Dual Fluorescent Staining Research Articles

Trypsin and Chymotrypsin are Involved in the Progesterone‐Induced Acrosome Reaction in Canine Spermatozoa

Acrosomal proteases allow the spermatozoon not only to cross the cumulus cells and penetrate the zona pellucida of the oocyte, but also they are needed for the acrosome reaction process (AR). The present study evaluated in vitro the role of trypsin and chymotrypsin in the acrosome reaction of canine spermatozoa by means of protease inhibitors. Spermatozoa obtained from the second fraction of the ejaculate and devoid of seminal plasma were re-suspended in canine capacitation medium (CCM) and incubated at 38.5 degrees C in 5% CO(2). After 2 h (period of sperm capacitation), aliquots of sperm suspension were incubated separately with trypsin inhibitor NPGB (p-nitrophenyl-p'-guanidino-benzoate); TI (Trypsin inhibitor I-S Type from soybean) and with chymotrypsin inhibitor TPCK (N-tosyl-L-phenylalanine-chloromethyl-ketone) for 30 min. The AR was induced with progesterone and evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. Acrosomal exocytosis levels were statistically significant higher in the samples treated with progesterone than in the control without inducer. However, the trypsin inhibitors NPGB, TI and the chymotrypsin inhibitor TPCK reduced the percentage of AR when compared with the control with progesterone and without inhibitor (p < 0.001), where the AR values were 45.63 +/- 3.8%, 51.63 +/- 2.8%, 58.38 +/-4.1% and 71.25 +/- 4.9%, respectively. These results show that trypsin and chymotrypsin inhibitors are effective in blocking the acrosome reaction induced by progesterone in canine; in addition, they suggest the participation of respective proteases in the AR process in this species.

Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions

Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil–C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems.

Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions

Understanding the infection biology of fungi is the key step in devising suitable control strategies for plant diseases. Recently, the Arabidopsis-Colletotrichum higginsianum (causal agent of anthracnose) system has emerged as a seminal paradigm for deciphering the infection biology underlying fungus-plant interactions. We describe here three staining methods coupled with confocal microscopy: trypan blue, aniline blue and dual trypan blue-aniline blue fluorescence staining. Trypan blue and aniline blue staining were employed to scan the infection structures of the hemibiotrophic fungus C. higginsianum and host response in A. thaliana leaf tissues. The two techniques then were combined to observe the contrast between in planta fungal infection structures, i.e., infection vesicles, primary hyphae and secondary hyphae, and the host plant defense responses, i.e., papilla formation and hypersensitive response. These staining techniques also were applied to the lentil-C. truncatum pathosystem to demonstrate their applicability for multiple pathosystems.

Autocrine Actions of Matrix Metalloproteinase (MMP)-2 Counter the Effects of MMP-9 to Promote Survival and Prevent Terminal Differentiation of Cultured Human Keratinocytes

Cultured human keratinocytes produce matrix metalloproteinase (MMP)-2 and MMP-9. In this study, using small interfering RNA (siRNA) for MMP-2 or MMP-9, we investigated the functions of these two gelatinases in the regulation of survival by measuring growth, differentiation, apoptosis, and migration of cultured keratinocytes. MMP-2 siRNA treatment significantly decreased keratinocyte growth and migration, and stimulated apoptosis fourfold. In addition, MMP-2 siRNA caused a 70% reduction in keratin-14 (K14) and a fourfold increase in K10. In contrast, MMP-9 siRNA treatment exerted opposite effects on cell growth, apoptosis, and K10 expression. MMP-2 appears to act through the ERK MAP kinase and caspase-3 signaling pathways as evidenced by the 53% reduction in the level of phosphorylated ERK1/2 and threefold increase in phosphorylated p38 and stronger staining for active caspase-3 in response to MMP-2 siRNA. Dual fluorescent staining revealed that almost all cultured cells stained positive for MMP-2, with only a few scattered cells being positive for MMP-9. There were considerably more BrdU-positive cells following MMP-9 siRNA treatment, indicating that MMP-9 inhibited proliferation. In conclusion, MMP-2 stimulates keratinocyte survival whereas MMP-9 promotes terminal differentiation.

Survival of Honey Bee (Hymenoptera: Apidae) Spermatozoa Incubated at Room Temperature from Drones Exposed to Miticides

We conducted research to examine the potential impacts ofcoumaphos, fluvalinate, and Apilife VAR (Thymol) on drone honey bee, Apis mellifera L. (Hymenoptera: Apidae), sperm viability over time. Drones were reared in colonies that had been treated with each miticide by using the dose recommended on the label. Drones from each miticide treatment were collected, and semen samples were pooled. The pooled samples from each treatment were subdivided and analyzed for periods of up to 6 wk. Random samples were taken from each treatment (n = 6 pools) over the 6-wk period. Sperm viability was measured using dual-fluorescent staining techniques. The exposure of drones to coumaphos during development and sexual maturation significantly reduced sperm viability for all 6 wk. Sperm viability significantly decreased from the initial sample to week 1 in control colonies, and a significant decrease in sperm viability was observed from week 5 to week 6 in all treatments and control. The potential impacts of these results on queen performance and failure are discussed.

Upregulation of EMMPRIN after permanent focal cerebral ischemia

Elevated activities of matrix metalloproteinases (MMPs) following ischemic stroke have been shown to mediate ischemic injury as well as neurovascular remodeling. The extracellular MMP inducer (EMMPRIN) is a 58-kDa cell surface glycoprotein, which has been known to play a key regulatory role for MMP activities. The roles of EMMPRIN in stroke injury are not clearly understood. In this study, we investigated changes of EMMPRIN in a mouse model of permanent focal cerebral ischemia, and examined potential association between EMMPRIN and MMP-9 expression. Adult male CD-1 mice were subjected to permanent focal ischemia by intraluminal occlusion of the left middle cerebral artery (MCAO) under anesthesia. EMMPRIN expression was markedly upregulated in the peri-infarct area at 2–7 days after ischemia compared to the contralateral non-ischemic hemisphere by Western blot analysis. Immunofluorescent double staining demonstrated that EMMPRIN signals co-localized with vwF-positive endothelial cells and GFAP-positive peri-vascular astrocytes. In contrast, EMMPRIN signal did not co-localize with NeuN-positive neurons, or MPO-positive neutrophils. Dual fluorescent staining revealed that EMMPRIN co-localized with MMP-9. Our data also demonstrated that increased EMMPRIN expression correlated with increased MMP-9 levels in a temporal manner. In summary, we report for the first time that EMMPRIN expression was significantly increased in a mouse model of permanent focal cerebral ischemia. The spatial and temporal association between increased EMMPRIN expression and elevated MMP-9 levels suggest that EMMPRIN may modulate MMP-9 activity, and participate in neurovascular remodeling after ischemic stroke.

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage

Rose Bengal (RB) is a very efficient photosensitizer which undergoes inactivation of its photophysical and photochemical properties upon addition of a quencher group-i.e. acetate-to the xanthene rings. The resulting RB acetate (RB-Ac) derivative behaves as a fluorogenic substrate: it easily enters the cells where the native photoactive molecule is restored by esterase activities. It is known that the viability of RB-Ac-loaded cells is strongly reduced by light irradiation, attesting to the formation of intracellular RB. The aim of this study was to identify the organelles photodamaged by the intracellularly formed RB. RB-Ac preloaded rat C6 glioma cells and human HeLa cells were irradiated at 530 nm. Fluorescence confocal imaging and colocalization with specific dyes showed that the restored RB molecules redistribute dynamically through the cytoplasm, with the achievement of a dynamic equilibrium at 30 min after the administration, in the cell systems used; this accounted for a generalized damage to several organelles and cell structures (i.e. the endoplasmic reticulum, the Golgi apparatus, the mitochondria, and the cytoskeleton). The multiple organelle damage, furthermore, led preferentially to apoptosis as demonstrated by light and electron microscopy and by dual-fluorescence staining with FITC-labelled annexin V and propidium iodide.

No Significant Endothelial Apoptosis in the Radiation-Induced Gastrointestinal Syndrome

This report addresses the incidence of vascular endothelial cell apoptosis in the mouse small intestine in relation to the radiation-induced gastrointestinal (GI) syndrome. Nonanesthetized mice received whole-body irradiation at doses above and below the threshold for death from the GI syndrome with 250 kVp X-rays, (137)Cs gamma rays, epithermal neutrons alone, or a unique approach for selective vascular irradiation using epithermal neutrons in combination with boronated liposomes that are restricted to the blood. Both terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining for apoptosis and dual-fluorescence staining for apoptosis and endothelial cells were carried out in jejunal cross-sections at 4 h postirradiation. Most apoptotic cells were in the crypt epithelium. The number of TUNEL-positive nuclei per villus was low (1.62 +/- 0.03, mean +/- SEM) for all irradiation modalities and showed no dose-response as a function of blood vessel dose, even as the dose crossed the threshold for death from the GI syndrome. Dual-fluorescence staining for apoptosis and endothelial cells verified the TUNEL results and identified the apoptotic nuclei in the villi as CD45-positive leukocytes. These data do not support the hypothesis that vascular endothelial cell apoptosis is the cause of the GI syndrome.

94 CRYOPRESERVATION OF HONEY BEE SPERMATOZOA

Our project investigated a new method for the cryopreservation of honey bee (Apis mellifera) sperm cells (SC). Few methods have been developed and none achieve normal sex ratios in progeny. Recently, honey bee colonies have been decimated by colony collapse disorder and infestation by varroa bee mites. A bank of preserved SC might enable the creation of a seed stock for restoration of genetic diversity through AI (Cobey 1983 Am. Bee J. 123, 389–395). We investigated two freezing rates using two diluents and their effect on post-thaw survival of the SC. The slower freezing rate was chosen from a report with the highest success to date (Harbo 1983 Ann. Entomol. Soc. Am. 76, 890–891). The rapid freezing rate was a method developed by us. We reasoned that the small volumes of ejaculate made it potentially suitable for vitrification. The SC were frozen either in 40% Harbo's DMSO diluent containing 25% DMSO, 25% egg yolk, 50% buffer (1.1% NaH2PO4�H2O (w/v) and 0.845% Na2HPO4�2H2O (w/v)), and 60% semen; or in 50% glycerol-based diluent containing 9% glycerol, 24% egg yolk, 67% buffer (5.9% Tris (w/v), 0.8% glucose (w/v), and 3.2% citric acid (w/v)), and 50% semen. Ejaculates were collected by applying bilateral pressure to the abdomens of the drones causing endophallus eversion. About 1 µL (8 � 106 SC) of ejaculate was drawn into siliconized 50-µL capillary tubes fitted to a Hamilton threaded-plunger syringe preloaded with Fluorinert (Sigma, St. Louis, MO, USA). Micro-glass cryostraws (µC) were constructed by pulling Pasteur pipettes to a 230-µm internal diameter and keeping what was the tip end of the pipette as the large end of the µC. The large end was fitted with Silastic tubing to act as a bulb for drawing and expelling fluid. Three µC per treatment were filled with 5 µL of diluted ejaculate and sealed with Critoseal. The µC were inserted into 500-µL Cassou straws (IMV Technologies, L'Aigle, France), immersed in a water bath, and cooled from 21�C to 5�C over 2 h. A Freeze Control� programmable cryochamber (CryoLogic Pty. Ltd., Mulgrave, Victoria, Australia) was used to cool samples slowly from 5�C to –40�C at 3�C min–1. At –40�C, the cryostraws were plunged into liquid N2 (LN2). Rapid freezing was done by plunging µC into a LN2 vortex, created using a magnetic stir bar. The µC were reinserted into the Cassou straws, while still under LN2, for storage in an LN2 tank. The µC were thawed by removal from the Cassou straws and immediate immersion in a 35�C H2O bath. Survival rates were evaluated using a dual fluorescent staining system (Molecular Probes, Eugene, OR, USA) and fluorescent microscopy. The largest portion of live staining cells (93.18%) were treated with DMSO diluent using the rapid freezing. The remaining treatments ranked as follows: slow freezing with DMSO (78.84%), rapid freezing with glycerol (38.9%), and slow freezing with glycerol (26%). All treatments differed significantly (P &lt; 0.01). Other studies state that queens inseminated with greater than 50% viable SC have a good probability of producing normally throughout a season. Therefore, our technique of rapid freezing in DMSO diluent might be useful to apiculturists.

A comparison of high cell density fed‐batch fermentations involving both induced and non‐induced recombinant Escherichia coli under well‐mixed small‐scale and simulated poorly mixed large‐scale conditions

In this work, multi-parameter flow cytometric techniques, coupled with dual colour fluorescent staining, have been used to study the metabolic consequences of inclusion body formation in high cell density fed-batch cultures of the recombinant E. coli strain MSD3735, producing the IPTG inducible model mammalian protein, AP50. Further, we report on the development of the scale-down, two compartment (STR + PFR) experimental simulation model to study, for the first time, the effect of a changing microenvironment with respect to three of the major spatial heterogeneities that may be associated with large-scale bioprocessing (pH, glucose and dissolved oxygen concentration) on a recombinant bacterial system. Using various time points for induction and various scale-down configurations, it has been shown that inclusion body formation is followed immediately by a detrimental progressive change in individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability, resulting in a lower final biomass yield. However, the extent of this change was found to be dependent on whether the AP50 protein was induced or not, on the time of induction and on which combination of heterogeneities was being simulated. From this and previous work, it is clear that the scale-down two-compartment model can be used to study the impact of genetically modifying an organism to produce inclusion bodies and any range and combination of potential heterogeneities known to exist at the large scale.

Clinical Scale Preparation of B Lymphoblastoid Cell Line-Tumor Cell Hybridoma Cells for Vaccination in Patients with Relapsed Ewing's Sarcoma and Neuroblastoma.

Despite improvements in survival for pediatric patients with cancer, for those with recurrent, high risk Ewing's sarcoma (ES) or neuroblastoma (NB) treatment options are limited. Tumor-specific antigens in these malignancies are poorly characterized, and expression of human leukocyte antigen (HLA) and co-stimulatory molecules may be down-regulated, imparing a cellular immune response. Several groups have studied the use of tumor cell-dendritic cell (DC) hybridoma vaccines in murine tumor models as well as in phase I clinical trials in human cancer. One problem in implementing these vaccine strategies on a clinical scale is the requirement for repeat blood collections or leukapheresis to expand a limited number of DC. B lymphoblastoid cell lines (BLCL) are effective APC expressing HLA as well as key co-stimulatory molecules, and can be rapidly expanded from relatively few peripheral blood mononuclear cells. We have studied the ability of BLCL to be fused to a series of NB and ES cell lines using polyethylene glycol, and the ability to propagate these hybrid cells long term in culture. Using monoclonal antibodies specific for tumor markers (anti-GD2 and CD56 for NB), BLCL (CD19), and by labeling ES tumor cells with fluorescent dye prior to fusion, 3–11% of the resulting cell products represented true fusion cells. This was confirmed in studies in which BLCL transduced to express green fluorescent protein (GFP) were fused with adherent tumor cells, resulting in a population of large green adherent cells with abnormal morphology. Dual fluorescent staining and flow cytometry of green fluorescent dye labeled tumor cells and unlabelled BLCL consistently revealed a mixed population of unfused BLCL, unfused tumor cells, and cells with irregular morphology expressing both CD19 and green fluorescent dye. Despite these findings, these hybrid cells could not be expanded in long term culture, and adherent hybrid cells did not survive in cell culture over 2 weeks. In addition, these hybrid cells could not be selected when tumor cells and BLCL were transduced with different antibiotic resistance genes and then subjected to selection media. Despite the inability to selectively expand tumor cell-BLCL hybridomas for long-term culture, the choice of BLCL as an APC for vaccine permits the preparation of relatively large numbers of fusion cells for serial vaccination or for in vitro stimulation of antigen specific CTL.

酸化ストレスで変動するリン酸化タンパク質の蛍光 2-DE ディファレンシャル解析 とMALDI-TOF-MSによる同定

酸化ストレスで変動するリン酸化タンパク質の蛍光 2-DE ディファレンシャル解析 とMALDI-TOF-MSによる同定

Sources of variation in the viability of honey bee, Apis mellifera L., semen collected for artificial insemination

Summary Viability of spermatozoa in honey bee semen from individual drones was highly variable, based on an assay using dual fluorescent staining. Temperature, physical manipulation and the saline used during artificial insemination were evaluated for their impact on sperm viability. Temperature of the assay buffer in which semen was collected had much less effect on viability than the way in which the semen was collected. Washing semen into the buffer did less harm than sucking it up into a syringe. The highest viability levels were seen when semen was collected directly from seminal vesicles. Given that obtaining semen from the seminal vesicles is difficult, collection of semen with the highest viability is best done by washing semen directly from an ejaculated drone into a buffer, rather than using a syringe. The sodium chloride/antibiotic solution used for prophylactic purposes during artificial insemination improved sperm viability slightly.

Cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human corpus cavernosum penis

Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the corpus cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the corpus cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1, 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1. Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the corpus cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.

The application of multi-parameter flow cytometry to the study of recombinant Escherichia coli batch fermentation processes.

Multi-parameter flow cytometric techniques coupled with dual colour fluorescent staining were used to study the physical and metabolic consequences of inclusion body formation in batch cultures of the recombinant Escherichia coli strain MSD3735. This strain contains a plasmid coding for the isopropylthiogalactopyranoside-inducible model eukaryotic protein AP50. It is known that the synthesis of foreign proteins at high concentrations can exert a severe metabolic stress on the host cell and that morphological changes can occur. In this work, using various points of induction, it was shown that inclusion body formation is followed immediately by measurable changes in the characteristic intrinsic light scatter patterns for the individual cell (forward scatter, 90 degrees side scatter) and a concomitant progressive change in the individual cell physiological state with respect to both cytoplasmic membrane polarisation and permeability. This work establishes flow cytometry as a potentially valuable tool for monitoring recombinant fermentation processes, providing important information for scale-up. Further, we discuss the possibility of optimising inclusion body formation by manipulating the fermentation conditions based on these rapid "real-time" measurements.

Plasmodium falciparum-Infected Erythrocytes Adhere Both in the Intervillous Space and on the Villous Surface of Human Placenta by Binding to the Low-Sulfated Chondroitin Sulfate Proteoglycan Receptor

In pregnant women infected with Plasmodium falciparum, the parasite-infected red blood cells (IRBCs) sequester in the placenta through chondroitin 4-sulfate (C4S)-mediated adherence. The pattern of IRBC adherence in P. falciparum-infected placenta has been controversial. Moreover, the identity of the chondroitin sulfate proteoglycan (CSPG) receptor, that mediates IRBC adherence, and its location in the placenta have not been established. This study, using immunohistochemical techniques, clearly shows, for the first time, that the low-sulfated CSPGs of the placenta are localized predominantly in the intervillous space. Ex vivo IRBC adherence analyses demonstrate that the IRBCs are adhered to the CSPG receptors in the placenta in a C4S-dependent manner. This IRBC binding pattern was similar to that observed in P. falciparum-infected placentas. These data and the results of dual-fluorescence staining of the endogenous RBCs and syncytiotrophoblasts, and co-localization of CSPG and IRBC adherence unequivocally establish that the low-sulfated CSPGs are the major natural receptors for IRBC adherence in the placenta. Further, it was found that IRBCs adhere mainly in the intervillous space and also at significant levels to the syncytiotrophoblasts. Finally, the ex vivo IRBC adherence method described herein provides a reliable procedure for future studies for the assessment of the efficacy of C4S inhibitors and adhesion inhibitory antibodies.

Non-toxic thermotropic liquid crystals for use with mammalian cells

This paper reports the results of a study that aimed to identify thermotropic liquid crystals that are not toxic to mammalian cells. Mesogenic compounds were mixed to create eight liquid crystalline phases, each with a unique set of functional groups. We investigated the toxicity of each liquid crystalline phase using two mammalian cell lines—3T3 fibroblast and SV-40 transformed human corneal epithelial (HCEC) cells. Using dual fluorescent staining assays based on calcein acetoxymethylester (Calcein-AM) and ethidium homodimer, we measured correspondingly the number of viable and dead cells following immersion of the cells in the liquid crystals. It was found that most of the liquid crystals, such as commonly used 5CB and E7, caused cell death after contact with cells for four hours. However, we identified a class of liquid crystals containing fluorophenyl groups that possess minimal or no toxicity (as indicated by results of assays based on Calcein-AM and ethidium homodimer) to cells. Following immersion in fluorophenyl liquid crystals for four hours, the cells were observed to proliferate in culture medium at rates similar to control cells exposed to phosphate-buffered saline (PBS) for four hours. In contrast, treatment of cells for 24 hours with either PBS or liquid crystals (media containing no nutrients and growth factors) resulted in cell death.

90EFFECTIVE VIABILITY THRESHOLD FOR PRESERVED HONEY BEE SEMEN

The honey bee, Apis mellifera, is one of very few insects for which artificial insemination is possible, and preservation of semen has been attempted. Honey bee queens normally mate with 7–20 males early in life, store the semen in the spermatheca and release approximately 30 sperm to fertilize each egg. Fertilized eggs are females (queens or workers) and unfertilized eggs are males (drones). The queen controls release of spermatozoa, laying only worker eggs across large areas of comb, and drone eggs in small separate groups. As stored spermatozoa are depleted, a queen will begin to lay mixed groups of workers and drones. Semen cryopreserved following existing protocols (Harbo JR 1983 Annals Entomol. Soc. Amer. 76, 890–891) has less than 20–25% live spermatozoa (based on percentage of workers reared). This viability level is insufficient to successfully fertilize all of the 1000–1500 eggs per day from an active queen, and the colony slowly dwindles and dies. Using dual fluorescent staining (Collins AM and Donoglye AM 1999 Theriogenology 51, 1513–1523). I have determined that semen with 46% live spermatozoa (50% fresh and 50% freeze-killed semen v/v) or more, produces consistent laying patterns with all fertilized eggs (workers) from inseminated queens (Collins AM 2000 Apidologie 31, 421–429). The current study was done to determine how long queens inseminated with mixed fresh:frozen semen would continue to produce normal brood (collective term for all eggs, larvae and pupae), as compared to those inseminated with all fresh semen, or naturally mated in flight. In spring, sister queens were reared and inseminated with [1] all fresh semen, [2] half fresh and half freeze-killed semen, or [3] ¼ fresh and ¾ freeze-killed semen, or [4] were allowed to mate naturally. The queens were evaluated monthly in individual colonies for percentage of worker offspring v. drone offspring and area of comb with eggs, larvae and pupae, until the queen failed. As expected, the inseminated queens produced less brood than did the naturally mated [4] queens. All of the queens with only fresh semen [1] produced normal brood with 100% workers, and lived well into the winter. Eighty-eight percent of the queens inseminated with half freeze-killed semen [2] performed as well, although some of those failed within a few months. The remaining 22% began laying with mixed worker and drone brood. In treatment [3], 60% of the queens produced variable but high levels of drones in reduced areas of egg laying. Those queens in this group that did lay well (40%) also survived into the winter. These results mean that preserved semen that has 50% or better viable sperm has a good probability of producing inseminated queens that will lay normally for a complete beekeeping season. This is sufficient time for breeders to rear daughter queens from such matings, and incorporate desirable genotypes into a breeding program.

