in this case, although protecting activity, did not prevent modification of carboxy groups; in fact an increase in the number of modified residues was seen. This effect could be imitated by performing the reaction in 8m-urea. In both cases the number of carboxy groups modified was close to the total number of free carboxy groups as determined by the method of Hoare & Koshland [(1967) J. Biol. Chem. 242, 2447–2453]. Guanidination of lysine residues after carboxy-group modification gave less homoarginine than did the unmodified lectin under the same conditions, suggesting the formation of intramolecular cross-links during carbodi-imide activation. 6. It is suggested from the results presented that amino, arginyl, methionyl, histidyl and carboxyl groups are not involved in the activity of the lectin and that tyrosyl and tryptophyl groups are very closely involved. These findings are similar to those reported for other proteins that bind N-acetylglucosamine oligomers and also fit the general trend in other lectins.     

Arthrobacter pokkalii sp nov,a Novel Plant Associated Actinobacterium with Plant Beneficial Properties,Isolated from Saline Tolerant Pokkali Rice,Kerala, India

A novel yellow colony-forming bacterium, strain P3B162^T was isolated from the pokkali rice rhizosphere from Kerala, India, as part of a project study aimed at isolating plant growth beneficial rhizobacteria from saline tolerant pokkali rice and functionally evaluate their abilities to promote plant growth under saline conditions. The novel strain P3B162^T possesses plant growth beneficial traits such as positive growth on 1-aminocyclopropane-1-carboxylic acid (ACC), production of indole acetic acid (IAA) and siderophore. In addition, it also showed important phenotypic characters such as ability to form biofilm and utilization of various components of plant root exudates (sugars, amino acids and organic acids), clearly indicating its lifestyle as a plant rhizosphere associated bacterium. Taxonomically, the novel strain P3B162^T was affiliated to the genus Arthrobacter based on the collective results of phenotypic, genotypic and chemotaxonomic analyses. Moreover, molecular analysis using 16S rRNA gene showed Arthrobacter globiformis NBRC 12137^T, Arthrobacter pascens DSM 20545^T and Arthrobacter liuii DSXY973^T as the closely related phylogenetic neighbours, showing more than 98% 16S rRNA similarity values, whereas the recA gene analysis displayed Arthrobacter liuii JCM 19864^T as the nearest neighbour with 94.7% sequence similarity and only 91.7% to Arthrobacter globiformis LMG 3813^T and 88.7% to Arthrobacter pascens LMG 16255^T. However, the DNA-DNA hybridization values between strain P3B162^T, Arthrobacter globiformis LMG 3813^T, Arthrobacter pascens LMG 16255^T and Arthrobacter liuii JCM 19864^T was below 50%. In addition, the novel strain P3B162^T can be distinguished from its closely related type strains by several phenotypic characters such as colony pigment, tolerance to NaCl, motility, reduction of nitrate, hydrolysis of DNA, acid from sucrose, cell wall sugars and cell wall peptidoglycan structure. In conclusion, the combined results of this study support the classification of strain P3B162^T as a novel Arthrobacter species and we propose Arthrobacter pokkalii sp.nov.as its name. The type strain is P3B162^T (= KCTC 29498^T = MTCC 12358^T).     

Rethinking Opsins

Opsins, the protein moieties of animal visual photo-pigments, have emerged as moonlighting proteins with diverse, light-dependent and -independent physiological functions. This raises the need to revise some basic assumptions concerning opsin expression, structure, classification, and evolution.     

Factors Associated with Nursing Home Placement of All Patients Admitted for Inpatient Rehabilitation in Singapore Community Hospitals from 1996 to 2005: A Disease Stratified Analysis

Objectives

To (1) identify social and rehabilitation predictors of nursing home placement, (2) investigate the association between effectiveness and efficiency in rehabilitation and nursing home placement of patients admitted for inpatient rehabilitation from 1996 to 2005 by disease in Singapore.

Design

National data were retrospectively extracted from medical records of community hospital.

Data Sources

There were 12,506 first admissions for rehabilitation in four community hospitals. Of which, 8,594 (90.3%) patients were discharged home and 924 (9.7%) patients were discharged to a nursing home. Other discharge destinations such as sheltered home (n = 37), other community hospital (n = 31), death in community hospital (n = 12), acute hospital (n = 1,182) and discharge against doctor’s advice (n = 24) were excluded.

Outcome Measure

Nursing home placement.

Results

Those who were discharged to nursing home had 33% lower median rehabilitation effectiveness and 29% lower median rehabilitation efficiency compared to those who were discharged to nursing homes. Patients discharged to nursing homes were significantly older (mean age: 77 vs. 73 years), had lower mean Bathel Index scores (40 vs. 48), a longer median length of stay (40 vs. 33 days) and a longer time to rehabilitation (19 vs. 15 days), had a higher proportion without a caregiver (28 vs. 7%), being single (21 vs. 7%) and had dementia (23 vs. 10%). Patients admitted for lower limb amputation or falls had an increased odds of being discharged to a nursing home by 175% (p<0.001) and 65% (p = 0.043) respectively compared to stroke patients.

Conclusions

In our study, the odds of nursing home placement was found to be increased in Chinese, males, single or widowed or separated/divorced, patients in high subsidy wards for hospital care, patients with dementia, without caregivers, lower functional scores at admission, lower rehabilitation effectiveness or efficiency at discharge and primary diagnosis groups such as fractures, lower limb amputation and falls in comparison to strokes.     

