Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.     

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies

A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

Synthesis and antitumor activity of 2,5-bis(3′-indolyl)-furans and 3,5-bis(3′-indolyl)-isoxazoles,nortopsentin analogues

A series of novel 2,5-bis(3′-indolyl)furans and 3,5-bis(3′-indolyl)isoxazoles were synthesized as antitumor agents. The antiproliferative activity was evaluated in vitro toward diverse human tumor cell lines. Initially 5 isoxazoles and 3 furan derivatives were tested against a panel of 10 human tumor cell lines and the most active derivatives 3c and 4a were selected to be evaluated in an extended panel of 29 cell lines. By exhibiting mean IC₅₀ values of 17.4 μg/mL (3a) and 20.5 μg/mL (4c), in particular 4c showed a high level of tumor selectivity toward the 29 cell lines.     

Evolutionary origin of the T-lymphocyte receptor. II. Production and partial characterization of antisera to the immunoglobulin-like cell membrane protein from thymocytes of the carp

An immunoglobulin-like protein from Triton X-100 solubilized cell surface proteins from thymocytes can be isolated by specific precipitation using anti-IgM antibodies. Immunizing rabbits with those immune complexes we succeeded to elicit anti-thymocyte mIg sera reacting with both thymocyte mIg and IgM. SDS-PAGE analysis revealed that anti-IgM and anti-thymocyte mIg sera recognize the same thymocytic surface protein. The cross-reactivity between thymocyte mIg and IgM is caused by protein determinants very similar to each other. Anti-thymocyte mIg sera absorbed with IgM lose their capacity to bind thymocyte mIg. However, following absorption with thymocytes anti-IgM sera still possess amounts of antibodies reacting with humoral IgM.     

Central connections of the olfactory bulb in the goldfish,Carassius auratus

Summary The central connections of the goldfish olfactory bulb were studied with the use of horseradish peroxidase methods. The olfactory bulb projects bilaterally to ventral and dorsolateral areas of the telencephalon; further targets include the nucleus praeopticus periventricularis and a caudal olfactory nucleus near the nucleus posterior tuberis in the diencephalon, bilaterally. The contralateral bulb and the anterior commissure also receive an input from the olfactory bulb. Contralateral projections cross in rostral and caudal portions of the anterior commissure and in the habenular commissure. Retrogradely labeled neurons are found in the contralateral bulb and in three nuclei in the telencephalon bilaterally; the neurons projecting to the olfactory bulb are far more numerous on the ipsilateral side than in the contralateral hemisphere. Afferents to the olfactory bulb are found to run almost entirely through the lateral part of the medial olfactory tract, while the bulb efferents are mediated by the medial part of the medial olfactory tract and the lateral olfactory tract. Selective tracing of olfactory sub-tracts reveals different pathways and targets of the three major tract components. Reciprocal connections between olfactory bulb and posterior terminal field suggest a laminated structure in the dorsolateral telencephalon.     

Effect of acute exercise on glutathione deficient heart

The role of glutathione (GSH) in myocardial antioxidant defense was investigated in Swiss-Webster mice either performing swim exercise to exhaustion or rested in both the GSH adequate (GSH-A) and GSH deficient (GSH-D) states. GSH deficiency was accomplished by injecting mice with L-buthionine [S,R]sulfoximine (BSO; 2 nmol/kg body wt, i.p.) and providing BSO (20 mM) in drinking water for 12 days. GSH and glutathione disulfide (GSSG) contents in the GSH-D hearts were decreased to 10 and 8%, respectively, of those in the GSH-A mice. This decrease was associated with a significant decline of the total glutathione level in the liver, skeletal muscle and plasma. Myocardial GSH peroxidase and GSH sulfur-transferase activities decreased significantly following GSH deficiency, whereas superoxide dismutase activity was significantly elevated. GSH deficiency did not affect exercise endurance performance. However, exhaustive exercise decreased GSH content in the myocardium of the GSH-A and GSH-D mice by 22 and 44% (p < 0.05), respectively. The GSH:GSSG ratio was not altered significantly following exercise because of a concomitant decrease in GSSG (p < 0.05). -Glutamyltranspeptidase activity was significantly increased after exercise, especially in the GSH-D hearts (72%; p < 0.05). GSH content after exercise correlated negatively with exercise time in both GSH-A and GSH-D mice (p < 0.05). These data indicate that GSH is actively used in the myocardium during prolonged exercise at moderate intensity and that GSH deficiency is tolerated by the heart, possibly compensated for by an increased GSH uptake from the plasma.