BIoecology of Oncideres cervina Thomson (Coleoptera: Cerambycidae) on Persea americana Mill. (Lauraceae).

Cerambycidae beetles limit production and establishment of forest and fruit trees. Oncideres cervina Thomson, 1868 (Coleoptera: Cerambycidae) is one of the most important species. The objective was to record O. cervina girdling branches of Persea americana Mill. (Lauraceae) for the first time, check the number of oviposition incisions (Noi) as a function of the diameter of branch sections, period of emergence, and describe the larval-pupal chamber. Individuals of O. cervina were observed, for the first time, in P. americana orchards in Santa Maria, Rio Grande do Sul, Brazil. The middle section of branches (40-60 cm interval) had higher number of incisions. Girdled branches with a diameter of 40-50 mm had higher number of them. Adults emerged from November through January. Larval-pupal boreholes had diameters between 9 and 11 mm, and average tunnel length was 28 mm, with a mean volume of consumed wood of 4.3 mL. This information is useful for establishing integrated pest management practices against O. cervina in P. americana since this crop has a high added value and can be significantly compromised by attack by Cerambycidae beetles.

LIGHT (TNFSF14) inhibits adipose differentiation without affecting adipocyte metabolism.

OBJECTIVE: The member of the tumor necrosis factor family LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells; TNFSF14 (tumor necrosis factor super family protein 14) is primarily expressed in lymphocytes, in which it induces the expression of pro-inflammatory cytokines and alterations of lipid homeostasis. Recently, the protein was shown to be upregulated in obesity and to induce cytokine secretion from adipocytes. RESEARCH METHODS AND PROCEDURES: Using an automated complementary DNA (cDNA) screen, LIGHT was identified to inhibit adipose differentiation. As cellular models for adipogenesis mouse 3T3-L1, human SGBS (Simpson-Golabi-Behmel syndrome) and primary human preadipocytes differentiated in vitro were used as well as primary human adipocytes to study adipocyte functions. Analysis of lipid deposition by Oil Red O staining, mRNA expression by quantitative reverse transcriptase-PCR, nuclear factor (NF)-κB activation as well as protein secretion by enzyme linked immunosorbent assay and Luminex technology was performed. RESULTS: LIGHT was found to inhibit lipid accumulation in the three models of preadipocytes in a dose-dependent manner without cytotoxic effects. This inhibition of differentiation was probably because of interference at early steps of adipogenesis, as early exposure during differentiation showed the strongest effect, as assessed by decreased peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα) mRNA expression. In contrast to TNFα, basal and insulin-stimulated glucose uptake and lipolysis of terminally differentiated mature adipocytes were not altered in the presence of LIGHT. At a concentration sufficient to inhibit differentiation, secretion of proinflammatory cytokines was not significantly induced and NF-κB activity was only modestly induced compared with TNFα. CONCLUSION: LIGHT is a novel inhibitor of human adipocyte differentiation without adversely influencing central metabolic pathways in adipocytes.

Upregulation of interleukin 6 and granulocyte colony-stimulating factor receptors by transcription factor CCAAT enhancer binding protein alpha (C/EBP alpha) is critical for granulopoiesis.

Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein alpha (C/EBPalpha)-deficient mice. This phenotype is distinct from that of G-CSF receptor-/- mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPalpha. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6-responsive colony-forming units (CFU-IL6) in C/EBPalpha-/- mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPalpha-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPalpha target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPalpha-deficient mice.

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.

Increased granulocyte colony-stimulating factor responsiveness but normal resting granulopoiesis in mice carrying a targeted granulocyte colony-stimulating factor receptor mutation derived from a patient with severe congenital neutropenia.

The role of mutations of the granulocyte colony-stimulating factor receptor (G-CSFR) in the pathogenesis of severe congenital neutropenia (SCN) and the subsequent development of acute myeloid leukemia (AML) is controversial. Mice carrying a targeted mutation of their G-CSFR that reproduces the mutation found in a patient with SCN and AML have been generated. The mutant G-CSFR allele is expressed in a myeloid-specific fashion at levels comparable to the wild-type allele. Mice heterozygous or homozygous for this mutation have normal levels of circulating neutrophils and no evidence for a block in myeloid maturation, indicating that resting granulopoiesis is normal. However, in response to G-CSF treatment, these mice demonstrate a significantly greater fold increase in the level of circulating neutrophils. This effect appears to be due to increased neutrophil production as the absolute number of G-CSF-responsive progenitors in the bone marrow and their proliferation in response to G-CSF is increased. Furthermore, the in vitro survival and G-CSF-dependent suppression of apoptosis of mutant neutrophils are normal. Despite this evidence for a hyperproliferative response to G-CSF, no cases of AML have been detected to date. These data demonstrate that the G-CSFR mutation found in patients with SCN is not sufficient to induce an SCN phenotype or AML in mice.

