Mechanically Tunable Hollow Silica Ultrathin Nanoshells for Ultrasound Contrast Agents.

Perfluoropentane (PFP) gas filled biodegradable iron-doped silica nanoshells have been demonstrated as long-lived ultrasound contrast agents. Nanoshells are synthesized by a sol-gel process with tetramethyl orthosilicate (TMOS) and iron ethoxide. Substituting a fraction of the TMOS with R-substituted trialkoxysilanes produces ultrathin nanoshells with varying shell thicknesses and morphologies composed of fused nanoflakes. The ultrathin nanoshells had continuous ultrasound Doppler imaging lifetimes exceeding 3 hours, were twice as bright using contrast specific imaging, and had decreased pressure thresholds compared to control nanoshells synthesized with just TMOS. Transmission electron microscopy (TEM) showed that the R-group substituted trialkoxysilanes could reduce the mechanically critical nanoshell layer to 1.4 nm. These ultrathin nanoshells have the mechanical behavior of weakly linked nanoflakes but the chemical stability of silica. The synthesis can be adapted for general fabrication of three-dimensional nanostructures composed of nanoflakes, which have thicknesses from 1.4-3.8 nm and diameters from 2-23 nm.

Exploration of chemically cross-linked metal-organic frameworks.

A series of geometrically constrained, cross-linked benzene dicarboxylic acid (bdc) derivatives have been synthesized and incorporated into the canonical isoreticular metal-organic framework (IRMOF) lattice. Only certain cross-links, which allow for the proper relative orientation of the bdc subunits, form the desired IRMOF. Design criteria from these cross-linked ligands allowed for the rational design of two oligomeric ligands composed of three bdc monomers tethered together. These oligomeric ligands were also readily incorporated into an IRMOF lattice with a high degree of crystallinity and porosity, providing a new dimension to rational ligand design for metal-organic frameworks.

Malignant potential in a Brunner's gland hamartoma.

Brunner's gland hamartomas are rare tumours of the duodenum. These lesions have previously been described as being benign, with no malignant potential. A case report is presented of a Brunner's gland hamartoma, whose histology revealed a focus of well marked epithelial dysplasia. This case suggests a dysplastic stage in the natural history of Brunner's gland hamartoma, and questions the malignant potential of these lesions.

In vitro and in vivo assessment of Salmonella enterica serovar Typhimurium DT104 virulence.

Multidrug-resistant Salmonella enterica serovar Typhimurium phage type DT104 has become a widespread cause of human and other animal infection worldwide. The severity of clinical illness in S. enterica serovar Typhimurium DT104 outbreaks has led to the suggestion that this strain possesses enhanced virulence. In the present study, in vitro and in vivo virulence-associated phenotypes of several clinical isolates of S. enterica serovar Typhimurium DT104 were examined and compared to S. enterica serovar Typhimurium ATCC 14028s. The ability of these DT104 isolates to survive within murine peritoneal macrophages, invade cultured epithelial cells, resist antimicrobial actions of reactive oxygen and nitrogen compounds, and cause lethal infection in mice were assessed. Our results failed to demonstrate that S. enterica serovar Typhimurium DT104 isolates are more virulent than S. enterica serovar Typhimurium ATCC 14028s.

The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice.

The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB:lacZ fusion to wild-type levels. A sigma32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41 degrees C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

A mitochondrial model for premature ageing of somatically cloned mammals.

Cloned sheep have recently been discovered to have an unexpectedly advanced biological age. We propose that the explanation is a simple consequence of inheritance of acquired, free radical-induced cellular damage with somatic mitochondria that contribute to the mitochondrial population of cloned cells but not to zygotes produced by fertilization in normal sexual reproduction. Each increment of ageing in cloning experiments is therefore predicted to be maternally inherited. The hypothesis suggests practical ways of decreasing the effect. The hypothesis is itself a prediction of the recent proposal that mitochondria of the female germ line function primarily as genetic templates.

Transposon-derived Brucella abortus rough mutants are attenuated and exhibit reduced intracellular survival.

