Bone marrow angiogenesis and proliferation in B-cell chronic lymphocytic leukemia

OBJECTIVE: To assess angiogenesis and the proliferative activity of bone marrow in patients with chronic lymphocytic leukemia (CLL) in relation to the bone marrow infiltration pattern. STUDY DESIGN: Bone marrow samples were obtained by trephine biopsy from 46 patients with B-cell CLL (B-CLL). Infiltration pattern was diffuse in 20 patients and nondiffuse--i.e., nodular, interstitial or mixed--in the remaining 26 patients. Ten normal bone marrow samples were used as a control group. Studies were carried out by immunohistochemical staining of paraffin-embedded bone marrow samples. Angiogenesis was assessed in the zones of highest vascular density (hot spots), visualized by the expression of endothelial antigen CD34 and expressed as a number of microvessels per high-powerfield (hpf) (final magnification, 400x). Proliferative activity was estimated by the expression of nuclear protein Ki-67, cyclin A and mean number of nucleolar organizer regions (AgNORs). RESULTS: Microvessel density was higher in B-CLL marrow than in normal marrow (30.1 and 16.44 per hpf, respectively) and was higher in the diffuse than nondiffuse pattern (33.6 and 27.5 per hpf, respectively). B-CLL bone marrow also showed higher proliferative activity, as assessed by mean number of AgNORs, than did normal marrow (1.52 and 1.25, respectively) and a higher mean percentage of cyclin A-positive cells (7.5 and 6.8, respectively). In contrast, mean Ki-67 expression was similar in B-CLL and the control group. Mean AgNORs number, Ki-67 and cyclin A-positive cell percentage were significantly higher in B-CLL marrow with a diffuse as compared to nondiffuse involvement pattern (AgNORs, 1.75 and 1.35; cyclin A, 9.27% and 3.95%; Ki-67, 34.9% and 23.3%, respectively). CONCLUSION: Our results indicate enhancement of bone marrow angiogenesis in B-CLL and a relationship between microvessel density and the bone marrow infiltration pattern. The study points also to a possible relationship between the bone marrow infiltration pattern and proliferative activity of bone marrow cells.     

Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion

To seek for novel rare and/or sporadic mutations within genomic neighborhood of common −344 C>T (rs2427827) FCER1A proximal promoter polymorphism, which by its prevalence could have masked the presence of less frequent genetic variants in our previous single-stranded conformational polymorphism (SSCP) mutational search study, SSCP screening was repeated using primers fixing −344 C>T variant. The genomic region surrounding −344 C>T polymorphism was confirmed to be fairly conservative and only a single sporadic mutation (present in 1 out of 196 chromosomes), a 6-bp deletion −262 to 257 del CTAGAC, was found. From the methodological point of view, we demonstrated a successful detection of a short-length polymorphism using SSCP screening in a population, in which the same genomic region contained frequent single-nucleotide polymorphic variant. In conjunction with subsequent cloning of aberrantly migrating PCR products and SSCP-driven indirect sequencing of the clones, this method offers a superior (to direct sequencing of PCR products) detection of rare mutations. The cost-effective method applied by us enables a reliable characterization of infrequent (thus present only in heterozygotic form) short-length variance of the sequence which are difficult to interpret by direct sequencing.     

Protein kinase Calpha regulates insulin receptor signaling in skeletal muscle

Certain PKC isoforms are stimulated by insulin and interact with IR as well as with IRS, but it is still not clear if specific PKC isoforms regulate IR signaling directly or through IRS-1. PKCalpha may regulate IRS activity in response to insulin. We investigated the possibility that PKCalpha may be important in insulin signaling. Studies were conducted on skeletal muscle in adult mice and on L6 skeletal cells. PKCalpha is constitutively associated with IRS-1, and insulin stimulation of PKCalpha causes disassociation of the two proteins within 5 min. Blockade of PKCalpha inhibited insulin-induced disassociation of PKCalpha from IRS1. Selective inhibition of PKCalpha increased the ability of insulin to reduce blood glucose levels. Insulin stimulation activates PKB and increases the association of PKCalpha with PKB. Blockade of PKCalpha increased threonine phosphorylation of PKB. We suggest that PKCalpha regulates insulin signaling in skeletal muscle through its disassociation from IRS-1 and association with PKB.     

Age-dependent flea (Siphonaptera) parasitism in rodents: a host's life history matters

We studied age-dependent patterns of flea infestation in 7 species of rodents from Slovakia (Apodemus agrarius, A. flavicollis, A. sylvaticus, A. uralensis, Clethrionomys glareolus, Microtus arvalis, and M. subterraneus). We estimated the age of the host from its body mass and expected the host age-dependent pattern of flea abundance, the level of aggregation, and prevalence to be in agreement with theoretical predictions. We expected that the mean abundance and the level of aggregation of fleas would be lowest in hosts of smallest and largest size classes and highest in hosts of medium size classes, whereas pattern of variation of prevalence with host age would be either convex or asymptotic. In general, mean abundance and species richness of fleas increased with an increase in host age, although the pressure of flea parasitism in terms of number of fleas per unit host body surface decreased with host age. We found 2 clear patterns of the change in flea aggregation and prevalence with host age. The first pattern demonstrated a peak of flea aggregation and a trough of flea prevalence in animals of middle age classes (Apodemus species and C. glareolus). The second pattern was an increase of both flea aggregation and flea prevalence with host age (both Microtus species). Consequently, we did not find unequivocal evidence for the main role of either parasite-induced host mortality or acquired resistance in host age-dependent pattern of flea parasitism. Our results suggest that this pattern can be generated by various processes and is strongly affected by natural history parameters of a host species such as dispersal pattern, spatial distribution, and structure of shelters.     

Toll-like receptors modulate adult hippocampal neurogenesis

Neurogenesis - the formation of new neurons in the adult brain - is considered to be one of the mechanisms by which the brain maintains its lifelong plasticity in response to extrinsic and intrinsic changes. The mechanisms underlying the regulation of neurogenesis are largely unknown. Here, we show that Toll-like receptors (TLRs), a family of highly conserved pattern-recognizing receptors involved in neural system development in Drosophila and innate immune activity in mammals, regulate adult hippocampal neurogenesis. We show that TLR2 and TLR4 are found on adult neural stem/progenitor cells (NPCs) and have distinct and opposing functions in NPC proliferation and differentiation both in vitro and in vivo. TLR2 deficiency in mice impaired hippocampal neurogenesis, whereas the absence of TLR4 resulted in enhanced proliferation and neuronal differentiation. In vitro studies further indicated that TLR2 and TLR4 directly modulated self-renewal and the cell-fate decision of NPCs. The activation of TLRs on the NPCs was mediated via MyD88 and induced PKCalpha/beta-dependent activation of the NF-kappaB signalling pathway. Thus, our study identified TLRs as players in adult neurogenesis and emphasizes their specified and diverse role in cell renewal.     

Growth and Mycorrhizal Community Structure of Pinus sylvestris Seedlings following the Addition of Forest Litter

We report the effects of pine and oak litter on species composition and diversity of mycorrhizal fungi colonizing 2-year-old Pinus sylvestris L. seedlings grown in a bare-root nursery in Lithuania. A layer of pine or oak litter was placed on the surface of the nursery bed soil to mimic natural litter cover. Oak litter amendment appeared to be most favorable for seedling survival, with a 73% survival rate, in contrast to the untreated mineral bed soil (44%). The concentrations of total N, P, K, Ca, and Mg were higher in oak growth medium than in pine growth medium. Relative to the control (pH 6.1), the pH was lower in pine growth medium (5.8) and higher in oak growth medium (6.3). There were also twofold and threefold increases in the C content of growth medium with the addition of pine and oak litter, respectively. Among seven mycorrhizal morphotypes, eight different mycorrhizal taxa were identified: Suillus luteus, Suillus variegatus, Wilcoxina mikolae, a Tuber sp., a Tomentella sp., Cenococcum geophilum, Amphinema byssoides, and one unidentified ectomycorrhizal symbiont. Forest litter addition affected the relative abundance of mycorrhizal symbionts more than their overall representation. This was more pronounced for pine litter than for oak litter, with 40% and 25% increases in the abundance of suilloid mycorrhizae, respectively. Our findings provide preliminary evidence that changes in the supply of organic matter through litter manipulation may have far-reaching effects on the chemistry of soil, thus influencing the growth and survival of Scots pine seedlings and their mycorrhizal communities.     

Asymmetric deceleration of ClpB or Hsp104 ATPase activity unleashes protein-remodeling activity

Two members of the AAA+ superfamily, ClpB and Hsp104, collaborate with Hsp70 and Hsp40 to rescue aggregated proteins. However, the mechanisms that elicit and underlie their protein-remodeling activities remain unclear. We report that for both Hsp104 and ClpB, mixtures of ATP and ATP-gammaS unexpectedly unleash activation, disaggregation and unfolding activities independent of cochaperones. Mutations reveal how remodeling activities are elicited by impaired hydrolysis at individual nucleotide-binding domains. However, for some substrates, mixtures of ATP and ATP-gammaS abolish remodeling, whereas for others, ATP binding without hydrolysis is sufficient. Remodeling of different substrates necessitates a diverse balance of polypeptide 'holding' (which requires ATP binding but not hydrolysis) and unfolding (which requires ATP hydrolysis). We suggest that this versatility in reaction mechanism enables ClpB and Hsp104 to reactivate the entire aggregated proteome after stress and enables Hsp104 to control prion inheritance.     

The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant phenotype

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1^w (hp-1^w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2^j (hp-2^j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1^w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A⁹³¹-to-T⁹³¹ base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1^w mutant, on the other hand, a single G²³⁹²-to-A²³⁹² transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F₂ population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R²=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1^w mutant phenotypes.Communicated by R. Hagemann     

Oxidative Damage of U937 Human Leukemic Cells Caused by Hydroxyl Radical Results in Singlet Oxygen Formation

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.     

Juvenile hormone biosynthesis and oocyte development in adult female Blattella germanica: Effects of grouping and mating

The roles of grouping and mating in modulating the activity of the corpora allata (CA) in adult female cockroaches were investigated using the in vitro radiochemical assay of juvenile hormone (JH) biosynthesis. Isolated virgin females have longer, asynchronous cycles of CA activity and oocyte maturation than do isolated females mated on day 8. Three factors were identified as the major contributors to this difference: (1) an experimental artifact of selection for sexually receptive females, (2) a positive effect of grouping on JH synthesis and oocyte maturation, and (3) a positive effect of copulation on oviposition and retention of the ootheca. Mated females constitute a subpopulation of receptive females that differ significantly from other females by having higher rates of JH synthesis prior to mating. The relative importance of such selection is substantial when the rate of mating is low, as in experiments with isolated females that are exposed to males for a short period of time. Long-term exposure of females to males introduces a grouping effect, which obscures any additional effect of mating on CA activity and oocyte development. However, mating influences ootheca formation and its retention. The effect of grouping can be mimicked in isolated females by transection of the nerves connecting the CA–corpora cardiaca complex to the brain, suggesting that in this insect isolation causes brain inhibition of the CA, and grouping provides disinhibitory stimuli that release the CA from brain inhibition.