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.

A novel c-fgr exon utilized in Epstein-Barr virus-infected B lymphocytes but not in normal monocytes.

The fgr proto-oncogene encodes a nonreceptor protein-tyrosine kinase, designated p55c-fgr. In this study, we have isolated human fgr cDNA molecules from normal monocyte mRNA templates. Nucleotide sequence analysis of the longest fgr cDNA revealed a 5' untranslated region of 927 bp which included two Alu-like repeats as well as three translation stop codons immediately upstream of the initiator for p55c-fgr synthesis. Within genomic DNA, these sequences were distributed over 13 kbp as three distinct 5' untranslated exons. Previous studies have shown that Epstein-Barr virus (EBV) increases c-fgr mRNA levels in B lymphocytes. By comparing the nucleotide sequence reported for transcripts isolated from EBV-infected B lymphocytes with those of our monocyte cDNA as well as genomic DNA, we identified a novel untranslated exon utilized only in EBV-infected cells. The transcriptional initiation sites of fgr mRNA expressed in EBV-converted cells were mapped and shown to reside within a region identified as an intron for fgr mRNA that is expressed in normal myelomonocytic cells. Furthermore, the region of the fgr locus upstream of the novel exon displayed properties of a transcriptional promoter when transfected into heterologous cells. We conclude from all of these findings that activation of the fgr gene by EBV is achieved by mechanisms distinct from those normally regulating its programmed expression in myelomonocytic cells.

Dynamic changes in the clonal structure of MDS and AML in response to epigenetic therapy.

Traditional response criteria in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are based on bone marrow morphology and may not accurately reflect clonal tumor burden in patients treated with non-cytotoxic chemotherapy. We used next-generation sequencing of serial bone marrow samples to monitor MDS and AML tumor burden during treatment with epigenetic therapy (decitabine and panobinostat). Serial bone marrow samples (and skin as a source of normal DNA) from 25 MDS and AML patients were sequenced (exome or 285 gene panel). We observed that responders, including those in complete remission (CR), can have persistent measurable tumor burden (that is, mutations) for at least 1 year without disease progression. Using an ultrasensitive sequencing approach, we detected extremely rare mutations (equivalent to 1 heterozygous mutant cell in 2000 non-mutant cells) months to years before their expansion at disease relapse. While patients can live with persistent clonal hematopoiesis in a CR or stable disease, ultimately we find evidence that expansion of a rare subclone occurs at relapse or progression. Here we demonstrate that sequencing of serial samples provides an alternative measure of tumor burden in MDS or AML patients and augments traditional response criteria that rely on bone marrow blast percentage.

Characterization of the 5' untranslated region of the human c-fgr gene and identification of the major myelomonocytic c-fgr promoter.

In this study, we have characterized the 5' region of the human c-fgr proto-oncogene and identified the major myelomonocytic c-fgr promoter. Seven distinct 5' untranslated exons were identified and localized to a region extending 13 kb upstream from the first coding exon. Two major promoters were identified, one utilized exclusively in Epstein-Barr virus (EBV)-infected B-lymphocyte cell lines, and the other functional only in myelomonocytic cells. Differential promoter utilization and alternative splicing of the 5' untranslated exons give rise to at least six distinct c-fgr mRNA species that differ only in their 5' untranslated regions. Two major mRNAs were identified, c-fgr A and c-fgr 4; these two mRNAs were detected exclusively in EBV-infected B-lymphocyte cell lines and myelomonocytic cells respectively. We have previously demonstrated that c-fgr is transcriptionally activated in U937 cells treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or cycloheximide (CHX). We now show that a DNA fragment extending from -772 to +97 (with respect to the transcription initiation site upstream from exon M4) is responsive to TPA but not CHX treatment in U937 cells. These results suggest that TPA and CHX induce c-fgr mRNA accumulation by different mechanisms.

Methotrexate-induced renal impairment: clinical studies and rescue from systemic toxicity with high-dose leucovorin and thymidine.

Four separate groups of patients have been studied: (1) The effect of high-dose methotrexate (MTX) administration on glomerular filtration rate was determined by pre- and posttreatment inulin and creatinine clearances in nine patients. Measurements were made prior to and 24-40 hr after drug administration. Inulin and creatinine clearances both decreased a mean of 43%. No signs of systemic toxicity occurred. (2) Three other patients given high-dose courses of MTX developed MTX toxicity. Their creatinine clearance decreased an average of 61%. (3) In a separate group of five patients undergoing weekly MTX treatment, comparison of serum MTX pharmacokinetics with and without alkalinization of the urine demonstrated no significant difference in peak serum MTX levels or serum MTX decay. (4) Eight additional patients with severe renal dysfunction secondary to MTX were treated with increased doses of leucovorin and a continuous infusion of thymidine (8 g/m2/day) once renal failure was recognized. When high-dose leucovorin and thymidine were begun 48-72 hr after the MTX infusion, severe toxicity in the form of leukopenia, thrombocytopenia, diffuse mucositis, stomatitis, or skin rash was averted. We concluded the following: (1) high-dose MTX causes a subclinical decrease in glomerular filtration rate with each administration, even in nontoxic courses; (2) alkalinization of the urine with sodium bicarbonate does not alter plasma MTX decay, while volume expansion (hydration) is maintained constant; and (3) rigorous monitoring of serum creatinine and serum MTX levels 24-48 hr after MTX administration allows for the institution of rescue measures, including leucovorin and thymidine, which will abort the systemic toxicity that accompanies MTX-induced renal failure.

1,25-Dihydroxyvitamin D3 induces monocytic differentiation of the PLB-985 leukemic line and promotes c-fgr mRNA expression.

1,25-dihydroxyvitamin D3 [1,25(OH)2D] is known to stimulate maturation of the human promyelocytic line HL-60 and murine bone marrow precursor cells along a monocyte/macrophage pathway. In this study, the steroid's effects on the PLB-985 leukemic line were examined. We found that 1,25(OH)2D induces monocytic differentiation of PLB-985 as manifested by morphological appearance, histochemical staining, and changes in cell surface antigen expression. Additionally, there was acquisition of functional monocyte characteristics including phagocytic activity and superoxide anion production via the respiratory burst pathway. Steady state levels of mRNA derived from the leukocyte-specific proto-oncogene c-fgr were also increased by the steroid. Thus, 1,25(OH2)D effectively differentiates PLB-985 cells along a monocytic pathway, providing a useful model of macrophage differentiation.

Binge eating and purging in young women with IDDM.

Case reports and empirical studies suggest that young women with insulin-dependent diabetes mellitus (IDDM) may be at high risk for developing eating disorders. In this study, self-reports of binge eating and purging from 59 IDDM women (aged 18-30 yr) were obtained. Most participants (58%) reported that they went on eating binges, and 12% met the DSM-III criteria for a diagnosis of bulimia. Nearly 40% admitted to controlling their weight by insulin purging, and 13.5% reported purging by other means. A group of bulimic participants had mean scores on an eating disorder questionnaire in the pathological range. Bulimic symptoms were positively related to reports of hospitalizations, episodes of ketoacidosis, and psychological symptoms. Implications of these results on the medical management of young women with IDDM are discussed.

Mechanisms of granulocyte colony-stimulating factor-induced hematopoietic progenitor-cell mobilization.

Hematopoietic progenitor cells (HPC) can be mobilized from the bone marrow into the peripheral circulation in response to diverse stimuli, including hematopoietic growth factors, cytotoxic agents, and certain chemokines. Despite significant differences in their biologic activities, these stimuli result in the mobilization of HPC with a similar phenotype, suggesting that a common mechanism for mobilization may exist. To explore the mechanisms of granulocyte colony-stimulating factor (G-CSF)-induced mobilization, we examined HPC mobilization in mice that are genetically deficient for the G-CSF receptor (G-CSFR). Their response was determined to each of three major types of mobilizing stimuli: cytotoxic agents (cyclophosphamide), chemokines (Interleukin-8[IL-8]), and hematopoietic growth factors (G-CSF, fit-3 ligand, and IL-12). These studies demonstrate that the G-CSFR is required for mobilization in response to cyclophosphamide and IL-8, but not fit-3 ligand or IL-12, and suggest that the G-CSFR may play an important and previously unexpected role in HPC migration.

Use of monoclonal antibody to increase sensitivity and specificity in quantitative immunodiffusion assays.

A mouse monoclonal antibody has been produced which recognizes human liver gamma-cystathionase, Radioiodination of the monoclonal antibody facilitated its use in combination with non-specific precipitating rabbit antisera in classical immunodiffusion assays. The technique may have broad applicability in the detection and quantitation of rare antigens which are difficult to purify but easily recognizable in immunodiffusion assays. It may also be used for the initial detection of monoclonal antibodies to unique antigens of interest.

Hydrogel embolic agents. Theory and practice of adding radio-opacity.

RATIONALE AND OBJECTIVES: Spheres of hydrogel have been developed as embolic material with the ability to incorporate radio-opaque materials. To optimize particle design for radiographic or fluoroscopic visualization, we have examined the theoretical determinants of particle contrast. In addition, loaded hydrogel particles were tested in a rabbit model. METHODS: Computer simulations of particle subject contrast were examined regarding particle composition, particle size, patient thickness, and x-ray beam kilovoltage. Embolizations in the rabbit kidney were used to test the practical aspects of the materials. RESULTS: Tantalum and tungsten offer some theoretical and practical advantages over other materials. With this particular hydrogel preparation, contrast material loading was limited to 20% of the volume as loaded contrast agent. The soft particles passed through catheters as small as 3 French; they were usually injected as a suspension of saline/contrast material. Tantalum/hydrogel particles as large as 2 mm could be forced through the 140 cm/3-Fr catheter with a guide wire. CONCLUSIONS: Radio-opacity of embolic material should add an element of control in embolization procedures that is lacking with the current agents. The heavy metals, tungsten and tantalum, are suitable additives for radiopaque material for hydrogel emboli. The input relationship appears predictable with computer monitoring techniques. Initial results in a study of these radiopaque particles are very encouraging. Further studies are underway to evaluate the long-term effects in renal and hepatic circulations.

New videodensitometric method for measuring renal artery blood flow at routine arteriography: validation in the canine model.

A contrast medium was injected in the aortic arch and selectively in a renal artery to estimate the renal blood flow as a percentage of the cardiac output by the videodensitometric (VD) method. Twenty-six paired VD measurements in four mongrel dogs were obtained and the results compared to electromagnetic (EM) flow readings from the aortic arch and a renal artery. The relative renal blood flow estimated by the VD method averaged 9.1% and correlated with the EM flow average of 9.6% with r = 0.96. Previous in vitro investigations of relative flow in a model have now been validated in vivo. These results suggest that videodensitometry could be a clinical tool for measuring renal blood flow in conjunction with routine arteriography.

Assessment of peripheral blood flow in man by video dilution technique: a preliminary report.

Video dilution technique is now available for clinical use in evaluating patients with peripheral vascular disease. The measurements can easily be performed in any modern angiographic suite. The only additional equipment required is a videodensitometer, video tape recorder, and a strip chart recorded. The new technique has been developed and tested in a hydrodynamic model and compared to volumetric flows. Further, the video dilution technique has been compared to electromagnetic flow readings using a canine model to measure the cerebral, renal, splanchnic, and extremity circulation and has proven to be extremely accurate (n = 389; r = 0.99). By applying the technique to patients with peripheral vascular disease, it is possible to evaluate the hemodynamic significance of stenotic lesions and arteriovenous shunts. Other uses of video dilution technique include evaluating the effects of vasoactive drugs and the adequacy of transluminal angioplasty.

Assessment of cerebral blood flow in man by video dilution technique: a preliminary report.

Video dilution technique (VDT) is currently performed in conjunction with routine cerebral angiography to determine carotid blood flow in humans. Preliminary results indicate that the blood flows (as a percentage of cardial output) of the common, internal, and external carotid arteries are 8.5%, 5.5%, and 3.0%, respectively (SD less than 1%). In contradistinction to previous techniques, VDT provides a safe and highly accurate method of determining carotid blood flow in human subjects. The usefulness of this technique in normal and pathologic states is discussed.

Bronchial blood flow measured by video dilution technique in sheep.

Bronchial blood flow was studied with the video dilution technique (VDT) in seven sheep. All animals were anesthetized (thiamylal and halothane) and ventilated. A videodensitometer and a videotape replay of the fluoroscopic image of dye moving through the common bronchial artery were used to construct dye mass vs. time curves. The areas under the curves were inversely proportional to flow in the bronchoesophageal artery, the site of dye injection. At thoracotomy, an electromagnetic flow probe (EMFP) was placed on the common bronchial artery (the major branch of the bronchoesophageal artery) to measure blood flow changes simultaneously by EMFP and by VDT. These two methods of measurement of blood flow to the airways were compared to validate the use of VDT in this circulation. Common bronchial artery blood flow was increased by injection of radiocontrast dye into the fluoroscopically positioned bronchoesophageal artery catheter causing hyperosmotically induced hyperemia. In 160 simultaneous measurements in five sheep, the percent change in flow as measured by EMFP and VDT correlated closely (r = 0.96). When flow changed because of spontaneous aortic pressure changes or pharmacologic intervention (28 simultaneous measurements in five sheep), the percent change in flow by EMFP and VDT also correlated well (r = 0.98). Bronchial blood flow changes in sheep can be measured accurately using the video dilution technique.