Bone marrow angiogenesis and angiogenic factors in multiple myeloma treated with novel agents

Introduction. An increased bone marrow (BM) angiogenesis is associated with poor outcome in multiple myeloma (MM). Objective. Angiogenesis study in MM treated with novel antimyeloma agents: thalidomide, lenalidomide, bortezomib, and with dexamethasone. Patients and methods. Forty-four patients with MM (14 newly diagnosed, 30 refractory/relapsed) were treated with novel agents at our institution. A BM biopsy was obtained before the initiation of therapy in 19. Angiogenesis was assessed by microvessel density (MVD) estimation in BM biopsies stained with the monoclonal anti-CD34 antibody, and by serum levels of angiogenic factors (VEGF, bFGF, and HGF) and cytokines (IL-6 and TNF-α). Results. A positive correlation was found between BM plasma cell involvement and MVD estimation (p = 0.01). However, MVD was not significantly correlated with either disease phase (p = 0.065) or response to therapy (p = 0.79). Neither baseline serum levels of angiogenic cytokines correlated to response to treatment. No significant correlation was found between BM MVD and serum levels of angiogenic cytokines. Serum levels of angiogenic cytokines before and after therapy showed a significant increase of bFGF (p = 0.008). Conclusion. There is no relationship between MVD estimation and baseline serum levels of angiogenic cytokines, neither between each of them and response to therapy.     

Changes in cell volume and internal sodium concentration in HeLa cells during exponential growth and following lonidamine treatment

Cell volumes decreased in HeLa cells as a function of time after seeding during exponential growth. Cell volume distributions revealed the presence of two cell populations in all stages of growth. When cells approached confluence, the ratio of the two populations abruptly shifted towards that characterised by the smallest volume. Percentages of G1-, S- and G2 + M-phase cells were also measured and it was found that G1 frequency increased as a function of cell density during exponential growth. Intracellular sodium concentration, [Na]i was monitored by 23Na NMR in the presence of 5 mM dysprosium (III) tripolyphosphate. [Na]i increased from 22.8 to 59.0 mM in cells from the second to the seventh day after seeding. Treatment with lonidamine, an antitumoral drug that it is known to slow down cell growth by affecting aerobic glycolysis, produced a complete block of cell progression after a few days of treatment. The progression of cell volume distributions towards smaller volumes and the increase in internal sodium concentration as a function of time after seeding were also affected by the drug. These phenomena were related to the existence of a subpopulation of mitotically inactive G1-phase cells during exponential growth, pointing out that a density-dependent cellular mechanism regulates the cell cycling in HeLa cells.     

Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate

Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20-fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol-anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.     

Response to thalidomide in multiple myeloma: impact of angiogenic factors

Thalidomide has antiangiogenic and immunomodulatory effects, mediated by several cytokines such as vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF-2), hepatocyte growth factor (HGF), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Although extramedullary plasmacytomas (EMP) have a high vascularization, the response of these patients to thalidomide is controversial. Thirty-eight patients with refractory/relapsed MM were treated with thalidomide. Eleven patients had EMP when therapy was initiated. Serum specimens were obtained in patients before treatment was started and at the time of maximum response in responding patients or at thalidomide discontinuation in non-responders. Serum levels of VEGF, HGF and FGF-2 were determined in 18 patients whereas IL-6 and TNF-alpha were measured in 19 patients. Sixteen of the 38 patients (42%) responded to thalidomide. The response rate was significantly higher in patients without EMP (59% vs 0%, p = 0.0006 ). VEGF serum levels were significantly higher in responding patients. In contrast, baseline serum levels of HGF were significantly lower in responders. Neither VEGF nor HGF serum levels showed correlation with the presence of EMP. Baseline TNF-alpha serum levels were significantly lower in responding patients and in those without EMP. The serum levels of FGF-2 and IL-6 did not correlate with response to treatment or presence of EMP.     

Microbiological Metabolism of Naphthyridines

Penicillium adametzi and seven other species convert nalidixic acid, 1,4-dihydro-1-ethyl-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid, to 1,4-dihydro-1-ethyl-7-hydroxymethyl-4-oxo-1,8-naphthyridine-3-carboxylic acid. Forty-seven other species from six orders of fungi seem to achieve the same conversion as judged by chromatographic and spectral evidence. Under special conditions, P. adametzi also produces a second metabolite which was identified as the corresponding 7-carboxylic acid. The metabolic attack on the ring substituent is identical with the pathway previously established with humans. No evidence was obtained for metabolic attack on the naphthyridine nucleus itself.     

Interaction of CacyBP/SIP with NPM1 and its influence on NPM1 localization and function in oxidative stress

Calcyclin (S100A6) binding protein/Siah‐1 interacting protein (CacyBP/SIP) is mainly a cytoplasmic protein; however, some literature data suggested its presence in the nucleus. In this work we examined more precisely the nuclear localization and function of CacyBP/SIP. By applying mass spectrometry, we have identified several nuclear proteins, among them is nucleophosmin (NPM1), that may interact with CacyBP/SIP. Subsequent assays revealed that CacyBP/SIP forms complexes with NPM1 in the cell and that the interaction between these two proteins is direct. Interestingly, although CacyBP/SIP exhibits phosphatase activity, we have found that its overexpression favors phosphorylation of NPM1 on S125. In turn, the RNA immunoprecipitation assay indicated that the altered CacyBP/SIP level has an impact on the amount of 28S and 18S rRNA bound to NPM1. The overexpression of CacyBP/SIP resulted in a significant increase in the binding of 28S and 18S rRNA to NPM1, whereas silencing of CacyBP/SIP expression decreased 28S rRNA binding and had no effect on the binding of 18S rRNA. Further studies have shown that under oxidative stress, CacyBP/SIP overexpression alters NPM1 distribution in cell nuclei. In addition, staining for a nucleolar marker, fibrillarin, revealed that CacyBP/SIP is indispensable for maintaining the nucleolar structure. These results are in agreement with data obtained by western blot analysis, which show that upon oxidative stress the NPM1 level decreases but that CacyBP/SIP overexpression counteracts the effect of stress. Altogether, our results show for the first time that CacyBP/SIP binds to and affects the properties of a nuclear protein, NPM1, and that it is indispensable for preserving the structure of nucleoli under oxidative stress.     

Recent trends in stream macroinvertebrates: warm-adapted and pesticide-tolerant taxa increase in richness

Recently, a plethora of studies reporting insect declines has been published. Even though the common theme is decreasing insect richness, positive trends have also been documented. Here, we analysed nationwide, systematic monitoring data on aquatic insect richness collected at 438 sites in Switzerland from 2010 to 2019. In addition to taxonomic richness, we grouped taxa in accordance with their ecological preferences and functional traits to gain a better understanding of trends and possible underlying mechanisms. We found that in general, richness of aquatic insects remained stable or increased with time. Warm-adapted taxa, common feeding guilds and pesticide-tolerant taxa showed increasing patterns while cold-adapted, rarer feeding guilds and pesticide-sensitive taxa displayed stable trends. Both climate and land-use-related factors were the most important explanatory variables for the patterns of aquatic insect richness. Although our data cover the last decade only, our results suggest that recent developments in insect richness are context-dependent and affect functional groups differently. However, longer investigations and a good understanding of the baseline are important to reveal if the increase in temperature- and pesticide-tolerant species will lead to a decrease in specialized species and a homogenization of biotic communities in the long term.     

Cranial Irradiation Alters the Brain’s Microenvironment and Permits CCR2+ Macrophage Infiltration

Therapeutic irradiation is commonly used to treat primary or metastatic central nervous system tumors. It is believed that activation of neuroinflammatory signaling pathways contributes to the development of common adverse effects, which may ultimately contribute to cognitive dysfunction. Recent studies identified the chemokine (C-C motif) receptor (CCR2), constitutively expressed by cells of the monocyte-macrophage lineage, as a mediator of cognitive impairments induced by irradiation. In the present study we utilized a unique reporter mouse (CCR2^RFP/+CX3CR1^GFP/+) to accurately delineate the resident (CX3CR1⁺) versus peripheral (CCR2⁺) innate immune response in the brain following cranial irradiation. Our results demonstrate that a single dose of 10Gy cranial γ-irradiation induced a significant decrease in the percentage of resident microglia, while inducing an increase in the infiltration of peripherally derived CCR2⁺ macrophages. Although reduced in percentage, there was a significant increase in F4/80⁺ activated macrophages in irradiated animals compared to sham. Moreover, we found that there were altered levels of pro-inflammatory cytokines, chemokines, adhesion molecules, and growth factors in the hippocampi of wild type irradiated mice as compared to sham. All of these molecules are implicated in the recruitment, adhesion, and migration of peripheral monocytes to injured tissue. Importantly, there were no measureable changes in the expression of multiple markers associated with blood-brain barrier integrity; implicating the infiltration of peripheral CCR2⁺ macrophages may be due to inflammatory induced chemotactic signaling. Cumulatively, these data provide evidence that therapeutic levels of cranial radiation are sufficient to alter the brain’s homeostatic balance and permit the influx of peripherally-derived CCR2⁺ macrophages as well as the regional susceptibility of the hippocampal formation to ionizing radiation.