A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.     

Episodes of Diversification and Isolation in Island Southeast Asian and Near Oceanian Male Lineages

Island Southeast Asia (ISEA) and Oceania host one of the world’s richest assemblages of human phenotypic, linguistic, and cultural diversity. Despite this, the region’s male genetic lineages are globally among the last to remain unresolved. We compiled ∼9.7 Mb of Y chromosome (chrY) sequence from a diverse sample of over 380 men from this region, including 152 first reported here. The granularity of this data set allows us to fully resolve and date the regional chrY phylogeny. This new high-resolution tree confirms two main population bursts: multiple rapid diversifications following the region’s initial settlement ∼50 kya, and extensive expansions <6 kya. Notably, ∼40–25 kya the deep rooting local lineages of C-M130, M-P256, and S-B254 show almost no further branching events in ISEA, New Guinea, and Australia, matching a similar pause in diversification seen in maternal mitochondrial DNA lineages. The main local lineages start diversifying ∼25 kya, at the time of the last glacial maximum. This improved chrY topology highlights localized events with important historical implications, including pre-Holocene contact between Mainland and ISEA, potential interactions between Australia and the Papuan world, and a sustained period of diversification following the flooding of the ancient Sunda and Sahul continents as the insular landscape observed today formed. The high-resolution phylogeny of the chrY presented here thus enables a detailed exploration of past isolation, interaction, and change in one of the world’s least understood regions.     

NLPR3 inflammasome inhibition alleviates hypoxic endothelial cell death in vitro and protects blood–brain barrier integrity in murine stroke

In ischemic stroke (IS) impairment of the blood–brain barrier (BBB) has an important role in the secondary deterioration of neurological function. BBB disruption is associated with ischemia-induced inflammation, brain edema formation, and hemorrhagic infarct transformation, but the underlying mechanisms are incompletely understood. Dysfunction of endothelial cells (EC) may play a central role in this process. Although neuronal NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome upregulation is an established trigger of inflammation in IS, the contribution of its expression in EC is unclear. We here used brain EC, exposed them to oxygen and glucose deprivation (OGD) in vitro, and analyzed their survival depending on inflammasome inhibition with the NLRP3-specific drug MCC950. During OGD, EC death could significantly be reduced when targeting NLRP3, concomitant with diminished endothelial NLRP3 expression. Furthermore, MCC950 led to reduced levels of Caspase 1 (p20) and activated Gasdermin D as markers for pyroptosis. Moreover, inflammasome inhibition reduced the secretion of pro-inflammatory chemokines, cytokines, and matrix metalloproteinase-9 (MMP9) in EC. In a translational approach, IS was induced in C57Bl/6 mice by 60 mins transient middle cerebral artery occlusion and 23 hours of reperfusion. Stroke volume, functional outcome, the BBB integrity, and—in good agreement with the in vitro results—MMP9 secretion as well as EC survival improved significantly in MCC950-treated mice. In conclusion, our results establish the NLRP3 inflammasome as a critical pathogenic effector of stroke-induced BBB disruption by activating inflammatory signaling cascades and pyroptosis in brain EC.Subject terms: Stroke, Preclinical research, Inflammasome     

Cardiogel: A biosynthetic extracellular matrix for cardiomyocyte culture

Summary Tissue-cultured neonatal cardiomyocytes can be successfully maintained in culture on a variety of extracellular matrix components such as laminin, fibronectin, and interstitial collagens (Types I and III).In vivo, however, cardiomyocytes (as well as many other cells) exist in a highly complex extracellular matrix environment composed of, in addition to the above three components, other proteins, proteoglycans, and growth factors. We have developed a procedure for culturing cardiomyocytes on a naturally occurring complete extracellular matrix, Cardiogel. This substrate, synthesized by cardiac fibroblasts, contains laminin, fibronectin, Types I and III collagen, and proteoglycans. When compared to cardiomyocytes grown on laminin alone or fibronectin alone, Cardiogel-supported cardiomyocytes adhere more rapidly after plating, exhibit spontaneous contractility earlier, undergo cytoskeletal and myofibrillar differentiation earlier, and grow larger than their counterparts. We suggest that their superior growth characteristics reflect the synergistic effect of numerous extracellular matrix components’ signals in Cardiogel transduced by the cardiomyocyte cytoskeletal elements.     

How many fish populations in Finland are affected by acid precipitation?

Synopsis The number of fish populations affected or lost from small lakes in southern and central Finland due to acid precipitation is estimated. Tolerance limits (pH and labile aluminum) of common fish species were obtained from a fish status survey of 80 lakes. These tolerance values were used to estimate the proportion of affected lakes from the water chemistry data of 783 statistically selected lakes. The proportion of anthropogenically acidified lakes was estimated by calculating pre-acidification pH and aluminum concentrations of the lakes, using a steady-state model based on water chemistry. The number of fish populations for which acidification has affected growth or population structure was estimated at between 2200 and 4400. Out of these, the number of fish populations that have disappeared due to acid precipitation would be about 1000–2000. Almost 60% of the affected or lost populations are roach, Rutilus rutilus, the most sensitive of the common fish species in small lakes in southern and central Finland. Less than 15% of the damaged population is European perch, Perca fluviatilis, the most common species. This is due to the substantially higher tolerance of perch to acidified water in comparison with roach.     

Die Lipide von Endomycopsis vernalis bei verschiedener Stickstoff-Ernährung

1. Endomycopsis vernalis was cultivated on media with different N supply: series A 1%, series B 0,125% asparagine. Sonified cells were extracted and yielded 14.3% (A) and 65.3 (B) total lipids/non lipid dry matter respectively.
2. Neutral and complex lipids were separated by rubber membrane dialysis. There is no difference in the percentage of complex lipids of both series. The increase of lipids in cells grown on low N level is due to a higher content of neutral lipids.
3. Components of the neutral lipids, analysed by DC, were diglycerides, triglycerides, free and esterified ergosterol. Their percentage is influenced by the nutritional conditions. There is a significant increase of triglycerides and of sterol esters in the high lipid cells of series B.
4. Methyl esters of component fatty acids of glycerides and sterol esters were analyzed by GLC. Saturated acids C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, monoenic acids C₁₆ and C₁₈, linoleic and linolenic acids were found to be present. Major acids were in all cases 18:1 (17–57%), 18:2 (18–50%) and 16:0 (10–18%). Linolenic acid is higher in di-and triglycerides of low lipid cells of series A than in high lipid cells of series B. Both qualitative and quantitative differences of fatty acids were found in sterol esters of series A and B respectively.
5. The major components of complex lipids, identified by DC and isolated by CC, in both series, were phosphatidyl choline (A:36.5, B:41.0%) and phosphatidyl ethanolamine (A:24.9, B:20.5%) in addition to small amounts of lysophosphatidyl choline, lysophosphatidyl ethanolamine, phosphatidyl serine, monophosphoinositide, diphosphatidyl glycerol and, possibly cerebroside like substances.
6. Methyl esters of the fatty acids of phosphatidyl choline and ethanolamine from both series were determined by GLC. In all samples 16:0, 18:0, 18:1, 18:2 and 18:3 acids were present besides of traces of 16:1 and 17:0. In contrast to neutral lipids the major acid of phospholipids is linoleic (53–58%), followed by oleic (8–24%) and linolenic acid (1–18%). The percentages of palmitic (4–8%) and stearic acids (tr.-1%) are small. Low lipid cells of series A differ from high lipid cells of series B by an increase of linolenic, and a decrease of linoleic acids, both in phosphatidyl choline and phosphatidyl ethanolamine.

     

The ocellus of the cockroach,Periplaneta americana (Blattariae)

Summary The ocelli of Periplaneta americana were studied by light and electron microscopy. The view that the ocellus of the cockroach represents a degenerated structure can no longer be supported. All organelles necessary for function are present.The club-shaped retinular cells lie homogeneously distributed in the cupule of the ocellus. Rhabdoms are seen as sickle-, y-, x-or star-shaped structures with up to six cells in formation. Cells were found which had formed two rhabdomeres.The mass of cell organelles lies in the cytoplasm between the cell nucleus and the rhabdom. Smooth endoplasmic reticulum is wound into a spindle formation of considerable size at the origin of the axon in some cells. A cylindrical body in which 10–40 microtubules are packed, as yet unknown in insect retinular cells, is described.The receptory area of the ocellus terminates in a tapetum which contains granules, soluble in alcohol. The axon bundles of the retinular cells run through the tapetum and immediately thereafter make synaptic contact with dendrites of the ocellar nerve cells, while still within the ocellus.The authors are indebted to Mrs. Margaret Weber-Wood for her linguistic assistance     

The liver isoform of carnitine palmitoyltransferase I is activated in neonatal rat cardiac myocytes by hypoxia

Fatty acids are the preferred substrate of ischemic, reperfused myocardium and may account for the decreased cardiac efficiency during aerobic recovery. Neonatal cardiac myocytes in culture respond to hypoxia/serum- and glucose-free medium by a slow decline in ATP which reverses upon oxygenation. This model was employed to examine whether carnitine palmitoyltransferase I (CPT-I) modulates high rates of -oxidation following oxygen deprivation. After 5 h of hypoxia, ATP levels decline to 30% control values and CPT- I activity is significantly stimulated in hypoxic myocytes with no alteration in cellular carnitine content or in the release of the mitochondrial matrix marker, citrate synthase. This stimulation was attributed to an increase in the affinity of hypoxic CPT-I for carnitine, suggesting that the liver CPT-I isoform is more dominant following hypoxia. However, there was no alteration in hypoxic CPT-I inhibition by malonyl-CoA. DNP-etomoxiryl-CoA, a specific inhibitor of the liver CPT-I isoform, uncovered identical Michaelis kinetics of the muscle isoform in control and hypoxic myocytes with activation of the liver isoform. Northern blotting did not reveal any change in the relative abundance of mRNA for the liver vs. the muscle CPT-I isoforms. The tyrosine phosphatase inhibitor, pervanadate, reversed the hypoxia-induced activation of CPT-I and returned the affinity of cardiac CPT-I for carnitine to control. Reoxygenation was also associated with a return of CPT-I activity to control levels. The data demonstrate that CPT-I is activated upon ATP depletion. Lower enzyme activities are present in control and reoxygenated cells where ATP is abundant or when phosphatases are inhibited. This is the first suggestion that phosphorylation may modulate the activity of the liver CPT-I isoform in heart.