An electron microscope study of nebenkern formation and differentiation in spermatids of Murgantia histrionica (Hemiptera, Pentatomidae)

Mitochondria in early spermatids of many insects aggregate and form a round body, the nebenkern. The nebenkern undergoes a structural differentiation and then divides into two separate equal-sized bodies. In the present study, nebenkerns of Murgantia histrionica, a Hemipteran insect, were reconstructed using electron micrographs of serial sections to determine how the mitochondria transform into the two separate bodies. Newly formed nebenkerns are made of one piece, an anastomosis of rod-like segments. Some segments interconnect to join networks of rings. Each network interlocks with another similar network, but networks which interlock are connected with each other by other segments of the nebenkern. Later, the entire nebenkern is made of two unconnected and interlocked networks of rings. The nebenkern appears to remain bipartite during subsequent differentiation. Since the two pieces are interlocked, breaks must occur before the pieces can separate. As breaks occur, each network transforms into a set of curved sheets, producing a nebenkern made of four concentric layers. The three outer layers are each made of two curved sheets which surround a bipartite central core. The surface sheets meet at a furrow in the surface of the nebenkern; segments in each layer are roughly symmetrical with each other about the plane in which the furrow lies. Rod-like segments join alternate segments. The number of layers then decreases to three, and later, to two. These nebenkerns resemble four-layered nebenkerns, but fewer connections between alternate segments are present. The two pieces constituting the nebenkern probably separate after most of the latter connections disappear. Hypotheses to account for the observed changes in nebenkern structure are presented.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Fusion protein-based epitope mapping of phytochrome. Precise identification of an evolutionarily conserved domain

Fusion proteins are used to define with precision an evolutionarily conserved domain on the carboxyl-terminal portion of the chromoprotein phytochrome. Simultaneously, assignments of two other epitopes are made with significantly greater precision, while the location of a fourth is confirmed. The epitope-mapping method that is described here is systematic, using complementary, overlapping nested sets of fusion proteins of predefined sequence rather than randomly generated peptides. Moreover, in contrast to previous methods, this approach yields rigorous and unambiguous assignments because it relies solely upon the ability of an antibody to detect a given polypeptide. A cDNA fragment encoding phytochrome amino acids 464-1129, which is its carboxyl terminus, was identified in lambda gt11 and subcloned in frame into the lacZ alpha sequence of pUC18. Four nested sets of subclones in pUC18 were created by digestion with selected restriction endonucleases and with the exonuclease Bal31. Fusion proteins were analyzed by immunoblotting following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The epitope for monoclonal antibody Oat-13 was confirmed to be between residues 551 and 617, while the epitopes for Oat-8 and Oat-28 were narrowed to 624-686 and 624-747, respectively. The epitope recognized by Pea-25, Pea-2, and Oat-15 was resolved unequivocally to a sequence of only seven residues (residues 765-771): N-Pro-Ile-Phe-Gly-Ala-Asp-Glu-C.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid