The ultrastructure of the parasitophorous vacuole formed by Leishmania major

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.     

The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in pulmonary host defense. Increased concentration of lysozyme in the airspaces of transgenic mice enhanced bacterial killing whereas lysozyme deficiency resulted in increased bacterial burden and morbidity. Lysozyme degrades peptidoglycan in the bacterial cell wall leading to rapid killing of Gram-positive organisms; however, this mechanism cannot account for the protective effect of lysozyme against Gram-negative bacteria. The current study was therefore designed to test the hypothesis that the catalytic activity (muramidase activity) of lysozyme is not required for bacterial killing in vivo. Substitution of serine for aspartic acid at position 53 (D53S) in mouse lysozyme M completely ablated muramidase activity. Muramidase-deficient recombinant lysozyme (LysM(D53S)) killed both Gram-positive and Gram-negative bacteria in vitro. Targeted expression of LysM(D53S) in the respiratory epithelium of wild-type (LysM(+/+)/LysM(D53S)) or lysozyme M(null) mice (LysM(-/-)/LysM(D53S)) resulted in significantly elevated lysozyme protein in the airspaces without any increase in muramidase activity. Intratracheal challenge of transgenic mice with Gram-positive or Gram-negative bacteria resulted in a significant increase in bacterial burden in LysM(-/-) mice that was completely reversed by targeted expression of LysM(D53S). These results indicate that the muramidase activity of lysozyme is not required for bacterial killing in vitro or in vivo.     

The interaction of fetuin with phosphatidylcholine monolayers. Characterization of a lipoprotein membrane system suitable for the attachment of myxoviruses

1. An artificial membrane system was formed by spreading at air/water and oil/water interfaces, by using phosphatidylcholine and the glycoprotein fetuin (mol.wt. 48400). 2. The plot of increase of interfacial pressure against amount of protein added beneath a monomolecular film of phosphatidylcholine showed two discontinuities, corresponding to the completion of two distinct layers of protein: (a) largely denatured and closely associated with the polar head groups of phosphatidylcholine, possibly with penetration of non-polar protein groups between the phosphatidylcholine molecules and (b) an additional adsorbed layer of substantially native fetuin in either a close-packed or open-lattice array. A more compactly organized membrane was apparently formed at pH7.4 with 1mm-Mg²⁺ in the aqueous phase than without Mg²⁺; at 15mm-Mg²⁺, more random adsorption of protein appeared to take place. Qualitatively similar results were obtained at pH5.1 with 1mm-Mg²⁺. Closer initial packing of the phosphatidylcholine layer decreased both the magnitude of the interfacial pressure change and the amounts of protein bound in the two layers. 3. The amount of N-acetylneuraminic acid released by neuraminidase (EC 3.2.1.18) in the subphase was measured at pH5.1; a mean distribution of 9.7×10¹³ residues/cm² was calculated for the completed second protein layer.     

Dynorphin (1-13): analgesia, hypothermia, cross-tolerance with morphine and beta-endorphin

Intracerebroventricular administration of 20, 40 and 60 nmol of dynorphin (1-13) produced analgesia, as assessed by flinch/jump response to footshock, and hypothermia in the rat. Rats developed tolerance to both the analgesic and thermic effects of the 20 nmol dose of dynorphin. Dynorphin and beta-endorphin showed cross-tolerance with respect to their analgesic but not their thermic effects. Dynorphin and morphine also produced cross-tolerant analgesic effects. Naloxone (10 mg/kg, IP) completely blocked the barrel rolling produced by 20 nmol dynorphin but did not alter its analgesic or thermic effects.     

Copper-Binding Compounds from Methylosinus trichosporium OB3b

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO^C) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L₁ and CBC-L₂, respectively. The amino acid sequence of CBC-L₁ is S?MYPGS?M, and that of CBC-L₂ is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L₁ showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g_⊥ = 2.087, g_∥ = 2.42 G, |A_∥| = 128 G). The EPR spectrum of CBC-L₂ was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu²⁺ center (g_⊥ = 2.073, g_∥ = 2.324 G, |A_∥| = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g_⊥ = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMO^C mutants PP319 and PP359 regardless of the copper concentration in the culture medium.In methanotrophs, the relationship between the concentration of copper and expression of the two different methane monooxygenases (MMOs) is well characterized (8, 11, 45, 49, 50). Under low copper-to-biomass ratios, methane oxidation activity is observed in the soluble fraction, and the enzyme is referred to as the soluble methane monooxygenase (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is observed in the membrane fraction, and the enzyme is referred to as the membrane-associated or particulate methane monooxygenase (pMMO). The polypeptides and structural genes for both enzymes have been characterized (4, 18–22, 24, 25, 32, 34–40, 43–45, 47–49, 51, 62, 63). In addition to expression of the two MMOs, four other physiological traits have been identified in cells expressing the pMMO that are affected by the copper concentration in the culture medium. First, the concentration of copper in the culture media is directly related to pMMO activity in cell-free fractions, although the levels of expression of pMMO polypeptides vary in different methanotrophs (1, 8, 30, 36, 50, 63). For example, the expression levels of the three pMMO polypeptides in Methylococcus capsulatus Bath remained constant with varying copper concentrations (8, 36), whereas in Methylomicrobium albus BG8, the expression level of the putative pMMO polypeptides increased with increased copper in the culture medium (8). Second, the concentrations of membrane-associated copper and iron show a proportional increase as the copper concentration in the culture medium is increased (36, 63). Third, the formation and level of intracytoplasmic membranes in cells cultured in copper-supplemented media are dependent on the copper concentration in the culture media (8, 11, 40, 48). Lastly, the K_s for methane oxidation by pMMO is altered by the copper concentration in the culture media (33a).Berson and Lidstrom (1) have recently noted that in spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMO^C) isolated by Phelps et al. (42). Fitch et al. (17) found that in M. trichosporium OB3b, these sMMO^C mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 × 10¹⁶ M⁻¹ (55). Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath (63). During the isolation of the pMMO from M. capsulatus Bath, CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.This paper ties together and extends these observations on specific copper acquisition systems in M. trichosporium OB3b and M. capsulatus Bath. Here we describe the initial isolation and characterization of two copper-complexing agents, called CBC-L₁ and CBC-L₂, from the M. trichosporium OB3b wild type and sMMO^C mutant PP319. CBC-L₁ from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath. This paper is also the first report of a second CBC, CBC-L₂, which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al. (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO^C mutant strains of M. trichosporium OB3b.     

Automatic cell identification and enrichment in lung cancer. III. Light scatter and two fluorescence parameters.

Two fluorescence parameters and size are used in a flow through system to enrich sputum specimens for cancer cells. Human cells in sputum which are stained with acridine orange show a characteristic distribution of red and green fluorescence from which cancer cells can be localized. The peak enrichment is obtained by selectively sorting cells with the largest values of red and green fluorescence. Cancer cells located in other distribution regions having smaller fluorescence intensities show progressively diminished nuclear and cytoplasmic tinctorial features by Papanicolaou stain, consistent with the decreased intensity of red and green fluorescence.     

Amphitropic amphiantarctic disjunctions in Apiaceae subfamily Apioideae

Aim Four genera of the plant family Apiaceae subfamily Apioideae –Apium, Chaerophyllum, Daucus and Lilaeopsis– are characterized by amphitropic and amphiantarctic distribution patterns, and in Australasia the subfamily is also represented by the tribe Aciphylleae. We infer the molecular ages of achieving amphitropic distribution for these lineages, reconstruct the biogeographical histories of Apium, Chaerophyllum, Daucus and Lilaeopsis, and identify the sister group of Aciphylleae. Location Worldwide, with an emphasis on South America and Australasia. Methods Divergence times were estimated employing a Bayesian approach (beast ) with fossil pollen of basal apioids as calibration points and using a data set of nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) sequences from 284 accessions of Apioideae. Additionally, maximum‐likelihood analyses were performed for data subsets comprising Apium, Daucus and Lilaeopsis. For Chaerophyllum, maximum‐likelihood and beast analyses were carried out using combined chloroplast DNA and ITS data. Biogeographical scenarios were inferred using diva and lagrange . Results The sister group to Aciphylleae is the Sino‐Himalayan Acronema clade and the divergence between these two lineages is dated at 34.8 Ma, whereas the radiation of Aciphylleae started 11.0 Ma. A Northern Hemispheric origin was inferred for Apium, Chaerophyllum and Daucus, whereas Lilaeopsis probably originated in South America following a dispersal of its ancestor from North America. Chaerophyllum, Daucus and Lilaeopsis dispersed to the Southern Hemisphere at 5.3, 7.0 and 27.9 Ma, respectively. For Apium, two dispersals from Europe were inferred: to South America at 6.3 Ma, and to southern Africa at 3.9 Ma. The taxa migrated along the land masses of North and South America (Daucus, Lilaeopsis) and Africa (Apium) or by direct transoceanic dispersals through the Atlantic (Apium) or the Pacific (Chaerophyllum). Within the Southern Hemisphere they dispersed both westwards (Apium, Daucus, Lilaeopsis) and eastwards (Chaerophyllum, Lilaeopsis). For Chaerophyllum and Lilaeopsis, subsequent dispersal events to the Northern Hemisphere were also inferred. Main conclusions Similar timing, contrasted with the diversity of migration routes, suggests that the dispersal events of these umbellifer taxa (and many other amphitropic amphiantarctic genera) were facilitated by favourable ecological conditions in the Southern Hemisphere (climatic cooling of the late Palaeogene/early Neogene) rather than by increased dispersal opportunities.     

Acute burns

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the pathophysiology of burn injury. 2. Identify patient criteria for transfer to a burn center. 3. Calculate burn size and resuscitation requirements. 4. Treat inhalation injury in the acute setting. 5. Describe treatment options for burn injuries. 6. Describe preoperative selection, intraoperative procedures, and postoperative protocols for patients who require surgical care for their burn injuries. 7. Understand the survival and functional outcomes of burn injury. SUMMARY: The review article summarizes basic issues in the treatment of acute burn injury as practiced in 2008. The pathophysiology, treatment options, and expected outcomes for an acute burn are described and discussed. Special attention is directed to the nonoperative and surgical management of small to moderate-size burns that might be treated by the practicing plastic surgeon.