A case of foodborne listeriosis in Sweden

A 70-year-old woman fell seriously ill overnight with meningitis and was admitted to hospital. Cerebrospinal fluid culture yielded Listeria monocytogenes . One of the first problems in solving a human case of listeriosis suspected to be foodborne is to find the foods likely to have been transmitting L. monocytogenes . Two enrichment procedures and a direct plating procedure were used for isolation of the bacteria from different food items collected from the patient's refrigerator, local retail store and producer. Samples of vacuum-packed products of sliced pork brawn, sliced cooked medwurst and berliner wurst of the same brand harboured L. monocytogenes . Serotyping and restriction enzyme analysis (REA) with pulsed-field gel electrophoresis (PFGE) were used to characterize and compare 41 isolates, including the human strain. At least three clones were present in the foods investigated, and one of these was identical to the human clone. This clone was present in samples of medwurst from the patient's refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.     

Listeria monocytogenes in Faeces from Clinically Healthy Dairy Cows in Sweden

Faecal samples from 102 clinically healthy dairy cows, representing 34 farms in the Swedish province of Uppsala, were analysed for the presence of Listeria spp. using an enrichment procedure. Listeria monocytogenes was isolated from six (6%) and L. innocua from 2 (2%) cows. From each of the 6 samples positive for L. monocytogenes, 5 isolates were further characterised by restriction enzyme analysis using the 3 enzymes Apa I, Sma I, and Asc I, followed by pulsed-field gel electrophoresis. Three of the L. monocytogenes positive cows lived at the same farm, and they all harboured the same clonal type. One of these 3 cows also harboured a further clonal type of L. monocytogenes. The fact that one of the cows harboured 2 different clonal types of L. monocytogenes is important from an epidemiological point of view when routes of infection are to be investigated.     

Formation of vasculo-syncytial membranes in the human placenta.

Vasculo-syncytial membranes are localised areas of the placental villous membrane where the thickness of the barrier separating the maternal and fetal circulations is reduced to as little as 1-2 microns. Consequently, they are believed to be important sites for diffusional exchange. The morphological appearances suggest that they are caused by the obtrusion of locally dilated segments of the fetal capillaries into the trophoblast layer. This study sought quantitative evidence for the hypothesis by performing stereological analyses on vasculo-syncytial membranes at the electron microscopic level. The results confirmed that a strong relationship existed between the thickness of the capillary endothelium and that of the overlying stromal and trophoblastic tissue at these sites (r = 0.47, P < 0.001), indicating that some asymmetrical stretching or remodelling of the capillary wall was involved. Comparisons were also made between the thickness of the trophoblastic, stromal and endothelial components of the villous membrane in villi obtained from the central and from the peripheral parts of placental lobules, where vasculo-syncytial membrane formation is accentuated. The mean thickness of each component was lowest in the samples from the peripheral region, although the differences only proved to be statistically significant for the stromal layer (P = 0.01). Both sets of data lend quantitative support to the hypothesis that vasculo-syncytial membrane formation is the result of obtrusion of locally dilated segments of the fetal capillaries. The way in which this may be linked to changes in the dynamics of the fetal circulation as gestation advances is discussed.     

Prevalence and Fingerprinting of Listeria monocytogenes Strains Isolated from Raw Whole Milk in Farm Bulk Tanks and in Dairy Plant Receiving Tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml⁻¹. L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

Characterization of thePac25I Restriction-Modification Genes Isolated from the Endogenous pRA2 Plasmid ofPseudomonas alcaligenesNCIB 9867

Genes for the class IIPseudomonas alcaligenesNCIB 9867 restriction-modification (R-M) system,Pac25I, have been cloned from its 33-kb endogenous plasmid, pRA2. ThePac25I endonuclease and methylase genes were found to be aligned in a head-to-tail orientation with the methylase gene preceding and overlapping the endonuclease gene by 1 bp. The deduced amino acid sequence of thePac25I methylase revealed significant similarity with theXcyI,XmaI,Cfr9I, andSmaI methylases. High sequence similarity was displayed between thePac25I endonuclease and theXcyI,XmaI, andCfr9I endonucleases which cleave between the external cytosines of the recognition sequence (i.e., 5′-C↓CCGGG-3′) and are thus perfect isoschizomers. However, no sequence similarity was detected between thePac25I endonuclease and theSmaI endonuclease which cleaves between the internal CpG of the recognition sequence (i.e., 5′-CCC↓GGG-3′). Both thePac25I methylase and endonuclease were expressed inEscherichia coli.An open reading frame encoding a protein which shows significant similarity to invertases and resolvases was located immediately upstream of thePac25I R-M operon. In addition, a transposon designated Tn5563was located 1531 bp downstream of the R-M genes. The location on a self-transmissible plasmid as well as the close association with genes involved in DNA mobility suggests horizontal transfer as a possible mode of distribution of this family of R-M genes in various bacteria.     

Identification of Mycoplasma pirum genes involved in the salvage pathways for nucleosides.

Genes encoding enzymes involved in the salvage pathway for nucleosides have been cloned and sequenced from the mollicute Mycoplasma pirum. One of them, encoding deoxyriboaldolase, was functionally identified by complementation of an Escherichia coli mutant. These genes are clustered, suggesting an operon organization, and they are immediately followed by the putative gene for the triose phosphate isomerase, an enzyme used during glycolysis.     

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.     

Angiotensin II injures the arterial wall causing increased aortic stiffening in apolipoprotein E-deficient mice

Cardiovascular diseases, such as atherosclerosis and hypertension, are associated with arterial stiffening. Previous studies showed that ANG II exacerbated atherosclerosis and induced hypertension and aneurysm formation in apolipoprotein E-deficient (apoE-KO) mice. The aim of the present study was to examine the effects of chronic treatment of ANG II on the arterial elastic properties in apoE-KO mice. We hypothesized that ANG II will injure the arterial wall resulting in increased arterial stiffening. Male apoE-KO mice were infused with either ANG II (1.44 mg. kg(-1). day(-1)) or vehicle (PBS) for 30 days. ANG II treatment accelerated atherosclerosis in the carotid artery by sixfold (P < 0.001) and increased blood pressure by 30% (P < 0.05). Additionally, our data demonstrated that ANG II increased arterial stiffening using both in vivo and in vitro methods. ANG II significantly increased pulse wave velocity by 36% (P < 0.01) and decreased arterial elasticity as demonstrated by a more than 900% increase in maximal stiffening (high strain Young's modulus) compared with vehicle (P < 0.05). These functional changes were correlated with morphological and biochemical changes as demonstrated by an increase in collagen content (60%), a decrease in elastin content (74%), and breaks in the internal elastic lamina in the aortic wall. In addition, endothelium-independent vasorelaxation to sodium nitroprusside was impaired in the aortic rings of ANG II-treated mice compared with vehicle. Thus, the present data indicate that ANG II injures the artery wall in multiple ways and arterial stiffening may be a common outcome of ANG II-induced arterial damage.     

Laminin modulates morphogenic properties of the collagen XVIII endostatin domain

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204-1243 of the alpha chain, 932-1161 of the beta chain, and 150-965 of the gamma chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.     

Neuregulin signaling via ErbB receptor assemblies in the nervous system

Neuregulins (NRG) play important roles in the development, maintenance, and repair of the nervous system, with influences on neuronal migration, synaptogenesis, receptor subunit composition, and the proliferation/survival of oligodendrocytes and Schwann cells. However, the precise detail of how the NRGs signal through ErbB receptors, particularly at central synapses, is incomplete. The receptor kinase domain provides sites for association with adaptor proteins. In addition, evidence from recent reports suggests that ErbB2/4 receptors, through their C-terminal amino acids, can form specific associations with scaffolding proteins. The existence of such assemblies expands the range of signaling cascades available to the NRGs.