Characterization of Monilinia fructicola Strains Resistant to Both Propiconazole and Boscalid.

In 2011 and 2012, significant brown rot disease caused by Monilinia fructicola was observed in a peach orchard in Spartanburg County, SC, despite preharvest fungicide applications of demethylation inhibitor (DMI), quinone outside inhibitor (QoI), and succinate dehydrogenase inhibitor (SDHI) fungicides. All 22 isolates obtained in 2011 from this orchard were sensitive to the QoI fungicide, azoxystrobin, and the methyl benzimidazole carbamate (MBC) fungicide, thiophanate-methyl. Five were resistant to the DMI fungicide, propiconazole, and were selected, together with five propiconazole-sensitive isolates, for further investigations. One of the 10 isolates was resistant to propiconazole but sensitive to the SDHI fungicide, boscalid (EC50 = 0.42 µg/ml), 3 were resistant to propiconazole with intermediate sensitivity to boscalid (EC50 0.72 to 2.1 µg/ml); 2 were sensitive to propiconazole with intermediate sensitivity to boscalid; 3 were sensitive to propiconazole but resistant to boscalid (EC50 ≥ 2.1 µg/ml); and 1 (isolate MD22) was resistant to both propiconazole and boscalid. Disease incidence on detached fruit treated with formulated propiconazole or boscalid was significantly higher for MD22 compared to a sensitive control isolate. Continued monitoring of fungicide resistance in the same orchard in 2012 revealed an increase of isolates resistant to propiconazole from 22.7% in 2011 to 34.7%, and an increase of isolates resistant to both propiconazole and boscalid from 4.5% in 2011 to 18.4%. Propiconazole resistance was always associated with the presence of the 'Mona' mobile element located upstream of the sterol 14α-demethylase (MfCYP51) gene. To investigate whether mutations in the subunits of the succinate dehydrogenase enzyme were involved in boscalid resistance, significant portions of the M. fructicola SdhA, SdhB, SdhC, and SdhD genes were cloned and analyzed for 2 sensitive, 2 boscalid-resistant, and 6 dual-resistant isolates. Although sequence variation was found among the isolates, no single change correlated with resistance. Interestingly, analysis of isolates collected from orchards in 2001 and 2002, prior to the registration of boscalid, revealed a range of sensitivities to boscalid (EC50 0.03 to 3.46 µg/ml) including boscalid-resistant isolates. The presence of boscalid-resistant isolates in the baseline population was unexpected and requires further investigation.

Prospective association between food sensitization and food allergy: results of the LISA birth cohort study.

BACKGROUND: Food allergy is common, especially in childhood, where 6-8% of children are affected. Identification of early and efficient markers for later development of food allergy is very important. OBJECTIVE: We examined the ability of repeated measurements of food sensitization in early childhood to predict doctor-diagnosed food allergy (DDFA) at the age of 6 years. METHODS: The analysis was based on data from a prospective birth cohort study. Information was collected by parental questionnaires, and blood samples were obtained at 2 and 6 years of age. Children with repeated determination of sensitization to food allergens at 2 and 6 years of age were categorized into the sensitization phenotypes: no, early onset, late onset and persistent sensitization. The association between sensitization phenotypes and DDFA was prospectively investigated using multiple logistic regression analyses. RESULTS: Of 3097 children recruited at birth, a complete follow-up of IgE measurements and questionnaires at 1.5, 2 and 6 years were available for 1082 children. Early food allergen sensitization (fx5) was a strong risk for DDFA at 6 years [odds ratio (OR)=4.7; 95% confidence intervals (95% CI) 2.0-11.2] and for a new onset of DDFA at 6 years (OR=4.1; 95% CI 1.5-11.3). Additionally, persistent food allergen sensitization increased the risk of DDFA at 6 years (OR=6.1; 95% CI 2.7-13.7). Early sensitized children with a history of parental atopy showed the highest risk for DDFA at 6 years. CONCLUSION: Food-sensitized children during the first 2 years of life, especially with a family history of atopy, might be considered as a susceptible subgroup that requires specific attention concerning the development of food allergy-related symptoms.

The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

Novel N-glycosylation in eukaryotes: laminin contains the linkage unit beta-glucosylasparagine.

The linkage unit to protein of N-linked carbohydrate in eukaryotic glycoproteins consists of N-acetylglucosamine, coupled to the amido nitrogen of asparagine. Additional N-glycosyl linkage units have been unequivocally proven to exist only in the cell surface glycoproteins of various bacteria. Based on immunological analyses, isolation and chemical characterization, we report that one of these units, namely glucose linked to asparagine, exists in the mammalian protein laminin, an extracellular basement membrane component. This finding and the occurrence of identical disaccharide structures in archaebacterial cell surface glycoproteins and mammalian basement membrane protein complexes points towards a conserved and distinct function of these extracellular structural elements. In addition, a method is described to uncover a masked epitope in fixed tissues by chemical O-deglycosylation. This has allowed to morphologically localize the antigen beta-Glc-Asn by immunofluorescence to the basement membranes of kidney glomeruli.

Evaluation of a new point-of-care test for influenza A and B virus in travellers with influenza-like symptoms.

Point-of-care (POC) tests for influenza facilitate clinical case management, and might also be helpful in the care of travellers who are at special risk for influenza infection. To evaluate influenza POC testing in travellers, a new assay, the ImmunoCard STAT! Flu A and B, was used to investigate travellers presenting with influenza-like symptoms. Influenza virus infection was diagnosed in 27 (13%) of 203 patients by influenza virus-specific PCR and viral culture. The POC test had sensitivity and specificity values of 64% and 99% for influenza A, and 67% and 100% for influenza B, respectively. Combined sensitivity and specificity were 67% and 99%, respectively, yielding positive and negative predictive values of 95%, and positive and negative likelihood ratios of 117 and 0.34, respectively. The convenient application, excellent specificity and high positive likelihood ratio of the POC test allowed rapid identification of influenza cases. However, negative test results might require confirmation by other methods because of limitations in sensitivity. Overall, influenza POC testing appeared to be a useful tool for the management of travellers with influenza-like symptoms.

Semisynthetic engineering of proteinase inhibitor homologues.

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.

Proteolytic processing of pro-ACTH/endorphin begins in the Golgi complex of pituitary corticotropes and AtT-20 cells.

The intracellular sites where proteolytic processing of pro-ACTH/endorphin or POMC take place have not yet been reliably identified. We have used affinity-purified antisera that recognize only the products of POMC processing and immunoelectron microscopy to identify the compartments of rat pituitary corticotropes and mouse AtT-20 cells in which cleavage occurs. Immunoperoxidase labeling of cryostat sections and immunogold labeling of ultrathin frozen sections were used for localization of the processing sites. By both procedures we detected processed peptides in Golgi cisternae and secretion granules. Within the Golgi, labeling was limited to the last or transmost cisterna and was most concentrated in its dilated rims which contain condensing secretory protein. No labeling of other Golgi cisternae was seen. All Golgi cisternae were labeled, however, when antisera that recognize unprocessed POMC were used for immunolabeling. We conclude that in AtT-20 and rat pituitary cells: 1) processing of POMC through at least two endo- and exoproteolytic cleavage steps and alpha-amidation of joining peptide begin in the trans Golgi subcompartment; 2) no detectable processing takes place before POMC reaches the trans Golgi cisterna; and 3) this Golgi cisterna as well as secretion granules contain the active enzymes necessary for proteolytic processing and alpha-amidation.

Biogenesis of podocalyxin--the major glomerular sialoglycoprotein--in the newborn rat kidney.

The appearance and distribution of podocalyxin on the glomerular epithelium (podocytes) during glomerular development was determined in the newborn rat kidney using specific monoclonal and affinity-purified polyclonal antibodies. Kidneys from 2-day-old rats were perfusion-fixed and processed for immunofluorescence or immunoperoxidase localization or immunogold labeling on ultrathin frozen sections. Podocalyxin first appeared on the apical surfaces of the presumptive podocytes of the S-shaped body above the level of the junctional complexes that connect the cells at this stage. The latter consist of a shallow occluding zonule and a deeper adhering zonule. Early in the capillary loop stage, when the urinary spaces open and the junctional complexes migrate from the apex to the base of the cells, labeling for podocalyxin extended along the lateral plasmalemma above the migrating junctions. In the maturing glomerulus when the foot processes form and the occluding and adhering junctions give way to developing slit diaphragms, podocalyxin was found along all newly-opened surfaces above the occluding junctions or slit membranes. No labeling was found below the latter. Podocalyxin was also detected intracellularly throughout the entire exocytotic pathway--i.e., in the rough endoplasmic reticulum and perinuclear cisternae, in Golgi cisternae and associated vesicles, and in carrier vesicles presumably en route to the cell surface. It is concluded that 1) podocalyxin is synthesized at a high rate in the differentiating podocyte; 2) its distribution is restricted to the apical plus lateral plasmalemmal domain facing the urinary spaces above the migrating junctions; 3) its time of appearance and distribution during glomerular development are identical to that reported earlier for epithelial polyanion; and 4) its synthesis and insertion into the podocyte plasmalemma is closely coupled to the development of the foot processes and filtration slits.

Novel fibroblast-specific monoclonal antibodies: properties and specificities.

Specific detection of fibroblasts has been one of the unsolved problems in cell biology. Because monoclonal antibodies (MoAbs) might provide an easy and reproducible method of fibroblast detection, we have produced a panel of MoAbs raised against cell surface proteins of human dermal fibroblasts. Using flow cytometry and immunohistochemistry, we have shown that two of these MoAbs, FibAS01 and FibAS02, react exclusively with human fibroblasts. They do not react in vitro with human keratinocytes, endothelial cells, or blood cells. Immunohistologic experiments investigating the binding pattern of the MoAbs FibAS01 and FibAS02 in cryostat sections of different tissues confirmed the flow cytometric results. In human skin, the antibodies exclusively labeled fibroblasts. In other human tissues such as lymph nodes, placenta, kidney, muscle, thyroid gland, gall bladder, cartilage, and tendon, the specificity for fibroblasts was borne out. Neither antibody reacts with fibroblasts from mouse, rat, or pig. The isotype was defined as an IgG1 for both. By western blot analysis, both antibodies detected a molecule of 60-65 kDa under reducing and nonreducing conditions. By immunoelectron microscopy, we observed the antigens on the cell surface without any clustering at specific sites. These data demonstrate that the two MoAbs, FibAS01 and FibAS02, exclusively recognize human fibroblasts.

Morphometric studies of the extraglomerular mesangial cell field in volume expanded and volume depleted rats.

The extraglomerular mesangial cell field was studied by morphometric techniques in volume expanded and volume depleted rats. The volume density of the extraglomerular mesangial interstitium was found to be significantly different between the two conditions, 16.9 +/- 3.7% in volume depletion and 29.0 +/- 4.1% in volume expansion. No difference in the volume density of the peritubular interstitium could be detected under the same conditions. These findings are interpreted as indicating a specific sensitivity of the extraglomerular mesangial interstitium to changes in body fluid content, a phenomenon which may play a role in the mechanism of resetting the tubulo-glomerular feedback control.

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species.

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.

Instability of a pEA29 Marker in Erwinia amylovora Previously Used for Strain Classification.

We examined the use of previously observed restriction fragment length polymorphisms (RFLPs) of a polymerase chain reaction (PCR)-amplified fragment of plasmid pEA29 for differentiating strains of Erwinia amylovora. The PCR fragment from E. amylovora strain CA11 contains a region of 8-bp tandem repeats which is predicted to cause the RFLPs. Examination of a collection of 93 strains revealed the repeat sequence GATTACA(GAATTACA)nGAATTACA in pEA29 with n ranging from 3 to 14. Selected strains were examined after growth in liquid culture to establish the stability of this character. Four strains originally with n = 14, 13, 7, and 3 repeats were grown overnight in liquid culture and streaked onto agar plates to produce individual colonies. Respectively, 4, 10, 1, and 0 out of 17 colonies per strain had an altered copy number when retested. Considering the instability in the number of repeats, it is concluded that the polymorphism in this region of pEA29 is not useful as a marker for following the migration of E. amylovora.

[Soft oculopression]. / Softokulopression.

BACKGROUND: The method of so-called oculopression is used to reduce the intraocular tension before intraocular operations. For outpatient cataract operations we tested a gentle and, especially for the patient, pleasant alternative which combines an easy and safe treatment with good results for tension reduction. PATIENTS AND METHOD: For a total of 100 patients a routine examination glove was filled with 350 ml of warm tap water and after injection placed for 10 min on the eye scheduled for surgery. RESULTS: With this method, an excellent and self-calibrating reduction of the intraocular pressure could be shown (average intraocular pressure before injection: 18.94 mmHg, after injection: 31.34 mmHg and after soft oculopression: 20.31 mmHg). CONCLUSION: The method of soft oculopression can be recommended because of its excellent self-calibrating tension reducing effect combined with a pleasant and easy application.

[Ambulatory geriatric rehabilitation--scope, models, prospects]. / Ambulante geriatrische Rehabilitation--Rahmenbedingungen, Modelle, Perspektiven.

At present the further development of outpatient geriatric rehabilitation is characterized by contradictions. On the one hand the legal basis and political statements on this matter emphasize the two principles/aims "home care comes before institutional care" and "rehabilitation comes before nursing care". On the other hand deficits with regard to the infrastructure, insufficient cooperation and coordination among the relevant parties as well as the existing payment system for benefits provided hinder the realization of these principles/aims. This paper does not only describe those barriers in detail but also outlines framework conditions and specific models which would facilitate the further positive development of outpatient geriatric rehabilitation.

[Synthesis of conjugates of heptalysine and penicillins (author's transl)]. / Synthese von Konjugaten des Heptalysins mit Penicillinen

Heptalysine was synthesized as a non-immunogenic carrier for artificial antigens with penicilloyl-group specificity. The synthesis was carried out by conventional techniques via different routes by condensation of appropriately protected intermediates. Best results were obtained with the use of the benzyloxycarbonyl group for intermediate protection of the alpha-amino-groups and the tert.-butyloxycarbonyl-group together with the tert.-butylester for permanent blockage of the terminal alpha-amino group, the epsilon-amino groups and the terminal carboxyl group. Heptalysine and also lysine were reacted with benzylpenicillin, alpha-aminobenzylpenicillin and tert.-butyloxycarbonyl-alpha aminobenzylpenicillin--to prevent the alpha-amino groups of penicillin from reacting--in aqueous solutions at pH 10.6-11.6 according to Levine and Redmond. The products were isolated after precipitation with acid or dialysation against water by lyophilisation. The penicilloyl group content of the conjugates as estimated from elemental analysis, penamaldate tests and the NMR spectra proved to be rather high: 5-7 residues/mol.

[Synthesis of omega-carboxyacyl-L-phenylalanine-aryl esters and their use as substrates for cathepsin G and chymotrypsin]. / Synthese von omega-Carboxyacyl-L-phenylalanin-arylestern und ihre Verwendung als Substrate für Cathepsin G und Chymotrypsin.

The synthesis of 5-carboxyvaleryl- and 3-carboxypropionyl-L-phenylalanine beta-naphthyl ester (Adi-Phe-ONap, Suc-Phe-ONap) and 3-carboxypropionyl-L-phenylalanine p-nitrophenyl ester (Suc-Phe-ONp) is reported. The two latter compounds were obtained in good yields by 3-carboxypropionylation of the L-phenylalanine aryl esters with succinic anhydride at pH values below 6 in aqueous organic solutions. The beta-naphthyl esters in particular proved to be sensitive substrates for cathepsin G and chymotrypsin. They are not or only slightly hydrolyzed by other proteinases like elastases, kininogenases, e.g. kallikrein, plasmin, thrombin and trypsin. The spontaneous hydrolysis of the beta-naphthyl esters is relatively slow below pH 8. beta-Naphthol split-off during the enzyme reaction may be conveniently monitored at 328.5 nm (epsilon = 1730M-1 X cm-1) or with an at least 15-fold increase in sensitivity in a discontinuous assay after coupling with Fast Garnet at 520 nm (epsilon = 34800M-1 X cm-1). The increase in absorbance is linear with time and proportional to the amount of enzyme up to A 328.5 of at least 0.62. Adi-Phe-ONap is preferentially used for cathepsin G (at 328.5 nm 9.2-fold more sensitive than benzoyl-L-tyrosine ethyl ester, Bz-Tyr-OEt) whereas for chymotrypsin Suc-Phe-ONap is more advantageous (4.2-fold increase in sensitivity at 328.5 nm over Bz-Tyr-OEt). The influence of dimethyl sulphoxide and Brij 35 on the activity of cathepsin G and chymotrypsin was investigated using Suc-Phe-ONap as the substrate. The values of Km and kcat were determined for both enzymes and substrates. Because of the relatively high rates of spontaneous hydrolysis above pH 7.0 the use of Suc-Phe-ONp is less advantageous.

[Current personnel status and qualification requirements in nursing care]. / Gegenwärtige Personalsituation und Qualifizierungserfordernisse in der Pflege.

Efforts to improve the quality of care and to adapt the professional care giving structures to changing demands are mainly influenced by staff qualification. Based on a survey of empirical studies and data sets the present qualification structures on German home care services and nursing homes for the aged will be described. The results showed that the German care-giving system is characterized by a variety of professions and qualification structures. The changing paradigm of care-giving still showed no reflection in practical work. Furthermore, a variety of different educational and training programs of professional nurses impedes its professionalization.

[Status of terminal care in family practice]. / Zum Stellenwert der Betreuung Sterbender in der hausärztlichen Tätigkeit.

The changing paradigm in the health care system to focus stronger on community care also effects the care of the dying. In consequence, general practitioners are confronted with changing tasks and challenges. In an exploratory oriented study 80 general practitioners, who were recruited in one rural and one urban area were interviewed with regard to different variables of the care of the dying such as self concept, different aspects of burden, pain management, education and vocational training as well as cooperation with third parties. Main results show that age of the general practitioners and amount of burden experienced are interrelated. Further, the subject "death and dying" is hardly considered in education and training and the cooperation between the general practitioner and other health care professionals has to be extended and improved.

The PIN and LAX families of auxin transport genes in Medicago truncatula.

Auxin transport proteins may be involved in nodule development. As a prelude to investigating the roles of these proteins in nodule development, we took advantage of the genetic and molecular resources available in the legume Medicago truncatula to characterize the gene families encoding auxin efflux and influx carriers. We identified ten auxin efflux carrier sequences (MtPINs) and five auxin influx/permease sequences (MtLAXs). The genomic sequence of each of these fifteen genes was determined, the genes were mapped on the publicly available map of M. truncatula, and their expression was examined in shoot and root tissue of nodulating plants. With one exception, transcripts of all MtPIN genes were detected. The expression of MtPIN2 was limited to nodulating roots, while transcripts of all other expressed genes were detected in both shoots and roots. Both the PIN and LAX gene families contain more members in M. truncatula than in Arabidopsis, but the gene families are not significantly expanded. Sequence comparison of the M. truncatula PIN and LAX genes with PIN and LAX genes from other dicots and monocots indicates that both gene families share a common overall structure, with areas of high homology both within M. truncatula and across species boundaries. Molecular phylogenies of both the PIN and LAX gene families were constructed. Combined with intron position and expression data, the phylogenies were used to assign relationships between MtPIN and MtLAX genes and the orthologous Arabidopsis PIN and LAX genes. MtPIN2 and MtPIN7 appear to be the result of a recent gene duplication with subsequent divergence of expression patterns. These results set the stage for the use of these genes in research on the role of auxin in nodulation.