Evidence for protein kinase C independent activation of phospholipase D by phorbol esters in lymphocytes

Recently it was reported that tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in lymphocytes through the activation of phospholipase D (PLD). However, it remains unclear whether this activation is mediated through protein kinase (PKC). The study reported here shows that tumor promoters 12-0-tetradecanoylphorbol-13-acetate (TPA), phorbol dibutyrate (PDBU), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPPA) and mezerin activated PLD, as measured by the formation of PEt, whereas Concanavalin A (ConA) had no effect. Inhibitors of PKC, sphingosine (2 x 10(-6) M - 5 x 10(-6) M), H-7, HA1004 (5 x 10(-7) - 5 x 10(-6) M) and K252a (1 x 10(-7) - 1 x 10(-6) M) failed to block the PEt synthesis induced by TPA. In fact, sphingosine increased it. Other PKC activators, 1-oleoyl-2-acetylglycerol (OAG) and dioctanoylglycerol (DiC8) had no effect on lymphocyte PLD activity. Analysis of the phospholipid contents after stimulation by TPA showed that only phosphatidylcholine (PC) was significantly decreased. Interestingly, TPA activated PLD in intact cells but not in lysates or subcellular fractions. These observations suggest that stimulation of PLD-catalyzed PEt synthesis by TPA is not solely mediated through PKC activation.     

Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia,antibodies, and exoantigens

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using this conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in protein kinase C and cAMP-dependent kinase in lymphocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A: quantitation of activities with an in situ gel assay

Primary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP-dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP-dependent kinases were identified by partial purification of the enzymes, by binding of [20-3H(N)]7-phorbol-12, 13-dibutyrate (3H-PDBU), and by photoaffinity labelling with 8-azidoadenosine-3':5'-cyclic monophosphate (8-N3-[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases. We found that when the preparations from either TPA- or Con A-treated lymphocytes were assayed, protein kinase C activity increased three- to four-fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30-50% occurred in the cytosol. In contrast, cytosolic cAMP-dependent protein kinase II increased 1.4-fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.     

Iodination of plasma membrane proteins of BHK cells in different growth states

The surfaces of BHK cells in confluent monolayers, immediately after mechanical dispersal and in logarithmically growing suspension cultures have been iodinated with ¹²⁵I using the lactoperoxidase technique. Electrophoretic resolution of the labeled proteins revealed that the representation of plasma membrane proteins varies with the growth state. Trypsinization of the cells produced a drastic revision of the surfaces leaving behind root fragments of membrane components and exposing additional proteins for iodination. The rapid turnover of membrane proteins in growing BHK cells restored the plasma membrane to a state characteristic of the replicating cell within 10 h.     

Osteogenesis in vitro: from pre-osteoblasts to osteocytes

Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5 mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (≤10 mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.     

Chronic resistance training in women potentiates growth hormone in vivo bioactivity: characterization of molecular mass variants

This investigation determined the influence of acute and chronic resistance exercise on responses of growth hormone (GH) molecular variants in women. Seventy-four healthy young women (23 +/- 3 yr, 167 +/- 7 cm, 63.8 +/- 9.3 kg, 26.3 +/- 4.0% body fat) performed an acute bout of resistance exercise (6 sets of 10 repetition maximum squat). Blood samples were obtained pre- and postexercise. Resulting plasma was fractionated by molecular mass (fraction A, >60 kDa; fraction B, 30-60 kDa; and fraction C, <30 kDa) using chromatography. Fractionated and unfractionated (UF) plasma was then assayed for GH using three different detection systems (monoclonal immunoassay, polyclonal immunoassay, and rat tibial line in vivo bioassay). Subjects were then matched and randomly placed into one of four resistance exercise training groups or a control group for 24 wk. All experimental procedures were repeated on completion of the 24-wk resistance training programs. After acute exercise, immunoassays showed consistent increases in UF GH samples and fractions B and C; increases in fraction A using immunoassay were seen only in the monoclonal assay. No consistent changes in bioactive GH were found following acute exercise. Conversely, chronic exercise induced no consistent changes in immunoassayable GH of various molecular masses, whereas, in general, bioassayable GH increased. In summary, although acute exercise increased only immunoactive GH, chronic physical training increased the biological activity of circulating GH molecular variants. Increased bioactive GH was observed across all fractions and training regimens, suggesting that chronic resistance exercise increased a spectrum of GH molecules that may be necessary for the multitude of somatogenic and metabolic actions of GH.     

