Louis L. Pech

Isolation and identification of a plasmatocyte-spreading peptide from the hemolymph of the lepidopteran insect Pseudoplusia includens

Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect insects. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and identification of a peptide from the moth Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte-spreading peptide (PSP1), contains 23 amino acid residues in the following sequence: H-ENFNGGCLAGYMRTADGRCKPTF-OH. In vitro assays using the synthetic peptide at concentrations ≥2 nm induced plasmatocytes from P. includens to spread on the surface of culture dishes. Injection of this peptide into P. includenslarvae caused a transient depletion of plasmatocytes from circulation. Labeling studies indicated that this peptide induced 75% of plasmatocytes that were double-labeled by the monoclonal antibodies 49G3A3 and 43E9A8 to spread, whereas plasma induced significantly more plasmatocytes to spread. This suggests that only a certain subpopulation of plasmatocytes responds to the peptide and that other peptidyl factors mediate plasmatocyte adhesion responses.

Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens.

Microplitis demolitor polydnavirus (MdPDV) is associated with Microplitis demolitor, a parasitic wasp that attacks the larval stage of the lepidopteran Pseudoplusia includens. Previously, we observed that MdPDV induced several alterations in the granular cells and plasmatocytes of P. includens, the primary haemocytes involved in regulating the cellular immune response toward M. demolitor and other parasites. In examining the mechanisms underlying immunosuppression of this host, we found that MdPDV induced apoptosis of granular cells. Granular cells underwent apoptosis both when virus was injected into the haemocoel of P. includens larvae and after infection with MdPDV in vitro. Characteristics of MdPDV-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of DNA into a 200 bp ladder. Although MdPDV induced changes in the ability of plasmatocytes to adhere to foreign surfaces, apoptosis of this morphotype was not observed. Examples from the literature suggest that some viruses promote their own survival by suppressing apoptosis of host cells. However, since polydnaviruses are likely to be transmitted vertically, we suggest that MdPDV promotes its own survival by inducing apoptosis of host immune cells which would otherwise kill the developing M. demolitor egg.

Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens (Lepidoptera: Noctuidae) involves an RGD-dependent cell adhesion mechanism

Abstract We tested the hypothesis that an insect immune response, encapsulation, involves an RGD-dependent cell adhesion mechanism by examining the effects of the tetrapeptide RGDS on hemocyte spreading and encapsulation. Soluble RGDS at concentrations of 0.5–2 mM inhibited the spreading of the primary encapsulating hemocyte, the plasmatocyte, on the surface of plastic tissue culture plates. At concentrations of 5–10 mM, the spreading of granular cells was also inhibited. RGES did not inhibit plasmatocyte or granular cell spreading, indicating that the effect was specific for RGDS. RGDS-Sepharose beads were encapsulated by hemocytes in vitro, whereas RGES-Sepharose beads were not. Furthermore, soluble RGDS, but not RGES, inhibited in vitro encapsulation of RGDS-Sepharose. When injected into the hemocoel of P. includens larvae, RGDS-Sepharose was encapsulated in 3 h, whereas RGES-Sepharose was not encapsulated until 24 h. The only hemocyte morphotype to encapsulate RGDS-Sepharose was the plasmatocyte. Lastly, RGDS. but not RGES, inhibited plasmatocyte spreading in response to cell-free plasma. These results indicate that the molecular basis of cell adhesion mediating hemocyte spreading and encapsulation in insects involves cell adhesion molecules containing the RGD recognition sequence.

Separation and behavior in vitro of hemocytes from the moth, Pseudoplusia includens

Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera: Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.

Plasmatocytes from the moth Pseudoplusia includens induce apoptosis of granular cells

The primary immune response toward internal parasites and other large foreign objects that enter the insect hemocoel is encapsulation. Prior studies indicated that granular cells and plasmatocytes are the two hemocyte types required for capsule formation by the moth Pseudoplusia includens (Lepidoptera: Noctuidae). Capsules formed by P. includens also have a defined architecture with primarily granular cells attaching directly to the target, multiple layers of plasmatocytes adhering to this inner layer of granular cells, and a monolayer of granular cells attaching to the capsule periphery. Dye-exclusion assays indicated that granular cells die shortly after attaching to the capsule periphery, leaving a basal lamina-like layer around the capsule. In examining the mechanisms underlying granular cell death, we found that culture medium preconditioned by plasmatocytes induced apoptosis of granular cells. Characteristics of plasmatocyte-induced apoptosis included condensation of chromatin, cell surface blebbing and fragmentation of nuclear DNA. Plasmatocyte-conditioned medium did not induce apoptosis of other hemocyte types, and medium conditioned by other hemocyte types did not induce apoptosis of granular cells. The adhesive state of granular cells and plasmatocytes also affected levels of apoptosis. Conditioned medium from spread plasmatocytes induced higher levels of granular cell apoptosis than medium conditioned by unspread plasmatocytes. Reciprocally, spread granular cells underwent significantly higher rates of apoptosis than unspread granular cells in medium conditioned by spread plasmatocytes. In situ analysis indicated that granular cells on the periphery of capsules also undergo apoptosis. Collectively, our results suggest that spread plasmatocytes release one or more factors that induce apoptosis of granular cells, and that this response is important in the final phases of capsule formation.

Effects of basement membranes on the behavior of hemocytes from Pseudoplusia includens (Lepidoptera; Noctuidae): Development of an in vitro encapsulation assay

The effects of culturing hemocytes from Pseudoplusia includens on the basement membrane preparation, Matrigel, were examined. Hemocytes in mixed populations did not spread on tissue culture plates coated with Matrigel, whereas cells readily spread on uncoated plates. The two adhesive morphotypes, granular cells and plasmatocytes, also failed to spread when purified and cultured on Matrigel coated plates, although plasmatocytes formed homotypic aggregates. Granular cells cultured on Matrigel retained their ability to endocytose fluorescently labeled beads and to adhere to a variety of foreign surfaces. Mixed populations of hemocytes or purified populations of plasmatocytes readily encapsulated Dowex 1X2 beads when incubated on Matrigel coated plates. In contrast, individual beads were not encapsulated when hemocytes were cultured with Dowex 1X2 beads in uncoated tissue culture plates. Capsules formed in vitro on Matrigel were indistinguishable from capsules formed in vivo, suggesting this simple system will be valuable in characterizing factors mediating encapsulation in insects.

REGULATION OF CILIARY MOTILITY IN PARAMECIUM BY CAMP AND CGMP

Abstract Paramecium tetraurelia is a useful model system for the study of signal transduction mechanisms that couple changes in membrane potential to changes in ciliary motility, and cAMP and cGMP have been implicated as second messengers in this system. Changes in ciliary beat are correlated with changes in the membrane potential, V m , with hyperpolarizations resulting in increased beat frequency and increased forward swimming speed. The intercellular levels of cAMP and cGMP vary with V m , and increasing internal cAMP by microinjection or the use of membrane permeant analogs results in increased swimming speed. In addition, cAMP and cGMP modify the swimming behavior of permeabilized Paramecium , and the sensitivity of cilia to cAMP and cGMP depends upon the location of the cilia on the cell surface. However, increasing internal cAMP also results in hyperpolarization and increasing internal cAMP does not override the effect of V m . These results have lead to two conflicting models to describe the role of cAMP: (1) stimulus → hyperpolarization of V m → increased [cAMP] in → increased beat frequency, and (2) stimulus → increased [cAMP] in → hyperpolarization of V m → increased beat frequency. This review discusses the data that support these models and possible experimental approaches to resolve the paradox presented by these models.

High-resolution scanning electron microscopy of frozen-hydrated cells

Cryo‐fixed yeast Paramecia and sea urchin embryos were investigated with an in‐lens type field‐emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low‐temperature scanning electron microscope (LTSEM).

Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia

Calmodulin (lysine 115) N-methyltransferase was purified from the cytosolic fraction of Paramecium tetraurelia by sequential dialysis, cellulose phosphate chromatography, Reactive Red 120 agarose chromatography, and calmodulin-Sepharose affinity chromatography. The enzyme was purified 6800-fold with a 15% yield. SDS-PAGE analysis of the purified enzyme invariably revealed a major protein of 37 kDa that was reproducibly obtained and minor proteins of 35 and 28 kDa that were sometimes obtained in variable yields. The enzyme formed a mixture of mono-, di-, and trimethyllysine residues at lysine 115 of calmodulin in vitro, had a Km for the methyl donor, S-adenosyl methionine (AdoMet), of about 1 microM and a pH optimum of about 7.5. The purified enzyme had an absolute requirement for the reductant DTT for activity, whereas the enzyme in crude fractions did not. The enzyme is a monomer with an estimated molecular mass of 33 kDa. Ca2+, Mg2+, Mn2+, and Ni2+ stimulated calmodulin N-methyltransferase activity but Zn2+ did not. Calmodulin N-methyltransferase was inhibited by its reaction product S-adenosyl homocysteine (SAH), but not by sinefungin and tubercidin. The calmodulin antagonists calmidazolium and mellitin were inhibitory but W7 was not. The enzyme was not stimulated by Triton X-100 nor by NaCl. Only calmodulins with an unmethylated lysine at residue 115, including cam2 calmodulin, were substrates. Histones and calcium-binding proteins from Paramecium other than calmodulin did not act as substrates for the purified calmodulin N-methyltransferase and no other substrates in the cytosolic fraction were observed.