Integrin-linked kinase controls microtubule dynamics required for plasma membrane targeting of caveolae

Caveolae are specialized compartments of the plasma membrane that are involved in signaling, endocytosis, and cholesterol transport. Their formation requires the transport of caveolin-1 to the plasma membrane, but the molecular mechanisms regulating the transport are largely unknown. Here, we?identify a critical role for adhesion-mediated signaling through β1 integrins and integrin-linked kinase (ILK) in caveolae formation. Mice lacking β1 integrins or ILK in keratinocytes have dramatically reduced numbers of plasma membrane caveolae in?vivo, which is due to impaired transport of caveolin-1-containing vesicles along microtubules (MT) to the plasma membrane. Mechanistically, ILK promotes the recruitment of the F-actin binding protein IQGAP1 to the cell cortex, which, in turn, cooperates with its?effector mDia1 to locally stabilize MTs and to allow?stable insertion of caveolae into the plasma membrane. Our results assign an important role to the integrin/ILK complex for caveolar trafficking to the cell surface.     

基于钙黄绿素- 铜(Ⅱ)荧光体系测定乙酰半胱氨酸

目的:基于钙黄绿素-铜(Ⅱ)荧光体系测定乙酰半胱氨酸。方法:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,以492 nm为激发波长,520 nm为发射波长测定乙酰半胱氨酸溶液的荧光强度。结果:在pH=8.0的Na2HPO.412H2O-KH2PO4缓冲液中,二价铜离子与钙黄绿素配位引起荧光猝灭。由于乙酰半胱氨酸中巯基上的硫离子与Cu2+的亲和力很强,可从钙黄绿素-铜(Ⅱ)的络合物中夺取铜离子而使钙黄绿素游离出来,从而使体系的荧光得以恢复,并且荧光恢复的程度与加入乙酰半胱氨酸的量在一定范围内成线性。结论:建立了一种测定乙酰半胱氨酸的荧光分析新方法,该方法的线性范围为6.0 10-6～1.4 10-5 mol/L,检出限为4.010-6 mol/L。     

Evolution ofE.coli tRNATrp

Earlier studies (1) have shown there are direct correlations between the hydrophobicity ranking of most amino acids and their anticodonic nucleotides. However, four anticodonic assignments, i.e. those for Trp, Tyr, Ile and the XGA anticodons for Ser, did not correlate. It was our proposal that this failure to correlate was due to the fact that these assignments were made late, relative to the bulk of the assignments, in evolution through the mutation of existing tRNAs. We have shown (2) thatE. coli tRNA^{Ile 1} and tRNA^{Ile 2} were likely derived from tRNA^{Val 1} and tRNA^Lys respectively andE. coli tRNA^Tyr was possibly derived fromE. coli 5s rRNA or a common precursor with 5s rRNA (3). The fact that quite high homologies were observed in these comparisons is consistent with the late evolution of the tRNAs in question. We now examine the evolution ofE. coli tRNA^Trp by comparing its homology with otherE. coli tRNAs. The data suggest a possible evolutionary relationship withE. coli tRNA^Gly or tRNA^Arg. The data support the idea of the late assignment of anticodons to Trp.Deceased.     

A short duration of high-fat diet induces insulin resistance and predisposes to adverse left ventricular remodeling after pressure overload

Insulin resistance is an increasingly prevalent condition in humans that frequently clusters with disorders characterized by left ventricular (LV) pressure overload, such as systemic hypertension. To investigate the impact of insulin resistance on LV remodeling and functional response to pressure overload, C57BL6 male mice were fed a high-fat (HFD) or a standard diet (SD) for 9 days and then underwent transverse aortic constriction (TAC). LV size and function were assessed in SD- and HFD-fed mice using serial echocardiography before and 7, 21, and 28 days after TAC. Serial echocardiography was also performed on nonoperated SD- and HFD-fed mice over a period of 6 wk. LV perfusion was assessed before and 7 and 28 days after TAC. Nine days of HFD induced systemic and myocardial insulin resistance (assessed by myocardial 18F-fluorodeoxyglucose uptake), and myocardial perfusion response to acetylcholine was impaired. High-fat feeding for 28 days did not change LV size and function in nonbanded mice; however, TAC induced greater hypertrophy, more marked LV systolic and diastolic dysfunction, and decreased survival in HFD-fed compared with SD-fed mice. Compared with SD-fed mice, myocardial perfusion reserve was decreased 7 days after TAC, and capillary density was decreased 28 days after TAC in HFD-fed mice. A short duration of HFD induces insulin resistance in mice. These metabolic changes are accompanied by increased LV remodeling and dysfunction after TAC, highlighting the impact of insulin resistance in the development of pressure-overload-induced heart failure.     

Identification of stable fly attractant compounds in vinasse,a byproduct of sugarcane–ethanol distillation

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), is a worldwide pest of livestock. Recent outbreaks of stable flies in sugarcane fields in Brazil have become a serious problem for livestock producers. Larvae and pupae found inside sugarcane stems after harvesting may indicate that stable flies use these stems as potential oviposition or larval development sites. Field observations suggest that outbreaks of stable flies are associated with the vinasse and filter cake derived from biomass distillation in sugarcane ethanol production that are used as fertilizers in sugarcane fields. Adult stable flies are attracted to vinasse, which appears to present an ideal larval development site. The primary goal of the present study is to demonstrate the role of vinasse in influencing the sensory physiological and behavioural responses of stable flies, and to identify its associated volatile attractant compounds. Both laboratory and field studies showed that vinasse is extremely attractive to adult stable flies. Chemical analyses of volatiles collected revealed a wide range of carboxylic acids, alcohols, phenols and aldehydes as potential attractant compounds. These newly identified attractants could be used to develop a tool for the attractant‐baited mass trapping of stable flies in order to reduce infestations.     

Comparative placental steroid synthesis. II. C19 steroid metabolism by guinea-pig placentas and fetal adrenals in vitro

Placental homogenates from guinea-pigs at 16, 20, 35 and 55 days gestation were incubated with 7α-³H-dehydroepiandrosterone and 4-¹⁴C-androstenedione and analyzed for conversion products by reverse isotope dilution methods. ¹⁴C-3α-Hydroxy-5α-androstan-17-one, ¹⁴C-androstane-3α, 17β-diol and ³Handrost-5-ene-3β, 17β-diol were isolated from homogenates incubated with substrates for 2 hours. ³H, ¹⁴C-Testosterone was isolated from preparations incubated for 15 minutes or with high substrate: tissue ratios. Androst-4-ene-3, 17-dione, 5α-androstane-3, 17-dione, 5β-androstanedione derivative and C₁₈ steroid formation could not be demonstrated. These results demonstrate the capacity of guinea-pig placentas to convert dehydroepiandrosterone and androstenedione to testosterone and to derivatives reduced in ring A (5α) and at carbon 17. The activity of the Δ⁵-3β-hydroxysteroid dehydrogenase enzyme system appears to have been rate limiting.Homogenates of adrenals from 44–55 day old fetuses converted 4-¹⁴C-pregnenolone to androst-4-ene-3, 17-dione and 6β- and 11β-hydroxyandrostenedione. A guineapig fetal-placental unit is postulated, with steroid metabolic characteristics different from the human unit. Both permit reduction of fetal adrenal cortisol production and placental removal of C₁₉ steroids.     

Long-term effects of early parental loss due to divorce on the HPA axis

We investigated the long-term effects of divorce and early separation from one parent on HPA axis reactivity, in young adults without psychopathology. Participants were 44 young subjects, 22 whose parents divorced before they reached age 10, and 22 controls. Psychiatric symptomatology was measured with the Brief Symptom Inventory (BSI), family perceived stress by the Dyadic Adjustment Scale (DAS), and bonding by the Parental Bonding Instrument (PBI). Assessment of HPA axis function included baseline morning cortisol and ACTH and cortisol response to a CRH stimulation test. No baseline or stimulated group differences were observed for ACTH. Cortisol levels were consistently but insignificantly lower in the divorce group throughout the CRH stimulation reaching statistical significance only at 5 min (p<0.03). Group by time effect reached a trend level (p<0.06). A correlation was found between psychiatric symptomatology and PBI scores; however, both parameters did not correlate with HPA axis activity. A significant correlation was found between DAS scores and ACTH. A regression model revealed a contributing effect for both family stress and child-parent bonding to stimulated ACTH levels. These preliminary findings suggest that even in the absence of adult psychopathology, a history of childhood separation from one parent due to divorce may lead to detectable, albeit mild, long-term alterations in HPA axis activity. Furthermore, they suggest that level of stress at home and parental bonding are important determinants of this effect. It is likely that divorce has significant and sustained effects on children's HPA axis only in the context of a traumatic separation.     

cGMP-dependent protein kinase I interacts with TRIM39R, a novel Rpp21 domain-containing TRIM protein

Nitric oxide modulates vascular smooth muscle cell (SMC) cytoskeletal kinetics and phenotype, in part, by stimulating cGMP-dependent protein kinase I (PKGI). To identify molecular targets of PKGI, an interaction trap screen in yeast was performed using a cDNA encoding the catalytic region of PKGI and a human lung cDNA library. We identified a cDNA that encodes a putative PKGI-interactor that is a novel variant of TRIM39, a member of the really interesting new gene (RING) finger family of proteins. Although this TRIM39 variant encodes the NH(2)-terminal RING finger (RF), B-box, and coiled-coil (RBBC) domains of TRIM39, instead of a complete COOH-terminal B30.2 domain, this TRIM39 isoform contains the COOH-terminal portion of Rpp21, a component of RNase P. RT-PCR demonstrated that the TRIM39 variant, which we refer to as TRIM39R, is transcribed in the human fetal lung and in rat pulmonary artery SMC. Indirect immunofluorescence using an antibody generated against the conserved domains of TRIM39 and TRIM39R revealed the proteins in speckled intranuclear structures in human acute monocytic leukemia (THP-1) and human epidermal carcinoma line (HEp-2) cells. PKGI phosphorylated a typical PKGI/PKA phosphorylation domain in a conserved region of TRIM39 and TRIM39R. Additional studies demonstrated that PKGI interacts with both isoforms of TRIM39 in yeast cells and phosphorylates both isoforms of TRIM39 in human cell lines. Although PKGI has been observed to interact with proteins that regulate cytoskeletal function and gene expression, this investigation shows for the first time that PKGI interacts with tripartite motif (TRIM) proteins, which, through diverse molecular pathways, are often observed to regulate important aspects of cellular homeostasis.