Efficacy of trap and lure types for detection of Agrilus planipennis (Col., Buprestidae) at low density

Development of effective trapping tools for forest pests and evaluating the key components of these tools is necessary to locate early‐stage infestations and develop management responses to them. Agrilus planipennis Fairmaire (emerald ash borer) is an introduced pest of ash (Fraxinus spp. L.) in North America. The effectiveness of different trap and lure combinations were tested in areas with low and high density populations of A. planipennis. At low density sites, purple prism traps outperformed green traps and girdled ash trap trees in capture rates (adults per day) and rates of detection of A. planipennis. Also, manuka oil lures, used as a standard lure in a national survey programme, captured higher rates of A. planipennis than did previous standards of girdled ash trap trees. There was no logistic relationship between the detection of A. planipennis on a trap and the diameter of the ash tree from which the trap was suspended, possibly because of the use of artificial lures with these traps. There was also no difference in the mean number of A. lanipennis captured per day between ash species and between vigour rating of ash associated with the traps. However, traps placed in open grown and dominant trees captured more beetles than traps placed in lower canopy class trees. At sites defined as low and high density, there was no difference in the larval density per cm³ of phloem. This suggests that exposure time to A. planipennis has been shorter at those low density sites. By exploiting the trap and tree characteristics that improve A. planipennis capture rates and detection efficacy, there can be future improvement in management of this pest. If detection can occur before infested ash trees exhibit signs and symptoms, there may be a potential for reducing the mortality of ash within stands.     

Field observations of visual attraction of three European oak buprestid beetles toward conspecific and heterospecific models

Agrilus biguttatus Fabricius, Agrilus sulcicollis Lacordaire, and Agrilus angustulus Illiger (Coleoptera: Buprestidae) are three beetle species associated with oak trees [Quercus spp. (Fagaceae)] in Europe. In Hungary, all three species were observed in the foliage near freshly cut oak log piles. Agrilus biguttatus was active later in the afternoon, whereas the other species were observed earlier in the day. Dead female models of these three native Agrilus species, as well as the native species Agrilus cyanescens Ratzeburg and the non‐native Agrilus planipennis Fairmaire, were pinned onto adjacent leaves in direct sunlight to observe the visual mating approaches of the local male populations. Agrilus biguttatus and A. sulcicollis males flew toward and landed directly on the models from a distance of 1 m. Agrilus angustulus flew toward the models from a similar distance, but rather than landing directly on a model would alight on the leaf, 1–2 cm away, before walking closer to the model while antennating it. For all three species, there was substantial cross‐attraction to models of other species. Both A. biguttatus and A. sulcicollis males chose A. angustulus models less often than their respective conspecific models. Likewise, A. angustulus males approached A. sulcicollis models less often than their normal conspecific models. Agrilus biguttatus males attempted to copulate with both A. biguttatus and A. planipennis models, afterward remaining with them for several minutes. Agrilus biguttatus males spent more time on A. planipennis models than on conspecific models. Thus, there is substantial cross‐species attraction in visually mediated mating approaches and copulation behavior. These findings suggest a common behavioral template for visual mate‐finding among buprestids and a large degree of close‐range mating compatibility between A. biguttatus and A. planipennis.     

Optimization of pheromone dosage for gypsy moth mating disruption

The effect of aerial applications of the pheromone disparlure at varying dosages on mating disruption in low‐density gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), populations was determined in field plots in Virginia, USA during 2000 and 2002. Six dosages [0.15, 0.75, 3, 15, 37.5, and 75 g active ingredient (AI)/ha] of disparlure were tested during the 2‐year study. A strongly positive dose–response relationship was observed between pheromone dosages and mating disruption, as measured by the reduction in male moth capture in pheromone‐baited traps and mating successes of females. Dosages of pheromone 15 g AI/ha (15, 37.5, and 75 g AI/ha) reduced the mating success of females by >99% and significantly reduced male moth catches in pheromone‐baited traps compared to untreated plots. Pheromone dosages <15 g AI/ha also reduced trap catch, but to a lesser extent than dosages 15 g AI/ha. Furthermore, the effectiveness of the lower dosage treatments (0.15, 0.75, and 3 g AI/ha) declined over time, so that by the end of the study, male moth catches in traps were significantly lower in plots treated with pheromone dosages 15 g AI/ha. The dosage of 75 g AI/ha was initially replaced by a dosage of 37.5 g AI/ha in the USDA Forest Service Slow‐the‐Spread (STS) of the Gypsy Moth management program, but the program is currently making the transition to a dosage of 15 g AI/ha. These changes in applied dosages have resulted in a reduction in the cost of gypsy moth mating disruption treatments.