The O antigen of Brucella abortus has been described as a major virulence determinant based on the attenuated survival of fortuitously isolated rough variants. However, the lack of genetic definition of these mutants and the virulence of naturally occurring rough species, Brucella ovis and Brucella canis, has confused interpretation. To better characterize the role of O antigen in virulence and survival, transposon mutagenesis was used to generate B. abortus rough mutants defective in O-antigen presentation. Sequence analysis of DNA flanking the site of Tn5 insertion was used to verify insertion in genes encoding lipopolysaccharide (LPS) biosynthetic functions. Not surprisingly, each of the rough mutants was attenuated for survival in mice, but unexpected differences among the mutants were observed. In an effort to define the basis for the observed differences, the structure of the rough LPS and the sensitivity of these mutants to individual killing mechanisms were examined in vitro. All of the B. abortus rough mutants exhibited a 4- to 5-log-unit increase, compared to the smooth parental strain, in sensitivity to complement-mediated lysis. Little change was evident in the sensitivity of these organisms to hydrogen peroxide, consistent with an inability of O antigen to exclude relatively small molecules. Sensitivity to polymyxin B, which was employed as a model cationic, amphipathic peptide similar to defensins found in phagocytic cells, revealed survival differences among the rough mutants similar to those observed in the mouse. One mutant in particular exhibited hypersensitivity to polymyxin B and reduced survival in mice. This mutant was characterized by a truncated rough LPS. DNA sequence analysis of this mutant revealed a transposon interruption in the gene encoding phosphomannomutase (pmm), suggesting that this activity may be required for the synthesis of a full-length core polysaccharide in addition to O antigen. B. abortus O antigen appears to be essential for extra- and intracellular survival in mice.

The mammalian acrosome reaction: gateway to sperm fusion with the oocyte?

Mammalian sperm undergo discharge of a single, anterior secretory granule following their attachment to the zona pellucida surrounding the oocyte. This secretory discharge is known for historical reasons as the acrosome reaction. It fulfils a number of purposes and without it, sperm are unable to penetrate the zona pellucida and fuse with the oocyte. In this review, we focus on the role of the acrosome reaction in the development of fusion competence in sperm. Any naturally occurring membrane fusion has two major sequential steps: a docking or adhesion step, in which two membranes adhere, and a fusion step, in which their lipid bilayers are destabilized and merged and a cellular compartment is either created or destroyed. Recent evidence suggests that there is an important role for oocyte integrins and sperm-bound disintegrins in mammalian sperm/oocyte adhesion and fusion. The fusion mechanism employed by sperm remains poorly understood, however, and circumstantial evidence suggests it is more complex than the interaction between a single protein species and its target. Sperm/oocyte fusion is probably the most accessible eukaryotic model for intercellular fusion currently available, partly because it is temporally separated from gene expression. Elucidation of the mechanism of sperm/oocyte fusion may throw light on the mechanism of other intercellular fusions such as myoblast fusion, and the evolutionary relationship of intercellular membrane fusion to intracellular membrane fusion.

Improved nonthoracotomy defibrillation based on ventricular fibrillation waveform characteristics.

The heart has been shown to be more susceptible to defibrillation at a higher absolute ventricular fibrillation voltage (AVFV) measured on the surface ECG. This study evaluated in a closed-chest canine model (n = 7) the clinical applicability of using a real-time VF waveform analysis system using an electrogram defined between the generator can and an RV endocardial electrode. Under fluoroscopic guidance, superior vena cava and RV spring coil catheter electrodes were inserted through the external jugular vein. A subcutaneous patch was placed on the left chest. A two-parameter tracking algorithm was used to dynamically identify the high AVFV area, and a biphasic shock was triggered synchronously at the next peak. The performance of this new peak shock method (PSM) was compared to the conventional method of shocking at a fixed time in 175 paired trials. Five shocks per voltage and five voltages per animal were randomized between the two methods to permit the generation of sigmoidal dose response curves for the estimation of 50% (E50), 75% (E75), and 100% (E100) success energies. Induction of VF and discharge voltage were kept constant while energy delivered, impedance (R), and AVFV at the point of shock were measured. Energy (8.63 +/- 0.40 vs 8.64 +/- 0.40 J), R (48.60 +/- 0.30 vs 48.59 +/- 0.30 omega), and current (7.50 +/- 0.18 vs 7.51 +/- 0.16 A) were not significantly different between trials for either the conventional or the PSM. The time from the onset of VF until the defibrillation shock was 7.98 +/- 1.44 seconds. Higher overall successes (46.3% vs 33.1%; P < 0.01) and lower E50, E75, and E100 were observed for the PSM. Finally, the significantly higher AVFV (9.12 +/- 0.32 vs 4.73 +/- 0.34 mV; P < 0.0001) with the peak method suggests that the high VF voltage could be detected as it occurred in real-time. The improved defibrillation success supports the use of this method for nonthoracotomy defibrillation.

A critical period of ventricular fibrillation more susceptible to defibrillation: real-time waveform analysis using a single ECG lead.

Previous studies have suggested that variations in the underlying ventricular fibrillation (VF) waveform may be one of the factors responsible for the probabilistic nature of defibrillation. The heart appeared to be more susceptible to defibrillation at higher absolute VF voltages (AVFV). This study investigated in an open-chest canine model (n = 8), a newly developed system that analyzed the VF waveform in real-time, instantaneously determined the time to shock, and immediately delivered a fixed low energy DC shock. A two parameter tracking technique using a running long-term and short-term AVFV average was devised to automatically identify a high voltage peak area of the VF waveform, which has been hypothesized to represent a critical period susceptible to defibrillation. Using a DC shock estimated at the 50% success level, the performance using this technique in 58 defibrillation trials was compared to the performance of the conventional method of shocking at a fixed time (random shock method) in 62 trials. Patch size, electrode location, and discharge voltage were kept constant while VF duration, transmyocardial resistance (TMR), energy delivered, and AVFV at the point of shock were measured. Shock energy and current, TMR, and VF duration were similar with both shock methods. A significantly higher AVFV was observed for trials performed with the peak shock method (0.66 +/- 0.02 mV) as compared to trials performed with the random shock method (0.25 +/- 0.09 mV) (P < 0.003). Using lead II as the only sensing lead, the success rate was increased in 6 of 8 dogs (75%) with the new method. One animal showed identical performance, and one animal a worse performance. The overall increase in success rate was 24% using a single ECG lead (range 0%-100%; P < 0.04). Our data document that using this algorithm a period of high VF voltage can be detected in real-time. The improved success in the majority of animals supports the hypothesis that a critical period susceptible to defibrillation exists during VF. However, the high AVFV detected using a single ECG lead did not translate to an improved success rate in all animals. This suggests that other factors in addition to the VF voltage measured on a single lead of the ECG are important in characterizing this critical period.

Monoclonal antibodies which recognize equatorial segment epitopes presented de novo following the A23187-induced acrosome reaction of guinea pig sperm.

Acrosome-intact mammalian sperm can adhere to zona pellucida-free oocytes but are only capable of fusing if they have previously undergone the acrosome reaction. This suggests that the acrosome reaction results in presentation of at least one novel epitope which plays a role in sperm-oocyte fusion. Monoclonal antibodies were raised against unfixed acrosome-reacted guinea pig sperm and screened by indirect immunofluorescence for binding to the equatorial segment. They were back-screened against unfixed acrosome-intact sperm for absence of binding. Using this approach, two antibodies, G11 and M13, were identified which detect equatorial segment epitopes presented de novo by sperm following an A23187-induced acrosome reaction. The localization of these epitopes to the equatorial segment was confirmed at the ultrastructural level by indirect immunogold-labelling. Fluorescein isothiocyanate-labelled Fab fragments of these two antibodies also localized to the equatorial segment. Affinity chromatography and western blotting established that the two mAbs recognize the same proteins, which have M(r)s of 34, 46, 48 and 51 x 10(3). When sperm were induced to undergo the acrosome reaction with A23187 and incubated with their discharged acrosomal contents, a further band was produced with an M(r) of 30 x 10(3). Production of this band was inhibited in the combined presence of 100 microM phenylmethylsulphonyl fluoride and 100 microM p-aminobenzamidine even though these compounds do not inhibit acrosomal exocytosis. Neuraminidase and O-glycosidase were without effect on the proteins detected by antibodies G11 and M13. Endoglycosidase F, however, eliminated the bands of M(r) 46, 48 and 51 x 10(3) and replaced them with a strong band of M(r) 44 x 10(3) and two minor bands of M(r) 43 and 45 x 10(3). Formaldehyde fixation of acrosome-intact sperm caused partial rupture of the acrosome with loss of the characteristic rouleaux (stacks) of guinea pig sperm. Indirect labelling of these formaldehyde-fixed sperm with fluorescein isothiocyanate- or gold-labelled second antibody, with or without permeabilization with 0.05% Triton X-100, showed dense labelling on the cytoplasmic face of the plasma membrane overlying the convex surface of the acrosome but little labelling elsewhere. Cryosections of acrosome-intact sperm labelled indirectly with immuno-gold showed labelling consistent with the same location, as well as sporadic labelling at other intracellular sites overlying the acrosome. Since there is no evidence that sperm can translocate intact membrane protein from the cytoplasmic face to the extracellular face of the plasma membrane during the acrosome reaction, the evidence suggests that there are two isolated antigen pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Redox conditions specify the proteins synthesised by isolated chloroplasts and mitochondria.

