Phosphorus mitigation during springtime runoff by amendments applied to grassed soil.

Permanent grass vegetation on sloping soils is an option to protect fields from erosion, but decaying grass may liberate considerable amounts of dissolved reactive P (DRP) in springtime runoff. We studied the effects of freezing and thawing of grassed soil on surface runoff P concentrations by indoor rainfall simulations and tested whether the peak P concentrations could be reduced by amending the soil with P-binding materials containing Ca or Fe. Forty grass-vegetated soil blocks (surface area 0.045 m, depth 0.07 m) were retrieved from two permanent buffer zones on a clay and loam soil in southwest Finland. Four replicates were amended with either: (i) gypsum from phosphoric acid processing (CaSO × 2HO, 6 t ha), (ii) chalk powder (CaCO, 3.3 t ha), (iii) Fe-gypsum (6 t ha) from TiO processing, or (iv) granulated ferric sulfate (Fe[SO], 0.7 t ha), with four replicates serving as untreated controls. Rainfall (3.3 h × 5 mm h) was applied on presaturated samples set at a slope of 5% and the surface runoff was analyzed for DRP, total dissolved P (TDP), total P (TP), and suspended solids. Rainfall simulation was repeated twice after the samples were frozen. Freezing and thawing of the samples increased the surface runoff DRP concentration of the control treatment from 0.19 to 0.46 mg L, up to 2.6-3.7 mg L, with DRP being the main P form in surface runoff. Compared with the controls, surface runoff from soils amended with Fe compounds had 57 to 80% and 47 to 72% lower concentrations of DRP and TP, respectively, but the gypsum and chalk powder did not affect the P concentrations. Thus, amendments containing Fe might be an option to improve DRP retention in, e.g., buffer zones.

(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Prevalence of antibodies against Chlamydia trachomatis and incidence of C. trachomatis-induced reactive arthritis in an early arthritis series in Finland in 2000.

OBJECTIVE: To study the prevalence of different serotypes of Chlamydia trachomatis antibodies and the incidence of C. trachomatis-induced reactive arthritis (ReA) among patients with early arthritis in a defined population. METHODS: Serum samples were collected from a cohort of 122 adult patients in the age group 18-65 years included in the Kuopio 2000 Arthritis Survey. Antibodies against C. trachomatis serotypes C, E, and G were studied using enzyme immunoassay (EIA) tests among patients and in a control cohort of 78 adults without any joint symptoms. The incidence assessment for Chlamydia-induced ReA was based on a ligase chain reaction (LCR) test in urine and clinical symptoms and signs appropriate for ReA. RESULTS: Of 122 patients, with the baseline diagnosis of rheumatoid arthritis (RA) in 11, spondyloarthropathy (SpA) in 28, and undifferentiated arthritis (UA) in 83 cases, 42 (34%) showed immunoglobulin (Ig)G or IgA antibodies against at least one serotype C, E, or G. Among the patients with UA the prevalence was significantly increased compared with the controls (p = 0.010). C. trachomatis-induced ReA arthritis was diagnosed in only three patients with the LCR test. On this basis the incidence of C. trachomatis-induced arthritis was 5.4/100 000 [95% confidence interval (CI) 1.1-15.7] in the age group 18-65 years. CONCLUSION: Antibodies against C. trachomatis were most common in patients with UA reflecting the fact that cases with chlamydia-induced ReA are included in this subgroup.

Effect of temporin A modifications on its cytotoxicity and antimicrobial activity.

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.

Highly immunoreactive antigenic site in a hydrophobic domain of HIV-1 gp41 which remains undetectable with conventional immunochemical methods.

A synthetic pentadecapeptide (A15; env residues 599-613: SGKLICTTAVPWNAS), derived from a hydrophobic region in the transmembrane protein gp41 of HIV-1 and comprising a highly immunoreactive antigenic site in eliciting antibody responses during HIV-1 infection in humans, was used to purify, by affinity, the corresponding anti-peptide antibodies from HIV-1-infected patient sera. The purified antibodies to peptide A15 reacted specifically with the peptide in EIA, but not in whole virus EIA. These antibodies were immunoreactive with the corresponding peptide-albumin conjugates in immunoblotting but not with gp41 molecules. The results suggest that the peptide A15 sequence is not exposed in intact gp41, but will be exposed and is antigenic in the course of HIV-1 infection in humans.

Amino acid sequence homology between human placental protein 14 and beta-lactoglobulins from various species.

The primary structure of 22 N-terminal amino acid residues of placental protein 14 was determined by automated Edman degradation with a gas-phase sequencer. This protein, isolated from the human placenta and its membranes, was considered pure as evidenced by a single N-terminal amino acid sequence M D I P Q T K Q D L E L P K L A G T W H S M. It shows significant sequence homology with horse, bovine, buffalo, sheep and goat beta-lactoglobulins. We found 13 identities out of 22 possible matches with horse beta-lactoglobulin. beta-lactoglobulins from several animal species have been found to bind retinol. Among the identical residues there is one tryptophan at position 19 which is conserved in beta-lactoglobulins and is also found in the human retinol-binding protein at the corresponding position. These data suggest a common origin of PP14 and beta-lactoglobulins.

A monoclonal antibody against a synthetic peptide reveals common structures among spectrins and alpha-actinin.

A monoclonal antibody (Mab) against a synthetic peptide, SEDYGKDL, corresponding to one conserved sequence in the chicken alpha-fodrin repeats reacts in immunoblotting with avian alpha-spectrin and alpha-fodrin, both mammalian spectrins and with mammalian alpha-fodrin. This Mab also reacts with alpha-actinin in both chicken and human cells. Our results confirm the previously detected structural homology between spectrins and alpha-actinin and implicate their common evolutionary origin.

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides.

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.

Recognition of synthetic peptides with sequences of rubella virus E1 polypeptide by antibodies and T lymphocytes.

Antigenicity of rubella virus E1 polypeptide was analyzed using synthetic peptides with predicted amino acid sequences. Overlapping solid-phase bound peptides were used to define antibody binding domains and a panel of free peptides to study T-cell responsiveness. Several antibody-binding areas including those earlier described to contain major neutralizing epitopes were recognized by human sera positive for rubella antibodies. T-cell lines specific for rubella virus were established from 14 rubella immune subjects. All cell lines responded to rubella virion-derived antigen but only eight (57%) responded to one or more of the synthetic peptides. Individual patterns of peptide recognition were found but peptide 8 representing amino acids 402-422 was most often stimulatory to T-cells lines, either alone (3 subjects) or in combination with peptide 3 (amino acids 245-269) or 3 and 4 (amino acids 269-287). The response was HLA restricted but no single DR specificity for this restriction was identified.

Monoclonal antibody defining a human syncytiotrophoblastic polypeptide immunologically related to mammalian retrovirus structural protein p30.

Antigenic material previously detected in human placental trophoblastic cells by immunoperoxidase staining using a goat antiserum against the feline RD114 retrovirus structural protein p30 was isolated by immunochromatography from normal syncytiotrophoblast. The antigen was used to immunize mice, and of the monoclonal antibodies produced by murine hybridomas an IgG1 was selected which reacted in enzyme immunoassay with the syncytiotrophoblast antigen and with purified RD114. This antibody, designated HPS-1, stained normal and neoplastic syncytiotrophoblasts in a manner similar to that of the goat antibodies, detected in immunoblotting a Mr = 130 000 polypeptide in cultured human choriocarcinoma cells and reacted in spot immunoblotting tests with purified preparations of mammalian retroviruses but not with an avian retrovirus. The polypeptide antigen may represent activation of human endogenous retroviral genes in syncytiotrophoblast.

Chlamydia trachomatis seropositivity is associated both with stillbirth and preterm delivery.

The cause of stillbirth and preterm delivery is often unknown. We studied the prevalence of Chlamydia trachomatis antibodies in mothers with stillbirth and preterm labor. Serum specimens from 72 mothers with stillbirth after the 21st gestational week, and from 48 mothers with preterm delivery between gestational weeks 23 and 29, both from the greater Helsinki area, and cord blood from 96 consecutive liveborn deliveries at the Department of Obstetrics and Gynecology, the University of Helsinki, were studied for antibodies to C. trachomatis immunotypes CJHI, GFK and BED by microimmunofluorescence test. The prevalence of C. trachomatis antibodies was highest, 33.3%, in mothers with stillbirth, 18.8% in mothers with preterm delivery, and 10.4% in cord blood. The IgM seropositivity rate was high among mothers with preterm delivery (8.3%). We conclude that C. trachomatis IgG antibodies are frequently detected in sera from mothers with stillbirth, suggesting past infection, while mothers with preterm delivery often have serum IgM antibodies, suggesting of acute infection.

Comparison of a new immunochromatographic rapid test with a commercial EIA for the detection of Puumala virus specific IgM antibodies.

BACKGROUND: Hantaviruses are associated with two human diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonary syndrome (HPS). Puumala virus (PUUV), which is one of the Hantaviruses, is a causative agent of nephropathia epidemica (NE), a mild form of HFRS. OBJECTIVE: a new 5 min rapid test, POC PUUMALA (Erilab Ltd, Finland), for detecting IgM antibodies to PUUV was evaluated and compared with the commercially available Hantavirus (Puumala) IgM ELISA test (Progen, Germany). Discrepant test results between the two tests were confirmed by a mu-capture reference EIA. STUDY DESIGN: Two hundred and thirty five serum samples, which had earlier been analyzed with the Progen IgM ELISA, were assayed with the POC PUUMALA rapid test. Five persons, without knowing the Progen IgM ELISA test results, interpreted independently the rapid test results. In addition, a panel of 48 serum samples was analyzed in parallel with the rapid test and the Progen IgM ELISA by one technician in daily routine diagnostics in a clinical microbiology laboratory. RESULTS: the agreement between the results of the five interpreters was 95%, and the congruence of the results between individual readers and commercial ELISA test varied from 93 to 96%. Diagnostic efficacy of the rapid test varied between 98 and 99% compared with 96% of the Progen IgM ELISA. The POC PUUMALA rapid test showed higher or similar sensitivity compared with the Progen IgM ELISA, whereas both the tests had similar levels of specificity. CONCLUSIONS: the analytical performance of the POC PUUMALA rapid test was found to be as good or even slightly better than the analytical performance of the Progen IgM ELISA. In addition, the rapid and straightforward procedure makes the POC PUUMALA a feasible tool for the diagnosis of the acute PUUV infection.

Synthetic peptides in HIV antibody screening and typing.

We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that is immunoreactive with all studied HIV-1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections.

PIP: This article reviews the basic method used to define native epitopes from transmembrane proteins and the function of synthetic peptides in HIV screening and typing. Identification of continuous native epitopes from structural protein sequences of HIV-1 and HIV-2 involves the use of systematic scanning epitope technology. Scanning profiles of these two types of HIV demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 as well as in the corresponding region in HV-2 gp34. However, the number of highly immunoreactive regions differs in the structural proteins of the two types of HIV infections. These highly immunoreactive regions, when presented accurately as synthetic antigens in solid-phase enzyme immunoassay, can provide tests that are remarkably appropriate for the early and reliable diagnosis and type-specification of HIV-1 and HIV-2 infections.

Detection of Chlamydia trachomatis antibodies by 2 novel tests: rELISA and peptide EIA.

The performance of 2 newly developed enzyme immunoassays (EIAs) intended for the routine serological diagnosis of chlamydial infections was evaluated. rELISA is based on a recombinant lipopolysaccharide antigen which detects chlamydia genus-specific antibodies, and Chlamydia trachomatis EIA is based on a peptide derived from major outer membrane protein and is therefore species-specific. Both tests distinguished patients with tubal factor infertility (TFI) or pelvic inflammatory disease (PID) from the controls. The prevalence of IgA antibodies was higher for the PID patients than for the TFI patients; the finding indicates a more active state of infections for the PID patients. Furthermore, C. trachomatis EIA detected more IgG antibodies in the TFI patients than in patients with non-tubal factor infertility. In conclusion, rELISA detected chlamydial antibodies in general, and C. trachomatis EIA detected species-specific antibodies. These EIA tests may be useful in the serodiagnosis of chlamydial infection.

Patient evaluation of the care and rehabilitation process in geriatric hospital care.

PURPOSE: To gain a deeper understanding of how elderly persons experience and evaluate the care and rehabilitation process. METHOD: Qualitative interview data from elderly patients were analysed using a grounded theory approach. The patients were interviewed twice, at the beginning of geriatric hospital care and some weeks after discharge. RESULTS: The patient-perceived outcome of the care and rehabilitation process reflected two dimensions; the effect on their health and the quality of the process, i.e. how their needs were met. The analysis revealed that the patients' needs differed during the care and rehabilitation process. It also indicated that patients perceived their needs and the care differently based on their previous experience of the care unit, their perceived trajectory of illness and their "patient character" which represented the patient's definition of himself/herself and the situation. A hypothetical model of the patients' evaluation process has been derived. CONCLUSION: The results indicate the importance of using a process perspective in the assessment and the interpretation of patient-perceived outcome of care and rehabilitation, and that patient expectations, trajectories of illness and the patient character must be taken into consideration.

Development of novel peptide ligands modulating the enzyme activity of prostate-specific antigen.

Prostate-specific antigen (PSA) is a serine proteinase produced mainly by epithelial cells of the prostate. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. The major problem of the PSA determination in early diagnosis is the high false positive rate due to benign prostatic hyperplasia, but the clinical accuracy can be improved by determining the proportions of various molecular forms of PSA. The main biological function of PSA is liquefaction of the seminal gel formed after ejaculation, but PSA has also been suggested to regulate invasiveness and metastatic potential of prostatic tumors. Thus, agents binding to and affecting the function of PSA have the potential to be used for diagnosis and therapy of prostate cancer. We have developed peptides specific for PSA by using cyclic phage display peptide libraries. After deducing the amino acid sequence of the peptides by sequencing the relevant part of phage genome, the peptides were expressed as glutathione-S-transferase (GST) fusion proteins or produced by chemical synthesis. The peptides were shown to bind to PSA specifically as indicated by lack of binding to other related serine proteinases. The binding of the peptides with PSA was strongly inhibited by monoclonal antibodies specific for free PSA and they did not bind to PSA-inhibitor complexes indicating that they bind close to the active site of the enzyme. Most of the peptides enhanced the enzyme activity of PSA against a chromogenic substrate. The affinity of the peptides could be increased by including Zn2+ in the reaction mixture. These results show that peptides that bind to PSA and modulate its enzyme activity can be developed by phage display techniques. These peptides have the potential to be used for targeting of prostatic tumors and diagnostics of prostate cancer.

SPECT/CT imaging of baculovirus biodistribution in rat.

Non-invasive imaging provides essential information regarding the biodistribution of gene therapy vectors and it can also be used for the development of targeted vectors. In this study, we have utilized micro Single-photon emission computed tomography to visualize biodistribution of a (99m)Tc-polylys-ser-DTPA-biotin-labelled avidin-displaying baculovirus, Baavi, after intrafemoral (i.f.), intraperitoneal (i.p.), intramuscular (i.m.) and intracerebroventricular (i.c.v.) administration. The imaging results suggest that the virus can spread via the lymphatic network after different administration routes, also showing accumulation in the nasal area after systemic administration. Extensive expression in the kidneys and spleen was seen after i.p. administration, which was confirmed by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Additionally, transduction of kidneys was seen with i.m. and i.f. administrations. We conclude that baculovirus may be beneficial for the treatment of kidney diseases after i.p. administration route.

The major horse allergen Equ c 1 contains one immunodominant region of T cell epitopes.

BACKGROUND: Despite the fact that most significant mammalian respiratory allergens are lipocalin proteins, information on the human T cell reactivity to these allergenic proteins is largely missing. OBJECTIVE: Knowing the T cell epitopes in allergens is a prerequisite for developing novel preparations for allergen immunotherapy. METHODS: Specific T cell lines were generated with recombinant Equ c 1 from the peripheral blood mononuclear cells (PBMCs) of 10 horse-allergic subjects. For determining T cell epitopes, the lines were stimulated with 16mer synthetic Equ c 1 peptides overlapping by 14 amino acids. The binding capacity of Equ c 1 peptides to human leucocyte antigen class II molecules was determined by the competitive ELISA. RESULTS: The major horse allergen Equ c 1 resembles two other lipocalin allergens, the major cow allergen Bos d 2 and the major dog allergen Can f 1, in that it is weakly stimulatory for the PBMCs of sensitized subjects. Moreover, the T cell epitopes of Equ c 1 are clustered in a few regions along the molecule, as is the case with Bos d 2 and Can f 1. Similar to Bos d 2, Equ c 1 contains one immunodominant epitope region at the carboxy-terminal end of the molecule. The T cell lines of eight horse-allergic subjects out of 10 showed strong reactivity to one or both of the two overlapping peptides, p143-158 and p145-160, in this region. The region probably contains two overlapping epitopes. CONCLUSION: The 18mer peptide p143-160 from the immunodominant region of Equ c 1 is a potential candidate for the peptide-based immunotherapy of horse-sensitized subjects.

Magnetic resonance imaging of viral particle biodistribution in vivo.

We describe here a technique for the visualization of viral vector delivery by magnetic resonance imaging (MRI) in vivo. By conjugating avidin-coated baculoviral vectors (Baavi) with biotinylated ultra-small superparamagnetic iron oxide particles (USPIO), we are able to produce vector-related MRI contrast in the choroid plexus cells of rat brain in vivo over a period of 14 days. Ten microlitres of 2.5 x 10(10) PFU/ml nuclear-targeted LacZ-encoding Baavi with bUSPIO coating was injected into rat brain ventricles and visualized by MRI at 4.7 T. As baculoviruses exhibit restricted cell-type specificity in the rat brain, altered MRI contrast was detected in the choroid plexus of the injected ventricles. No specific signal loss was detected when wild-type baculoviruses or intact biotinylated USPIO particles were injected into the lateral ventricles. Cryosectioned brains were stained for nuclear-targeted beta-galactosidase gene expression, which was found to colocalize with MRI contrast. This study provides the first proof of principle for robust and non-invasive viral vector MRI by using avidin-displaying viruses in vivo. Considering the widespread use of MRI in current medical imaging, the approach is likely to provide numerous future applications in imaging of therapeutic gene transfer.

Purification, from cultured human choriocarcinoma cells, of a 75000-Mr protein reacting with antibodies to a synthetic peptide based on a cloned human endogenous provirus nucleotide sequence.

We previously detected in cultured choriocarcinoma cells a 75000-Mr polypeptide defined by immunoblotting with antibody to a synthetic peptide Sp23 (Cys-Glu-Asn-Pro-Ser-Gln-Phe-Tyr-Glu-Asp-Leu) based on a cloned human endogenous proviral nucleotide sequence. On immunohistological staining, anti-Sp23 stains antigen(s) in the syncytiotrophoblasts of first-trimester placentas and in renal-cell adenocarcinoma tissues. The present report describes purification to homogeneity of the protein from cultured choriocarcinoma cells. The procedure involves extraction with non-ionic detergent and h.p.l.c. using, sequentially, gel-permeation, anion-exchange and reverse-phase columns. The yield was 110 micrograms/g of total choriocarcinoma-cell protein. The results indicate that the purified protein is a monomeric and relatively hydrophilic molecule of Mr 75000.