Two new fossil woods from the early Miocene of Kutch,Gujarat, India and their significance

The Kutch region of western India (Gujarat State) is today arid to semiarid and characterised by mostly ephemeral streams which carry water during the monsoon. The uneven distribution of rainfall and disturbed topography are the result of climate change during the Cenozoic period. Two fossil woods, namely Bauhinium palaeomalabaricum Prakash and Prasad (Fabaceae) and Ebenoxylon indicum Ghosh and Kazmi (Ebenaceae), are described from Kutch in order to provide insights into the palaeovegetation and palaeoclimate. Because the modern representatives of the present and previously described taxa from the same horizon are thermophilic in nature and grow in evergreen to deciduous forests, a warm and humid climate is interpreted. Furthermore, the finding of some mangrove taxa in the assemblage indicates the lagoonal to intertidal environment at the time of deposition.     

MEKK4 sequesters RIP2 to dictate NOD2 signal specificity

The Crohn's-disease-susceptibility protein, NOD2, coordinates signaling responses upon intracellular exposure to bacteria. Although NOD2 is known to activate NFkappaB, little is known about the molecular mechanisms by which NOD2 coordinates functionally separate signaling pathways such as NFkappaB, JNK, and p38 to regulate cytokine responses. Given that one of the characteristics of Crohn's disease is an altered cytokine response to normal bacterial flora, the coupling of signaling pathways could be important for Crohn's-disease pathophysiology. We find that a MAP3K, MEKK4, binds to RIP2 to sequester RIP2 from the NOD2 signaling pathway. This MEKK4:RIP2 complex dissociates upon exposure to the NOD2 agonist, MDP, allowing NOD2 to bind to RIP2 and activate NFkappaB. MEKK4 thus sequesters RIP2 to inhibit the NOD2:RIP2 complex from activating NFkappaB signaling pathways, and Crohn's-disease-associated NOD2 polymorphisms cannot compete with MEKK4 for RIP2 binding. Lastly, we find that MEKK4 helps dictate signal specificity downstream of NOD2 activation as knockdown of MEKK4 in macrophages exposed to MDP causes increased NFkappaB activity, absent p38 activity, and hyporesponsiveness to TLR2 and TLR4 agonists. These biochemical findings suggest that basal inhibition of the NOD2-driven NFkappaB pathway by MEKK4 could be important in the pathogenesis of Crohn's disease.     

Phenotype Specific Analyses Reveal Distinct Regulatory Mechanism for Chronically Activated p53

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.     

The Low Fall as a Surrogate Marker of Frailty Predicts Long-Term Mortality in Older Trauma Patients

Background

Frailty is associated with adverse outcomes including disability, mortality and risk of falls. Trauma registries capture a broad range of injuries. However, frail patients who fall comprise a large proportion of the injuries occurring in ageing populations and are likely to have different outcomes compared to non-frail injured patients. The effect of frail fallers on mortality is under-explored but potentially significant. Currently, many trauma registries define low falls as less than three metres, a height that is likely to include non-frailty falls. We hypothesized that the low fall from less than 0.5 metres, including same-level falls, is a surrogate marker of frailty and predicts long-term mortality in older trauma patients.

Methods

Using data from the Singapore National Trauma Registry, 2011–2013, matched till September 2014 to the death registry, we analysed adults aged over 45 admitted via the emergency department in public hospitals sustaining blunt injuries with an injury severity score (ISS) of 9 or more, excluding isolated hip fractures from same-level falls in the over 65. Patients injured by a low fall were compared to patients injured by high fall and other blunt mechanisms. Logistic regression was used to analyze 12-month mortality, controlling for mechanism of injury, ISS, revised trauma score (RTS), co-morbidities, gender, age and age-gender interaction. Different low fall height definitions, adjusting for injury regions, and analyzing the entire adult cohort were used in sensitivity analyses and did not change our findings.

Results

Of the 8111 adults in our cohort, patients who suffered low falls were more likely to die of causes unrelated to their injuries (p<0.001), compared to other blunt trauma and higher fall heights. They were at higher risk of 12-month mortality (OR 1.75, 95% CI 1.18–2.58, p = 0.005), independent of ISS, RTS, age, gender, age-gender interaction and co-morbidities. Falls that were higher than 0.5m did not show this pattern. Males were at higher risk of mortality after low falls. The effect of age on mortality started at age 55 for males, and age 70 for females, and the difference was attributable to the additional mortality in male low-fallers.

Conclusions

The low fall mechanism can optimize prediction of long-term mortality after moderate and severe injury, and may be a surrogate marker of frailty, complementing broader-based studies on aging.     

Detecting Dysglycemia Using the 2015 United States Preventive Services Task Force Screening Criteria: A Cohort Analysis of Community Health Center Patients

BackgroundIn 2015, the United States Preventive Services Task Force (USPSTF) recommended targeted screening for prediabetes and diabetes (dysglycemia) in adults who are aged 40 to 70 y old and overweight or obese. Given increasing prevalence of dysglycemia at younger ages and lower body weight, particularly among racial/ethnic minorities, we sought to determine whether the current screening criteria may fail to identify some high-risk population subgroups.ConclusionsTargeted diabetes screening based on new USPSTF criteria may detect approximately half of adult community health center patients with undiagnosed dysglycemia and proportionately fewer racial/ethnic minorities than whites. Future research is needed to estimate the performance of these screening criteria in population-based samples.