Oviposition site and food preference of the green June beetle (Coleoptera: Scarabaeidae).

Relative preferences of green June beetle, Cotinis nitida (L.), adults and grubs for different organic fertilizers were determined in field and laboratory choice experiments. Six organic fertilizer treatments (low rate of broiler litter, high rate of broiler litter, cow manure, hay, Milorganite, or no fertilizer [the control]) were applied to sandy-loam soil and exposed to adults in 2.7 by 3.7 by 2.4-m screen cages. More eggs and larvae were found in pots treated with broiler litter (43%), cow manure (23%), and hay (30%) than in pots treated with Milorganite (4%) or no fertilizer (0%). Orientation preferences of third-instar grubs were tested in Y-tube and satellite olfactometers. Of the five treatments (broiler litter, cow manure, hay, Milorganite, and a blank control), preference was greatest for broiler litter and cow manure, but all organic fertilizer treatments were generally preferred over the blank control. These experiments suggest that use of organic fertilizers may result in higher densities of green June beetle grubs both by attracting the ovipositing females, and by acting as a food attractant for the mobile larvae.     

Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.     

Transforming growth factor-beta: multifunctional regulator of differentiation and development

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.     

Antibodies to peptide determinants in transforming growth factor beta and their applications

Polyclonal antibodies have been raised to a series of synthetic peptides which correspond to essentially all regions of the transforming growth factor beta 1 (TGF-beta 1) molecule. All antisera were evaluated for their abilities to react with TGF-beta 1 and TGF-beta 2 in either the native or reduced form in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. While all antisera demonstrated some ability to recognize TGF-beta 1 in these systems, there was limited cross-reactivity with TGF-beta 2, suggesting that substantial sequence or conformational differences exist between the two growth factors. On Western blots 5-10 ng of purified human platelet TGF-beta 1 could be detected when probed with affinity-purified peptide antisera generated against peptides corresponding to residues 48-77, 50-75, and 78-109 of the 112 amino acid TGF-beta 1 monomer. Antisera raised against peptides 50-75 and 78-109 were most effective in immunoprecipitating reduced and native 125I-TGF-beta 1, respectively. The antisera also were tested for their effectiveness in blocking the binding of 125I-TGF-beta 1 to its receptor. Anti-peptide 78-109 and anti-peptide 50-75 blocked 80% and 40% of the binding, respectively, while antibodies against amino-terminal peptides were without effect. These data suggest that the carboxyl-terminal region of TGF-beta 1 may play a significant role in the binding of the native ligand to its receptor.     

Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry

To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunodetection and quantitation of the two forms of transforming growth factor-beta (TGF-beta 1 and TGF-beta 2) secreted by cells in culture

Transforming growth factor beta (TGF-beta), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF-beta 1 and TGF-beta 2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF-beta 1 and TGF-beta 2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF-beta 1 and TGF-beta 2 in complex biological fluids. Using these assays we show that both TGF-beta 1 and TGF-beta 2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF-beta 1 or TGF-beta 2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF-beta is independently regulated.     

Linkage Analysis with Multiplexed Short Tandem Repeat Polymorphisms Using Infrared Fluorescence and M13 Tailed Primers

The use of short tandem repeat polymorphisms (STRPs) as marker loci for linkage analysis is becoming increasingly important due to their large numbers in the human genome and their high degree of polymorphism. Fluorescence-based detection of the STRP pattern with an automated DNA sequencer has improved the efficiency of this technique by eliminating the need for radioactivity and producing a digitized autoradiogram-like image that can be used for computer analysis. In an effort to simplify the procedure and to reduce the cost of fluorescence STRP analysis, we have developed a technique known as multiplexing STRPs with tailed primers (MSTP) using primers that have a 19-bp extension, identical to the sequence of an M13 sequencing primer, on the 5′ end of the forward primer in conjunction with multiplexing several primer pairs in a single polymerase chain reaction (PCR) amplification. The banding pattern is detected with the addition of the M13 primer-dye conjugate as the sole primer conjugated to the fluorescent dye, eliminating the need for direct conjugation of the infrared fluorescent dye tn the STRP primers. The use of MSTP for linkage analysis greatly reduces the number of PCR reactions. Up to five primer pairs can be multiplexed together in the same reaction. At present, a set of 148 STRP markers spaced at an average genetic distance of 28 cM throughout the autosomal genome can be analyzed in 37 sets of multiplexed amplification reactions. We have automated the analysis of these patterns for linkage using software that both detects the STRP banding pattern and determines their sizes. This information can then be exported in a user-defined format from a database manager for linkage analysis.     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.     

Transforming Growth Factor-β3 (TGF-β3) Knock-in Ameliorates Inflammation Due to TGF-β1 Deficiency While Promoting Glucose Tolerance

Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1^Lβ3/Lβ3). In the TGF-β1^Lβ3/Lβ3 mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1^−/− mice, the TGF-β1^Lβ3/Lβ3 mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1^Lβ3/Lβ3 mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1^Lβ3/Lβ3 mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease.