Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO4, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20-35 degrees C in both fungal species.     

The effect of oral glucose loads on tissue metabolism during angiotensin II receptor and beta-receptor blockade in obese hypertensive subjects.

AT1 receptor blockers and ACE inhibitors decrease the risk for new onset diabetes mellitus. The phenomenon could be related to a direct angiotensin II effect on tissue metabolism. To address the issue, we recruited eighteen obese hypertensive patients. Patients were randomized to double-blind treatment with either valsartan (n = 8) or atenolol (n = 10) for thirteen weeks. They underwent an oral glucose tolerance test before and during active treatment, while metabolism was monitored through subcutaneous and intramuscular microdialysis and indirect calorimetry. After glucose ingestion, venous glucose and insulin concentrations increased rapidly while systemic free fatty acid concentrations were suppressed. Dialysate glucose and lactate concentrations increased briskly in adipose tissue and in skeletal muscle. Dialysate glycerol decreased profoundly in both tissues. Respiratory quotient increased markedly after glucose ingestion. These responses were identical at baseline and during active treatment either drug. We conclude that AT1 receptor blockade in obese hypertensive patients has no effect on interstitial glucose supply, lipolysis, and substrate oxidation. One possible explanation is that angiotensin II levels in obese hypertensives are not sufficient to elicit the metabolic changes that have been observed after direct angiotensin II application. The exact mechanism by which inhibition of the renin-angiotensin-aldosterone system decreases the diabetes risk remains unresolved and requires further study.     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3–dimensional collagen sponges

Abstract. Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro , but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 × 10⁶ cells are loaded, mineralization as measured by ⁸⁵Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10⁷ cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phos-phatase positive cells. However, synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.     

Mechanism of centrosome positioning during the wound response in BSC-1 cells

Locomoting cells are characterized by a pronounced external and internal anterior-posterior polarity. One of the events associated with cell polarization at the onset of locomotion is a shift of the centrosome, or MTOC, ahead of the nucleus. This position is believed to be of strategic importance for directional cell movement and cell polarity. We have used BSC-1 cells at the edge of an in vitro wound to clarify the causal relationship between MTOC position and the initiation of cell polarization. We find that pronounced cell polarization (the extension of a lamellipod) can take place in the absence of MTOC repositioning or microtubules. Conversely, MTOCs will reposition even after lamellar extension and cell polarization have occurred. Repositioning requires microtubules that extend to the cell periphery and is independent of selective detyrosination of microtubules extending towards the cell front. Significantly, MTOCs maintain, or at least attempt to maintain, a position at the cell's centroid. This is most clearly demonstrated in wounded monolayers of enucleated cells where the MTOC closely follows the centroid position. We suggest that the primary response to the would is the biased extension of a lamellipod, which can occur in the absence of microtubules and MTOC repositioning. Lamellipod extension leads to a shift of the cell's centroid towards the wound. The MTOC, in an attempt to maintain a position near the cell center, will follow. This will automatically put the MTOC ahead of the nucleus in the vast majority of cells. The nucleus as a reference for MTOC position may not be as meaningful as previously thought.     

Apoplastic expression of yeast-derived invertase in potato : effects on photosynthesis, leaf solute composition, water relations, and tuber composition

In potato plants (Solanum tuberosum), a chimeric yeast-derived invertase gene fused to a 35S cauliflower mosaic virus promoter has been expressed. The protein was targeted to the cell wall by using the signal peptide of proteinase inhibitor II fused to the amino terminus of the yeast invertase. The transformed plants had crinkled leaves, showed a reduced growth rate, and produced fewer tubers. Although in the apoplast of the leaves of the transformed plants the content of glucose and fructose rose by a factor of 20, and that of sucrose declined 20-fold, 98% of the carbohydrate in the phloem sap consisted of sucrose, demonstrating the strong specificity of phloem loading. In the leaf cells of the transformed plants, glucose, fructose, and amino acids, especially proline, were accumulated. Consequently, the osmolality of the cell sap rose from 250 to 350 mosmol/kg. Our results show that the observed 75% decrease of photosynthesis is not caused by a feedback regulation of sucrose synthesis and is accompanied by an increase in the osmotic pressure in the leaf cells. In the transformed plants, not only the amino acid to sucrose ratio in the phloem sap, but also the amino acid and protein contents in the tubers were found to be elevated. In the tubers of the transformed plants, the protein to starch ratio increased.