In chloroplasts and mitochondria isolated from pea leaves, (35)S-methionine incorporation reveals that different subsets of proteins are selected for synthesis in the presence of the external redox reagents ferricyanide, ascorbate, duroquinol, dithiothreitol and dithionite, and in the presence of different electron transport inhibitors in the light (in chloroplasts) or with respiratory substrates (in mitochondria). Redox state of specific electron carriers may therefore regulate expression of specific genes in chloroplasts and mitochondria. The results are consistent with the hypothesis that chloroplast and mitochondrial genomes encode proteins whose synthesis must be regulated by electron transport in photosynthesis and respiration.

Expression of p53 protein in oesophageal carcinoma: clinicopathological correlation and prognostic significance.

Correlation of p53 expression with 5-year survival and histopathological parameters was examined immunohistochemically in two groups of 30 patients with oesophageal carcinoma (5-year survivors versus non-survivors). Tumour type, sex, operative procedure and age were matched. Some 64 per cent of squamous carcinomas and 79 per cent of adenocarcinomas were p53 positive. Normal squamous, normal glandular and metaplastic glandular epithelia were negative. Dysplastic squamous and glandular epithelium adjacent to tumours was positive when the tumour was positive and negative when it was not. Univariate analysis showed that nodal status (P = 0.001), and grade and depth of invasion (both P = 0.01) correlated with outcome. Correlation of tumour grade with outcome, when the most poorly differentiated area is used, is a novel finding for oesophageal carcinoma. The p53 status was not significantly associated with survival or any of these parameters.

Electrophysiologic consequences of hyperkalemic cardioplegia during surgical ischemia.

Myocardial protection strategies use cardioplegic solutions to reduce the injury induced by surgical ischemia and reperfusion. However, there is a high incidence of electrophysiologic abnormalities after cardioplegic arrest. A computerized epicardial mapping system in a porcine cardiopulmonary bypass model was used to measure the electrophysiologic consequences of different myocardial protection techniques. Both warm and cold, crystalloid and blood cardioplegic solutions were compared. The effects of hypothermia and prolonged cardiopulmonary bypass were examined in a control group that underwent a 2-hour period of hypothermia without cardioplegia or aortic cross-clamping, followed by 2 hours of normothermic reperfusion. Isochronous activation maps, unipolar electrograms, ventricular refractory periods, and pacing thresholds were measured before cardioplegic arrest and during reperfusion. Compared with the control group, crystalloid cardioplegia, but not blood cardioplegia, was accompanied by large changes in the pattern of ventricular activation and by persistent (> 2 hours) and significant slowing of the time required for complete ventricular activation. This was not the result of hypoxia. Moreover, the effective refractory period and the pacing threshold were unchanged by any cardioplegia. Our data suggest that crystalloid cardioplegia increases myocardial resistance to current flow leading to a derangement of electrical impulse propagation that may underlie arrhythmogenesis.

Classification of CEA-related positivity in primary and metastatic malignant melanoma.

Using a panel of antibodies to carcinoembryonic antigen (CEA), in paraffin-processed biopsy material patchy, predominantly membranous positivity was seen on tumour cells in 70 per cent of cases of superficial spreading melanoma, 60 per cent of nodular melanomas, and 75 per cent of secondary deposits studied with unabsorbed polyclonal anti-CEA only. No staining was seen using monoclonal anti-CEAs. Localization of CEA to the cell membrane was confirmed with confocal microscopy. Immunoblotting of fresh frozen material detected CEA of around 180 kD in both primary and metastatic melanomas migrating with an apparent molecular weight of between 150 and 200 kD, indicating variable glycosylation of the protein. Recognition of an adhesive role for CEA with roles in immunolocalization and immunotherapy emphasizes the importance of more precise classification of CEA-related positivity in human tumours.

Anti-inflammatory effects of LPS, MDP and FMLP on carrageenan pleurisy in the rat.

Bacterial products fmet-leu-phe (FMLP), muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were assayed for their ability to alter the inflammatory response to lambda carrageenan-induced pleurisy in Hooded Surgery rats. Continuously infused FMLP, or one initial i.v. dose of FMLP, MDP or LPS either ablated or partially suppressed the pleurisy. Total circulating leucocytes and neutrophils were suppressed by 55-65% when compared to the normal circulating leucocyte response to carrageenan pleurisy, excepting the protocol incorporating a single i.v. dose of FMLP where suppression was intermediate at 30%. There were also significant changes in the expression of FMLP receptors on circulating neutrophils. MDP and LPS induced a receptor number increase of 2 and 1.7 times initial value respectively, whilst a continuous FMLP infusion caused a receptor decrease to 0.3 times the initial value. The introduction of bacterial products at an alternative site to that of the pleurisy had an anti-inflammatory effect and the pleurisy was reduced.