Lactadherin binds to elastin--a starting point for medin amyloid formation?

Medin amyloid is found in the medial layer of the aorta in almost 100% of the Caucasian population over 50 years of age. The medin fragment is 5.5 kDa and derives from the C2-like domain of the precursor protein lactadherin. We have previously reported immunohistochemical findings showing that medin amyloid co-localizes with elastic fibers of arteries and herein we show that lactadherin also is associated with elastic structures of human aortic material. In addition, results from in vitro binding assays demonstrate that both medin and lactadherin bind to tropoelastin in a concentration-dependent fashion, suggesting that the lactadherin-tropoelastin interaction is mediated via the medin domain. It is possible that lactadherin, which is a cell adhesion protein, in this way connects smooth muscle cells to the elastic fibers of arteries. Given that both medin and lactadherin interact with elastic fibers, elastin is probably an important component in the formation of medin amyloid.     

KSHV viral cyclin binds to p27KIP1 in primary effusion lymphomas

Primary effusion lymphomas (PELs) represent a unique non-Hodgkin lymphoma that is consistently infected by Kaposi sarcoma herpesvirus (KSHV). PEL cells express high levels of the cell cycle inhibitor p27(KIP1) and yet proliferate actively. KSHV genome encodes a viral cyclin homolog, v-cyclin, which has previously been implicated in down-regulation of p27(KIP1) levels. To address how PEL cells can tolerate high p27(KIP1) levels, we investigated functional interactions between v-cyclin and p27(KIP1) using PEL-derived cell lines as a model system. Here we demonstrate that v-cyclin and p27(KIP1) stably associate in PEL cells in vivo suggesting an attractive model by which p27(KIP1) is inactivated in the actively proliferating PEL cells. Moreover, we show that v-cyclin and cyclin-dependent kinase 6 (CDK6) form an active kinase without p27(KIP1) and that CDK6 is the in vivo catalytic subunit of v-cyclin in PEL cells. These findings suggest that KSHV may promote oncogenesis in PEL by expressing v-cyclin, which both overrides negative cell cycle controls present in the PEL precursor cells and induces a strong proliferative signal via CDK6 kinase activity.     

Crystal structure of the guanylyl cyclase Cya2

Cyclic GMP (cGMP) is an important second messenger in eukaryotes. It is formed by guanylyl cyclases (GCs), members of the nucleotidyl cyclases class III, which also comprises adenylyl cyclases (ACs) from most organisms. To date, no structures of eukaryotic GCs are available, and all bacterial class III proteins were found to be ACs. Here we describe the biochemical and structural characterization of the class III cyclase Cya2 from cyanobacterium Synechocystis PCC6803. Cya2 shows high specificity for GTP versus ATP, revealing it to be the first bacterial GC, and sequence similarity searches indicate that GCs are also present in other bacteria. The crystal structure of Cya2 provides first structural insights into the universal GC family. Structure and mutagenesis studies show that a conserved glutamate, assisted by an interacting lysine, dominates substrate selection by forming hydrogen bonds to the substrate base. We find, however, that a second residue involved in substrate selection has an unexpected sterical role in GCs, different from its hydrogen bonding function in the related ACs. The structure identifies a tyrosine that lines the guanine binding pocket as additional residue contributing to substrate specificity. Furthermore, we find that substrate specificity stems from faster turnover of GTP, rather than different affinities for GTP and ATP, implying that the specificity-determining interactions are established after the binding step.     

Clinical Factors Associated with Biochemical Adrenal-cortisol Insufficiency in Hospitalized Patients

Background

Diagnosis of adrenal-cortisol insufficiency is often misleading in hospitalized patients, as clinical and biochemical features overlap with comorbidities. We analyzed clinical determinants associated with a biochemical diagnosis of adrenal-cortisol insufficiency in non-intensive care unit (ICU) hospitalized patients.

Methods

In a retrospective cohort study we reviewed 4668 inpatients with random morning cortisol levels ≤15 μg/dL hospitalized in our center between 2003 and 2010. Using serum cortisol threshold level of 18 μg/dL 30 or 60 minutes after Cortrosyn (250 μg; Amphastar Pharmaceuticals, Inc., Rancho Cucamonga, Calif) injection to define biochemical adrenal-cortisol status, we characterized and compared insufficient (n = 108, serum cortisol ≤18 μg/dL) and sufficient (n = 394; serum cortisol >18 μg/dL) non-ICU hospitalized patients.

Results

Commonly reported clinical and routine biochemical adrenal-cortisol insufficiency features were similar between insufficient and sufficient inpatients. Biochemical adrenal-cortisol insufficiency was associated with increased frequency of liver disease, specifically hepatitis C (P = .01) and prior orthotopic liver transplantation (P <.001), human immunodeficiency virus (HIV; P = .005), and reported pre-existing male hypogonadism (P <.001), as compared with the biochemical adrenal-cortisol sufficiency group. Forty percent of insufficient inpatients were not treated with glucocorticoids after diagnosis. Multivariable logistic analysis demonstrated that inpatients with higher cortisol levels (P = .0001) and higher diastolic blood pressure (P = .05), and females (P = .009) were more likely not to be treated, while those with previous short-term glucocorticoid treatment (P = .002), other coexisting endocrine diseases (P = .005), or who received an in-hospital endocrinology consultation (P <.0001), were more likely to be replaced with glucocorticoids.

Conclusions

Commonly reported adrenal-cortisol insufficiency features do not reliably identify hospitalized patients biochemically confirmed to have this disorder. Comorbidities including hepatitis C, prior orthotopic liver transplantation, HIV, and reported pre-existing male hypogonadism may help identify hospitalized non-ICU patients for more rigorous adrenal insufficiency assessment.     

Changes in marriage age and first birth interval in Huaning County, Yunnan Province, PR China

The timing of marriages, first birth interval and the prevalence of premarital conception (PMC) among women of successive birth cohorts in one rural county in Yunnan, China, were examined. Detailed pregnancy histories were collected for 1,336 women aged 15-64 years using a Life History Calendar. The rising marriage age and shorter first birth interval correspond to over-all changes in the Chinese society over the same time period. The mean age at first marriage for women born before 1950 was just below 20, and 22.5 among women born 1976-1980. The later marriage age was partly offset by the dramatic shortening of the first birth interval from over 30 months in the oldest cohort to 11-12 months in the youngest. One explanation of the shortening of the first birth interval may be the increase in premarital conception. Among the young women in our study almost one-third of first pregnancies were conceived before marriage.     

Monocyte CD14 and soluble CD14 in predicting mortality of patients with severe community acquired infection

Monocyte membrane CD14 (mCD14) and soluble CD14 (sCD14) both associate with poor outcome in sepsis. Because the value of combined use of the markers is unknown we measured both in patients with severe community acquired infections. The study comprised 142 acutely ill patients with community acquired pneumonia and/or blood culture-positive sepsis. Expression of mCD14 was measured, on admission to hospital, by whole blood flow cytometry and sCD14 by ELISA. There was no significant correlation between mCD14 and sCD14. Patients in the lowest tertile of mCD14 were 9.79 times (95% CI 1.31- >50, p =0.006) more likely to die than patients in the middle/highest tertiles. Survival rates in the highest and middle/lowest tertiles of sCD14 levels were comparable. After stratification by sCD14, patients in the lowest tertile of mCD14 were 14.4 times (95% CI 1.90-39.44) more likely to die than patients in the middle/highest tertiles. A significant positive correlation was detected between C-reactive protein and sCD14 levels, providing evidence that sCD14 may serve as an acute phase reactant. In conclusion, low monocyte mCD14 level, unlike the concurrent sCD14 level, predicts 28-d mortality in patients with community acquired infections.     

COX-2 and SCD, markers of inflammation and adipogenesis, are related to disease activity in Graves' ophthalmopathy.

CONTEXT: Inflammation and adipogenesis are two parallel processes with increased activity in severe Graves' ophthalmopathy. OBJECTIVE: The aim of this work was to define target genes for therapeutic intervention in adipogenesis and inflammation in Graves' ophthalmopathy. DESIGN: Orbital tissue was obtained from patients with ophthalmopathy in acute or chronic phase undergoing orbital surgery to study gene expression followed by the study of potential intervention mechanisms in preadipocytes. SETTING: Clinic of Endocrinology, University Hospital, Malm?, Sweden. PARTICIPANTS: Patients in acute severe or in chronic phase of ophthalmopathy. Interventions: Lateral orbital decompression in acute phase and restorative surgery in chronic phase. In vitro treatment of preadipocytes with rosiglitazone and diclofenac. MAIN OUTCOME MEASURE: Gene expression in intraorbital tissue or preadipocytes and differentiation of preadipocytes. Results: A marker of adipose tissue, stearoyl-coenzyme A desaturase (SCD), and the proinflammatory gene, cyclooxygenase-2 (COX-2), were overexpressed in patients in active phase compared to the chronic phase of ophthalmopathy. In growth-arrested preadipocytes stimulated with rosiglitazone, COX-2 expression increased temporarily within 1 hour and decreased to undetectable levels after 48 hours. In contrast, SCD and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression increased continuously from day 2 to day 7 during adipogenesis. Diclofenac, an inhibitor of cyclooxygenases with antagonistic effects on PPAR-gamma, reduced the number of mature adipocytes by approximately 50%. CONCLUSION: We conclude that inflammation and adipogenesis decrease with a decrease in activity of ophthalmopathy and that the nonsteroidal antiinflammatory drug diclofenac inhibits adipogenesis. This may represent a putative future treatment of endocrine ophthalmopathy.     