A scientific note on the effect of centrifugation on pooled honey bee semen

The ability to pool semen from many drones and then use it for artificial insemination (AI) is a valuable tool for honey bee, Apis mellifera, breeding as it reduces the rate of inbreeding and results in a greater effective breeding population (Page and Laidlaw, 1982). Several authors have reported pooling semen (Taber, 1961; Poole and Taber, 1969; Kaftanoglu and Peng, 1980), including some who also deliberately mixed it (Moritz, 1983; Harbo, 1990). In all cases, the semen was also centrifuged. Using a live:dead dual fluorescent staining technique (Collins and Donoghue, 1999), we found unacceptably low viability (34.1%, unpublished data) in semen mixed and centrifuged in a manner similar to Moritz and Harbo. Therefore, a study was undertaken to determine the optimum centrifugation conditions of pooled, honey-bee semen to be used for AI. Mature, free-flying drones were stimulated to ejaculate by the standard method and the semen collected with a Harbo syringe (Harbo, 1974) into glass capillary tubes. For each sample, the semen was mixed by inversion in Kiev buffer (Moritz, 1984) in an eppendorf tube, subdivided and centrifuged as dictated by experimental design, and the pellet resuspended in 800 μL of fresh buffer. Observations were made on the compactness of the pellet, the ease of removal of the supernatant and ease of resuspension of the pellet. A 200 μL aliquot was stained using the protocol of Collins and Donoghue (1999). Two observers counted two or three subsamples each, scoring one hundred cells as live or dead (percent live spermatozoa). Data were analyzed by Analysis of Variance, using Proc GLM (SAS Institute, 1988). Ten semen samples (5–8 drones) were used to determine that holding samples for 5 m, 30 m, 60 min or 120 min after the staining did not increase the proportion of dead spermatozoa (F = 0.35; df = 3; P = 0.7886; means ranged from 85.1– 96.9%). Seven larger samples (25–35 drones) were collected, diluted and mixed, and divided into 6 equal parts, each of which was centrifuged with a different published speed/time treatment (Tab. IA). A third experiment compared three gentle centrifugation levels across three times (5 replicates of 10 or 30 μL semen in 10 subsamples) (Tab. IB). Centrifugation at speeds of 8 160 g killed a significant number of spermatozoa and produced pellets that were difficult to resuspend. The best results were obtained with speeds of 82 or 250 g at 20–30 or 10–20 min, respectively. These pellets were easily separated from the supernatant and resuspended in buffer. The numeric differences of percent live spermatozoa seen between the two experiments is due to some aspect of collection and handling of the semen prior to centrifugation. What exactly caused the high mortality in Experiment B has not yet been determined. Collins (2000) has reported that even queens inseminated with only 50% live spermatozoa produced normal worker brood, at least early in their lives. Some of the dead spermatozoa were left behind in the vagina during migration to the spermatheca. If 30 or so spermatozoa are released with each egg just after mating (Harbo, 1979), enough live ones would have been present to fertilize the egg successfully. If the mixed semen reported by Harbo (1990), Moritz (1983), and Taber (1961) had levels of viability similar to the treatments here, the normal brood appearance would have hidden the presence of damaged spermatozoa. At least two commercial queen breeders who use mixed semen experienced problems (drone layers and supersedure) and shifted to mechanical stirring (Cobey, personal communication) or slower centrifugation (Kuhnert et al., 1989).

Tissue inhibitor of metalloproteinase-3 is associated with neuronal death in reperfusion injury.

Programmed cell death occurs in ischemia when cell surface death receptors (DRs) are stimulated by death-inducing ligands (DILs). Matrix metalloproteinases are extracellular matrix-degrading enzymes involved in the shedding of DRs and DILs from the cell surface. Tissue inhibitor of metalloproteinase-3 (TIMP-3), which is bound to the extracellular matrix, has been shown to promote apoptosis in cancer cell lines by inhibiting cell surface sheddases. Since apoptosis is an important mechanism of cell death in ischemia, the authors hypothesized that TIMP-3 would be expressed in ischemic neurons that are undergoing programmed cell death. Spontaneously hypertensive rats had a 90-minute middle cerebral artery occlusion with reperfusion. Transcription of TIMP-3 mRNA was measured by quantitative reverse transcription-polymerase chain reaction at 2, 6, 24 and 48 hours after reperfusion. Western blots were used to measure TIMP-3 protein expression. Spatial distribution and production of TIMP-3 was studied by immunohistochemistry at 3, 24, and 48 hours, 5 days, and 3 weeks. DNA fragmentation in cells dying by necrosis and apoptosis was identified with terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL). After 2 hours of reperfusion, TIMP-3 mRNA increased significantly in both ischemic and nonischemic hemispheres. Western blot analysis confirmed the identity of the TIMP-3, which appeared to be increased on the ischemic side. After 3 hours of reperfusion, TIMP-3 immunostaining was increased in neurons on the ischemic side, and by 24 hours the majority of the ischemic neurons were TIMP-3-positive. Dual-fluorescence staining for TUNEL and TIMP-3 showed that they were coexpressed in many neurons. The results suggest that ischemic neurons express TIMP-3, which may be inhibiting sheddases. The authors propose that TIMP-3 facilitates cell death in ischemic neurons. Further studies are needed to identify the sheddases inhibited by the TIMP-3, and on the effect of inhibition of matrix metalloproteinases on cell death mechanisms.