Differences in response to the toxin sirodesmin PL produced by Phoma lingam (Tode ex fr.) Desm. on protoplasts,cell aggregates and intact plants of resistant and susceptible Brassica accessions

Summary The selective property of sirodesmin PL, a toxin produced by Phoma lingam, was studied on protoplasts, cell aggregates, leaves and roots. Directly after isolation, protoplasts from all the different Brassica accessions were sensitive when treated with toxin in a concentration higher than 1 M. When more differentiated plant tissue. i.e. cell aggregates, leaves or roots, were investigated, insensitivity to the toxin was found in the plant material resistant to P. lingam, while the plant material susceptible to P. lingam was sensitive. The results reveal that a clear correlation between resistance to P. lingam and insensitivity to sirodesmin PL is present, and that the toxin can be used to distinguish resistant plant material from susceptible both in vivo and in vitro.     

The effect of different blood sampling sites and analyses on the relationship~ between exercise intensity and 4.0 mmol ·1? blood lactate concentration

The aim of the study was to examine whether the difference in lactate concentration in different blood fractions is of practical importance when using blood lactate as a test variable of aerobic endurance capacity. Ten male firefighters performed submaximally graded exercise on a cycle ergometer for 20-25 min. Venous and capillary blood samples were taken every 5 min for determination of haematocrit and lactate concentrations in plasma, venous and capillary blood. At the same time, expired air was collected in Douglas bags for determination of the oxygen consumption. A lactate concentration of 4.0 mmol.l-1 was used as the reference value to compare the oxygen consumption and exercise intensity when different types of blood specimen and sampling sites were used for lactate analysis. At this concentration the exercise intensity was 17% lower (P less than 0.01) when plasma lactate was compared to venous blood lactate, and 12% lower (P less than 0.05) when capillary blood lactate was used. Similar discrepancies were seen in oxygen consumption. The results illustrated the importance of standardizing sampling and handling of blood specimens for lactate determination to enable direct comparisons to be made among results obtained in different studies.     

Quantitation of glucagon by radioreceptorassay

Receptors for glucagon on rat liver membranes were characterized. They bound [125I] glucagon rapidly in a specific and saturable way. Addition of unlabelled glucagon displaced [125I] glucagon from the binding sites in a concentration dependent way. Concentrations from 10(-9) to 10(-8) M of glucagon caused a linear reduction of binding of labelled glucagon. This concentration interval was used for a three-point assay which fulfilled statistical requirements for validity. Individual assays normally resulted in potency estimates of high precision and statistical weight. Mean values for glucagon activity of preparations tested by receptor assay were within the fiducial limits (P = 0.95) for corresponding activity determined by the rabbit blood glucose method. The receptor assay is less time consuming and requires only part of one rat liver while the in vivo assay uses 16 rabbits. Thus, the receptor assay is less resource demanding and should serve well as a screening instrument for control of potency of glucagon preparations.     

An in vitro bioassay for quantitation of human interferons by measurements of antiproliferative activity on a continuous human lymphoma cell line

Interferons have, in addition to their antiviral effects, been shown to possess several non-antiviral activities. In this study, an in vitro bioassay for interferon alpha (IFN-alpha) preparations based on their antiproliferative effect in cultured Daudi cells has been developed. Briefly, about 10(5) cells per ml treated with different concentrations of IFN were incubated under standard culture conditions for 3 days. Two different end points, i.e. incorporation of [3H]thymidine and final cell density, were used and responses were evaluated according to established pharmacopoeial principles for quantification of biomolecules. Both methods gave similar results. However, measurement of final cell density yielded the most precise results. The proposed assay, with an effective assay range of 1-10 IU/ml (approximately 0.2-2 x 10(-12)M, had a high sensitivity and precision as well as a good reproducibility. Compared with antiviral assays, it is less resource demanding. In conclusion, the in vitro bioassay described is well suited for potency determinations of IFN-alpha and probably also IFN-beta preparations.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

Induction of an immune network cascade in cancer patients treated with monoclonal antibodies (ab1)

The antitumor effector functions of unconjugated monoclonal antibodies (mAb) in cancer therapy are not fully understood. Direct cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytolysis and apoptosis have been suggested. Induction of anti-idiotypic (ab₂) and anti-anti-idiotypic (ab₃) antibodies as well as the corresponding T cells (T₂ and T₃) has also been proposed to be of therapeutic significance. In this study induction of an immune network cascade in ten patients with colorectal carcinoma, treated with mAb 17-1A (ab₁) was assessed. After treatment, all ten patients had anti-idiotypic antibodies and anti-anti-idiotypic antibodies with ab₁-like binding specificity while only five of ten patients had T cells corresponding to ab₃ (T₃) as assessed by a proliferation assay (DNA synthesis), and an assay of interferon production (ELISPOT) (Enzyme-linked immuno SPOT) in vitro or by a delayed-type hypersensitivity reaction in vivo. Purified T cells from four of the five patients with a positive T₃ test responded with DNA synthesis after stimulation using human anti-mAb 17-1A anti-idiotypic monoclonal antibodies. These four patients had a clinical response showing a tumor reduction after therapy, while all six patients lacking a proliferative response failed to show tumor regression. Induction of a cell-mediated immune network cascade might accordingly be an important anti-tumor effector function of mAb and should be considered in the future design of mAb-based therapy protocols in cancer patients.     

The clinical significance of HAMA in patients treated with mouse monoclonal antibodies

Twenty-four patients were analyzed for the development of HAMA (human antimouse antibodies) after being treated with repeated doses (200–500 mg) of the mouse monoclonal antibody (MAb) 17-1A. All patients developed anti-17-1A IgG antibodies, and most of them also developed IgM antibodies. In only two patients could immune complexes be demonstrated. Allergic reactions were rare (1.9%). In an extended study, a further 19 patient were analyzed for an idiotypic response. Forty-one out of 43 patients developed antiidiotypic antibodies (ab₂), and 20 of these also antianti-idiotypic antibodies (ab₃). Ab₃ ⁺ patients responded significantly better (p=0.01) and survived longer (p<0.001) compared to ab₃ ⁻ patients. In this study, we showed that MAb 17-1A could be repeatedly given on a safe basis. The development of high titers of HAMA did not cause significant clinical problems when further repeated infusions of MAb 17-1A were given. The development of an idiotypic response also indicate that the induction of HAMA might be beneficial and not harmful to the patient.     

Esterase activity and interaction of human hemoglobin with diclofenac sodium: A spectroscopic and molecular docking study

To get an idea about the pharmacokinetics and pharmacodynamics, it is important to study the drug‐protein interaction. Therefore, herein, we studied the interaction of diclofenac sodium (DIC) with human hemoglobin. The binding study of nonsteroidal antiinflammatory drug, DIC with human hemoglobin (HHB) was done by utilizing fluorescence, UV–visible, time‐resolved fluorescence and far‐UV circular dichroism spectroscopy (CD). Various thermodynamic parameters such as enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) were also calculated. CD results showed that DIC induces secondary structure change in HHB. Fluorescence resonance energy transfer was also performed. Additionally, it was also observed that DIC inhibits the esterase‐like enzymatic activity of HHB via competitive inhibition.     

Variation in root morphology,enhancement in anti-oxidative enzyme responses and improved arbutin and bergenin levels in Bergenia ciliata (Haw.) Sternb. raised in vitro via EMS and gamma irradiations

Plant Cell, Tissue and Organ Culture (PCTOC) - Plant cells develop defence mechanisms in response to mutagen stress which leads to modulation of certain metabolic and defensive pathways. Owing to...     

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.