Microscopic colitis in patients with ulcerative colitis or Crohn’s disease: a retrospective observational study and review of the literature

Objectives: Onset of microscopic colitis (MC) in patients with ulcerative colitis (UC) or Crohn’s disease (CD), or vice versa, has been reported occasionally but the subject is not well described. We therefore report a retrospective observational study of such patients and review the literature.

Methods: Forty-six Swedish gastroenterology clinics were contacted about patients with diagnoses of both inflammatory bowel disease (IBD) and MC. Publications were searched on PubMed.

Results: We identified 31 patients with onset of MC after a median (range) of 20 (2–52) years after diagnosis of IBD, or vice versa; 21 UC patients developed collagenous colitis (CC) (n?=?16) or lymphocytic colitis (LC) (n?=?5); nine CD patients developed CC (n?=?5) or LC (n?=?4); one CC patient developed CD. Of the 21 UC patients, 18 had extensive disease, whereas no consistent phenotype occurred in CD. Literature review revealed 27 comprehensive case reports of patients with diagnoses of both IBD and MC. Thirteen MC patients developed IBD, of which four required colectomy. Fourteen IBD patients later developed MC. There were incomplete clinical data in 115 additional reported patients.

Conclusions: Altogether 173 patients with occurrence of both IBD and MC were found. The most common finding in our patients was onset of CC in a patient with UC. Although these are likely random associations of two different disorders, MC should be considered in the patient with UC or CD if there is onset of chronic watery diarrhoea without endoscopic relapse of IBD.     

Rapid initiation of cell cycle reentry processes protects neurons from amyloid-β toxicity

Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer’s disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-β (oAβ) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAβ challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aβ precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aβ toxicity.

Alzheimer’s disease (AD) is the most prevalent of all the neurodegenerative disorders. Distinct protein inclusions, amyloid-β (Aβ) plaques and neurofibrillary tangles (NFTs) characterize the AD brain (1). The exact causes of neuronal cell death in AD are still debated but several neuropathological studies have linked neuronal death to unexpected reappearance of cell cycle events (2–4). Accordingly, cell cycle activation, aberrant DNA replication, as well as aneuploidy have been found in neurons of AD brains (5–11). Furthermore, numbers of aneuploid neurons that are rare in control brains were increased in AD brains (5, 12, 13). However, mitosis itself has not been reported, and it appears that postmitotic neurons in AD patients cannot complete the cell cycle (14). In dividing cells, the coordination of the cell cycle requires interplay between cyclins and cyclin-dependent kinases (CDKs) at different checkpoints and the transitions between different phases are tightly regulated (15, 16). Therefore, it has been suggested that cell cycle reentry of differentiated mature neurons would have detrimental consequences, rendering them vulnerable and contribute to neurodegeneration (4, 8, 17). However, it remains unclear whether cell cycle events are a prerequisite for neuronal death, and why aneuploid neurons accumulate in AD brains.The introduction of the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system enabled live tracking of cell cycle progress in dividing cells (18) and has been valuable in understanding the implications of the cell cycle in disease pathogenesis, e.g., in cancer (19–21).Briefly, the FUCCI system allows live monitoring of the cell cycle in cells, visualized by the ectopic expression of red and green fluorescent-tagged truncated proteins, mKO2-hCdt1 (30-120) and mAG-hGem (1-110), respectively, distinguishing the G1 from S, G2, and M phases (22). While frequently used in cancer and developmental biology research (23–25) to our knowledge, the FUCCI system has not been used to study mature neurons, including in the context of neurodegenerative diseases.Here, we applied FUCCI to primary hippocampal neuronal cultures challenged with oligomeric Aβ (oAβ) and transgenic mice with expression of mutant human amyloid precursor protein (APP), which are both established models of AD (26–29). By obtaining quantitative and qualitative information about cell cycle events (CCEs) in response to Aβ, we show that increased FUCCI reporter activity was associated with resistance to Aβ-induced cell death, which is contrary to the current opinion of CCEs contributing to neurodegeneration in AD.