High-frequency ultrasound-based differentiation between nodular dermal filler deposits and foreign body granulomas.

BACKGROUND: The number of procedures involving dermal filler injection is still on the rise. Although their safety is improving, not all complications can be avoided. The late complications manifesting as nodules or granulomas pose a particular diagnostic and therapeutic challenge, due to the lack of uniform standards or guidelines. High-frequency, ultrasound imaging appears to be a useful method for distinguishing between granulomas and nodular dermal filler deposits. AIM: The aim of the paper was to evaluate the utility of high frequency ultrasound imaging for distinguishing between foreign body granulomas and nodular dermal filler deposits. MATERIAL AND METHODS: Eleven females aged 21-66 years (mean age of 43.6 years old) who had soft tissue fillers injected were enrolled. All patients had a high-frequency ultrasound scan of the involved skin area performed. The shape, margins, area, location and echogenicity of the lesions were assessed. Additionally, the lesions were evaluated histologically and photographs were taken. RESULTS: The analysis indicated differences between the ultrasound image of granulomas and dermal filler deposits. Characteristic ultrasound features of granulomas include oval shape and blurred, irregular outer edges. Small hyperechoic areas were seen inside the granulomas. The deposits were anechogenic, with sharp, regular borders. CONCLUSIONS: High-frequency ultrasound imaging enables distinguishing between granulomas and nodules-dermal filler deposits, which form after dermal filler injections.

Quantitative trait loci for leaf chlorophyll fluorescence parameters, chlorophyll and carotenoid contents in relation to biomass and yield in bread wheat and their chromosome deletion bin assignments.

Relatively little is known of the genetic control of chlorophyll fluorescence (CF) and pigment traits important in determining efficiency of photosynthesis in wheat and its association with biomass productivity. A doubled haploid population of 94 lines from the wheat cross Chinese Spring × SQ1 was trialled under optimum glasshouse conditions for 4 years to identify quantitative trait loci (QTL) for CF traits including, for the first time in wheat, JIP-test parameters per excited cross section (CSm): ABS/CSm, DIo/CSm, TRo/CSm, RC/CSm and ETo/CSm, key parameters determining efficiency of the photosynthetic apparatus, as well as chlorophyll and carotenoid contents to establish associations with biomass and grain yield. The existing genetic map was extended to 920 loci by adding Diversity Arrays Technology markers. Markers and selected genes for photosynthetic light reactions, pigment metabolism and biomass accumulation were located to chromosome deletion bins. Across all CF traits and years, 116 QTL for CF were located on all chromosomes except 7B, and 39 QTL were identified for pigments on the majority of chromosomes, excluding 1A, 2A, 4A, 3B, 5B, 1D, 2D, 5D, 6D and 7D. Thirty QTL for plant productivity traits were mapped on chromosomes 3A, 5A, 6A, 7A, 1B, 2B, 4B, 6B, 7B, 3D and 4D. A region on chromosome 6B was identified where 14 QTL for CF parameters coincided with QTL for chlorophyll content and grain weight per ear. Thirty-five QTL regions were coincident with candidate genes. The environment was shown to dominate in determining expression of genes for those traits.

Structure-function analysis of Yersinia pestis YopM's interaction with alpha-thrombin to rule on its significance in systemic plague and to model YopM's mechanism of binding host proteins.

The plague virulence protein YopM of Yersinia pestis KIM5 belongs to the large family of leucine-rich repeat (LRR) proteins. The only activity demonstrated so far for YopM is thrombin-binding, which could be a function of the small amount of YopM that is released into surrounding tissues by the bacteria. This study combined deletional and mutational analysis, chemical crosslinking assays, and in vitro functional tests with molecular modelling to identify key features of YopM necessary for interacting with thrombin. Two Y. pestis strains expressing YopM variants that differed in thrombin binding were used to assess the importance of thrombin-binding for lethality of plague. Both strains suffered a similar decrease in virulence by three orders of magnitude, indicating that thrombin-binding per se was not the major deficiency for lethality in the systemic disease model employed. It remains possible that extracellular YopM could contribute to plague pathology and to early events in peripheral tissues. The structural studies provided a model for how YopM may interact with thrombin and an insight into how YopM's LRR structure may assemble distinct regions for binding different targets.

Targeting of the Yersinia pestis YopM protein into HeLa cells and intracellular trafficking to the nucleus.

The YopM virulence protein of Yersinia pestis has been described as binding human alpha-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.

Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca2+ response.

Yersinia pestis contains a virulence plasmid, pCD1, that encodes many virulence-associated traits, such as the Yops (Yersinia outer proteins) and the bifunctional LcrV, which has both regulatory and antihost functions. In addition to LcrV and the Yops, pCD1 encodes a type III secretion system that is responsible for Yop and LcrV secretion. The Yop-LcrV secretion mechanism is believed to regulate transcription of lcrV and yop operons indirectly by controlling the intracellular concentration of a secreted repressor. The activity of the secretion mechanism and consequently the expression of LcrV and Yops are negatively regulated in response to environmental conditions such as Ca2+ concentration by LcrE and, additionally, by LcrG, both of which have been proposed to block the secretion mechanism. This block is removed by the absence of Ca2+ or by contact with eukaryotic cells, and some Yops are then translocated into the cells. Regulation of LcrV and Yop expression also is positively affected by LcrV. Previously, LcrG was shown to be secreted from bacterial cells when the growth medium lacks added Ca2+, although most of the LcrG remains cell associated. In the present study, we showed that the cell-associated LcrG is cytoplasmically localized. We demonstrated that LcrG interacts with LcrV to form a heterodimeric complex by using chemical cross-linking and copurification of LcrG and LcrV. Additionally, we found that small amounts of LcrV and YopE can be detected in periplasmic fractions isolated by cold osmotic shock and spheroplast formation, indicating that their secretion pathway is accessible to the periplasm or to these procedures for obtaining periplasmic fractions. We propose that the cytoplasmically localized LcrG blocks the Yop secretion apparatus from the cytoplasmic side and that LcrV is required to remove the LcrG secretion block to yield full induction of Yop and LcrV secretion and expression.

Interaction between Yersinia pestis YopM protein and human alpha-thrombin.

YopM, a 41.5 kDa virulence protein of Yersinia pestis is believed to have an anti-inflammatory role in bubonic plague. It has been shown previously that YopM binds human alpha-thrombin but not prothrombin and inhibits thrombin-induced platelet aggregation in vitro. In the present studies we carried out crosslinking reactions between purified YopM and alpha-thrombin or its blocked form FPR-alpha-thrombin in the presence of various competitors to identify where on thrombin YopM binds. We found that thrombin cleaves YopM at the C-terminus, indicating that this part of YopM must interact with thrombin's catalytic site. Hirudin, a 65 amino acid natural thrombin inhibitor, prevents both the YopM degradation and the formation of a ca. 75 kDa crosslinking complex between YopM and alpha-thrombin. A similar effect is observed when hirugen, a short peptide corresponding to hirudin's C-terminus (amino acids 58-64), is used as a synthetic thrombin inhibitor. A 15 bp long specific oligonucleotide known to block alpha-thrombin successfully competes with YopM for the thrombin-binding site, whereas a control, scrambled sequence aptamer does not. As these competitors block a fibrinogen binding site (also called anion binding exosite I), our crosslinking data indicate that YopM binds not only to the active site of alpha-thrombin but also to the abeI.

Differential effects of deletions in lcrV on secretion of V antigen, regulation of the low-Ca2+ response, and virulence of Yersinia pestis.

The Yersinia pestis V antigen is necessary for full induction of low-calcium response (LCR) stimulon virulence gene transcription, and it also is a secreted protein believed to have a direct antihost function. We made four nonpolar deletions in lcrV of Y. pestis to determine if secretion, regulation, and virulence functions could be localized within the V antigen (LcrV). Deletion of amino acids 25 to 40 caused secretion of LcrV to be decreased in efficiency; however, removal of residues 108 to 125 essentially abolished LcrV secretion. Neither mutation had a significant effect on LCR regulation. This showed that LcrV does not have to be secreted to have its regulatory effect and that the internal structure of V antigen is necessary for its secretion. Both mutants were avirulent in mice, showing that the regulatory effect of LcrV could be separated genetically from its virulence role and raising the possibility that residues 25 to 40 are essential for the virulence function. This study provides the best genetic evidence available that LcrV per se is necessary for the virulence of Y. pestis. The repressed LCR phenotype of a mutant lacking amino acids 188 to 207 of LcrV raised the possibility that the deleted region is necessary for regulation of LCR induction; however, this mutant LcrV was weakly expressed and may not have been present in sufficient amounts to have its regulatory effect. In double mutants containing this mutant lcrV and also lacking expression of known LCR negative regulators (LcrG, LcrE, and LcrH), full induction of the LCR occurred in the absence of functional LcrV, indicating that LcrV promotes induction not as an activator per se but rather by inhibiting negative regulators.

Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R-M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5' half site of the wild type site is repeated in inverse orientation. The additional base pair in the non-specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl-terminal DNA binding domain which recognizes the 3' half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino-terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R-M systems will be discussed.

Regulation by Ca2+ in the Yersinia low-Ca2+ response.

The Yersinia low-Ca2+ response (LCR) is a regulatory response in which a set of plasmid-borne operons is transcriptionally regulated at 37 degrees C in response to the presence or absence of mM concentrations of Ca2+. LCR-regulated operons encode secreted proteins with regulatory and virulence roles as well as non-secreted regulatory proteins and components of the secretion machinery. Downregulation by Ca2+ is imposed by a signalling cascade that includes secreted proteins and possibly also components of the secretion system and is hypothesized to act on membrane-bound inductive components. An important role in LCR induction is played by LcrD, an inner-membrane protein with homologues in several virulence-associated and flagella assembly-related systems in diverse bacterial species. The mechanism of signal transduction in response to Ca2+ is not known, and the proteins that bind DNA to downregulate transcription have not been identified.

New suicide vector for gene replacement in yersiniae and other gram-negative bacteria.

A suicide vector named pUK4134 was constructed to enlarge the repertoire of vectors available for allelic exchange of mutated sequences in gram-negative bacteria. This plasmid combines the properties of two previously described plasmid vectors, pJM703.1 and pRTP1. pUK4134 is a suicide vector, carrying the origin of replication of the plasmid R6K and thus requiring the product of the pir gene for its stable maintenance. The rpsL gene encoding Escherichia coli ribosomal protein S12 confers streptomycin sensitivity on streptomycin-resistant strains and provides a positive selection for bacteria that have lost the plasmid following allelic exchange. The bla gene provides for selection by Apr. Other features are a unique EcoRV cloning site, oriT of plasmid RK2, and the bacteriophage lambda cos sequence. This vector was successfully used several times to carry out allelic exchange in Yersinia pestis.

Relation between cardiovascular pathologic conditions and malignant tumours as seen by pathologist. II. Myocardial infarct and coronary atherosclerosis in the population suffering from malignant neoplasm as seen by pathologist.

In the autopsy material of 2932 men and 3051 women the frequency of fresh myocardial infarct and the severity of coronary atherosclerosis were highly significantly lower in the population suffering from malignancies than in that free of cancer. Lower frequency of myocardial infarct seems to result from lower severity of coronary atherosclerosis. The incidence rate of myocardial infarct was also significantly lower in the cancer--bearing group when both compared populations showed identical, severe (grade 4) coronary atherosclerosis. This observation seems to indicate lesser contribution of coronary spasm in cancerous population and justifies the discussion of possible significance of EDRF (endothelium-derived relaxing factor) and catecholamines.

[Clinical and histopathological observations of retinal sutures. Preliminary report]. / Kliniczne i histopatologiczne obserwacje szwów siatkówkowych. Doniesienie wstepne.

One of the therapeutical methods in the great hole and ora serrata retinal detachment are the retino-scleral sutures. The study evaluates the tolerance of this type of stitches. Experimental investigations have been performed in rabbits with sutures of Ethilon 8-0 or chromic collagen 8-0 put through the sclera. Both segments--the anterior and posterior one have been taken under consideration. The eye have been enucleated on the 1st, 2d, 7th, 14th and 60th day after surgery and evaluated macroscopically. The specimen from the spot of stitching have been executed after fixation in formaline. Clinical observations, macroscopic evaluation of the enucleated eyes and results of the histopathological examination showed good tolerance of the retinal stitches through the tissue of the rabbits eye and indicate the possibility of a clinical utilization of this method.

The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems.

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.

The EcoDXX1 restriction and modification system of Escherichia coli ET7. Purification, subunit structure and properties of the restriction endonuclease.

The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system. The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography. Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S. The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme. The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity. The enzyme has also methylase activity acting against non-modified DNA.