A novel hook-related protein family and the characterization of hook-related protein 1

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains.     

PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells

Rat lung microvascular endothelial cell monolayers were exposed to donor plasma from burned rats (25% total body surface area) at 1:3 dilution for 30 min. Immunofluorescence analysis revealed that concomitant with gap formation alterations were seen in the adherens junction (AJ) proteins beta-catenin and vascular endothelial-cadherin. Both of these components were shown to exist in a smooth, uniform arrangement at the cell periphery in untreated cells. However, upon exposure to burn plasma, this uniformity was lost, and the AJ proteins showed a disrupted, zipper-like pattern at the cells' edge. In addition, these proteins were absent from areas of gap formation between the cells, and an increase in punctate staining throughout the cells suggests they were internalized in response to burn plasma. Measurements of both transendothelial electrical resistance and FITC-albumin flux across the cell monolayer were used to assess barrier integrity. Our study found that exposure to burn plasma rapidly caused the electrical resistance across confluent monolayers to decrease and albumin flux to increase, phenomena associated with barrier dysfunction. Furthermore, all the above responses to burn plasma were attenuated when cells were pretreated with the PKC inhibitor bisindolylmaleimide, suggesting that PKC is required for burn-induced pulmonary endothelial dysfunction.     

Cell wall structure of the agarophytes Gracilaria tikvahiae and G. cornea (Rhodophyta) and penetration by the epiphyte Ulva lactuca (Chlorophyta)

Use of light, transmission, and scanning electronmicroscopes revealed that the epidermal cell wall ofthe red algal agarophytes Gracilaria tikvahiaeMcLachlan and G. cornea J. Agardh consists of adecklamelle and outer and inner wall layers. The twospecies differed, with G. cornea having asignificantly thicker outer wall and a more diffusedecklamelle. After induction, the zooids of Ulvalactuca would attach to glass slides and the twospecies of Gracilaria via an adhesion pad. Within a few days, 3–5 celled germlings penetrated thedecklamelle and outer wall layer of both basiphytes. By the time the epiphyte germlings reached the 15celled stage, they had penetrated the inner walllayer. The differences in epidermal cell wallconstruction between the two basiphytes may play arole in the ability of zooids of U. lactuca toattach in nature where epiphytization of G.cornea is infrequent.     

Neonatal necrotizing enterocolitis: the relation of age at the time of onset to prognosis.

A study was undertaken to evaluate the predisposing factors, age at the time of onset and prognosis of neonatal necrotizing enterocolitis in 62 patients treated in a neonatal intensive care unit during a 5-year period (1974-78). Because of a peak frequency during the first week of life, the cases were divided into those of early-onset illness (appearing within the first week of life) and those of late-onset illness (appearing after this week). The main differences between the two groups were in the age at the time of the first enteric feeding (1.6 +/- 0.9 d v. 40 +/- 2.4 d [mean +/- one standard deviation]; P less than 0.001) and the interval between this feeding and the onset of symptoms (3.0 +/- 1.5 d v. 10.6 +/- 6.0 d; P less than 0.01). Furthermore, the early-onset illness was more severe, more often necessitating surgical intervention and carrying a higher mortality, than the late-onset illness. Thus, this study demonstrates that there are two forms of neonatal necrotizing enterocolitis, differing in time of appearance after birth as well as in severity and prognosis.     

Bretylium Tosylate in the Management of Refractory Ventricular Fibrillation

Nine patients who had recurrent ventricular fibrillation following acute myocardial infarction or angina were given bretylium tosylate in a dose of 5 mg./kg. intramuscularly every eight hours after other measures had proved ineffective. Provided the patients were not in shock or in heart failure, there was a considerable reduction in the episodes of ventricular fibrillation.A second group of nine patients who developed recurrent ventricular fibrillation following open heart surgery were given bretylium intravenously, which controlled the arrhythmia in every instance.Bretylium did not completely abolish ventricular premature beats but the latter did not initiate ventricular fibrillation even when they occurred on the T wave.     

Macroclimate associated with urbanization increases the rate of secondary succession from fallow soil

To examine the impact of projected climate changes on secondary succession, we exposed the same fallow soil with a common seed bank to an in situ gradient of urban to rural macroenvironments that differed in temperature and CO₂ concentration ([CO₂]). This gradient was established at three locations: Baltimore city center (urban), a city park on the outskirts of Baltimore (suburban), and an organic farm 87 km from the Baltimore city center site (rural). Over a five-year period, the urban site averaged 2.1°C warmer and had a [CO₂] that was ~20% higher than at the rural location, indicating that this gradient was a reasonable surrogate for projected changes in those variables for this century. Previous work had demonstrated that other abiotic variables measured across the transect, including tropospheric ozone and nitrogen deposition, did not differ consistently. The first year of exposure resulted in (two- to threefold) greater aboveground biomass in the urban relative to the rural site, but with uniform species composition across sites. Simple regression of abiotic variables indicated that temperature and vapor pressure deficit (VPD) were the best predictors of plant biomass among locations. Stepwise multiple regressions were also performed to analyze the effect of more than one macroenvironmental variable on total plant biomass. The combination of daily CO₂ concentration and nighttime temperature explained 87% (P < 0.01) of the variability in total biomass between sites. After five years, the species demography of the plant communities had changed significantly, with a greater ratio of perennials to annuals for the urban relative to the rural location. Greater first-year biomass and litter accumulation at the urban site may have suppressed the subsequent seed germination of annual species, accelerating changes in species composition. If urban macroenvironments reflect future global change conditions, these data suggest a faster rate of secondary succession in a warmer, higher [CO₂] world.     

Tracing early stages of species differentiation: Ecological,morphological and genetic divergence of Galápagos sea lion populations

Background  

Oceans are high gene flow environments that are traditionally believed to hamper the build-up of genetic divergence. Despite this, divergence appears to occur occasionally at surprisingly small scales. The Galápagos archipelago provides an ideal opportunity to examine the evolutionary processes of local divergence in an isolated marine environment. Galápagos sea lions (Zalophus wollebaeki) are top predators in this unique setting and have an essentially unlimited dispersal capacity across the entire species range. In theory, this should oppose any genetic differentiation.     

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001