Total Tissue Weight Research Articles

Abstract P1048: Dual Myristic Acid And Trans Activator Of Transcription Conjugated Peptide Facilitates Delivery Of Protein Kinase C Beta II Peptide Inhibitor To Attenuate Myocardial Ischemia/Reperfusion Injury

Peptides conjugated to myristic acid (myr) or trans activator of transcription (tat) have long been used to increase cell permeability via simple diffusion and endocytosis, respectively. Previously myr-PKCβII inhibitor (myr-PKCβII-) exerted cardioprotective effects in myocardial ischemia/reperfusion (I/R) and inhibited superoxide (SO) release from rat leukocytes by 40% (20 μM). By contrast, dual conjugated myr-tat-PKCβII inhibitor (myr-tat-PKCβII-) inhibited SO release by 90% at the same concentration. The purpose of this study was to determine the potency of myr-tat-PKCβII- compared to myr-PKCβII- in mitigating infarct size and restoring cardiac function in an ex-vivo rat model of myocardial I/R injury. Isolated hearts from male Sprague-Dawley rats (~300g) were subjected to global I(30-min)/R(50-min) . Left ventricular cardiac function was recorded using a pressure transducer. Treatments were infused during the first 5 min of R. After R, the hearts were sectioned (2 mm) from base to apex and stained with 1% triphenyltetrazolium chloride. Infarcted tissue was excised to determine infarct size (i.e. infarct tissue weight/ total tissue weight). Data were analyzed using ANOVA Fisher’s LSD analysis. Compared to untreated controls (23.2±3.1%, n=17), myr-tat-PKCβII- exerted a significant and similar decrease in infarct size that was observed from 100 nM (9.7±0.59%, n=3, p<0.05), 1 nM (10.0±2.3%, n=5, p<0.05), to 100 pM (12.6±2.3%, n=5, p<0.05); but not at 10 nM (13.8±3.6%, n=5). Cardiac function for all treatment groups did not significantly improve from control. However, at 100pM (1070±170 mmHg/s), +dP/dt max was significantly improved compared to all other treatment groups (p<0.05). The addition of myr to tat-conjugated peptides improved the cardioprotective effects ~200-fold compared to myr-PKCβII- and ~2000-fold compared to native peptide presumably by enhanced intracellular delivery of cargo. The ~50% reduction in infarct size suggests that myr-tat-PKCβII- is cardioprotective in I/R. Future studies will test our most effective dose determined in ex vivo hearts to restore post-reperfusion cardiac function combined with significant cardiac salvaging effect (reduced infarct size) in a porcine myocardial I/R model.

Proximate Composition, Amino Acids, and Fatty Acids in the Adductor Muscle of the Giant Honeycomb Oyster Hyotissa hyotis (Linnaeus, 1758) from Jeju Island, Korea

The giant honeycomb oyster Hyotissa hyotis occurs at a high density in the shallow subtidal rocky bottom of Jeju Island off the south coast of Korea, where the sea surface temperature ranges from 14°C to 25°C seasonally. Unlike other oysters, H. hyotis has an extraordinarily large adductor muscle (AM) that accounts for more than 40% of the total tissue weight. This study analyzed the proximate composition, amino acids, and fatty acids of the AM of H. hyotis in Jeju Island to evaluate the nutritional potential. For the analyses, oysters were grouped based on their reproductive condition as prespawning (May–August), spawning (September–November), and postspawning (December–April). The AM contained a high level of protein (54.7%–69.4%) followed by carbohydrate (16.0%–25.3%) and lipid (4.5%–9.9%) throughout the year, indicating that the AM is enriched with protein. The total amino acids (TAA) in the AM ranged from 9.4 to 12.9 g/100 g dry weight annually, and essential amino acids accounted for up to 36.7% of the TAA. Like other marine bivalves, the AM contained a high level of taurine, which accounted for 26.5%–28.5% of the total free amino acids. The AM also included high levels of essential fatty acids, such as palmitic acid, docosahexaenoic acid, and eicosapentaenoic acid, as these fatty acids accounted for 35.9%–37.0% of the total fatty acids. The AM of prespawning oysters collected during May and August contained comparatively higher total proteins and carbohydrates. Results suggested that, like scallops and pen shells, the AM of the giant honeycomb oyster is highly suitable as seafood, and H. hyotis should be considered as a species of high potential for future aquaculture efforts.

The Metabolic Landscape in Osteoarthritis.

Articular cartilage function depends on the temporal and zonal distribution of coordinated metabolic regulation in chondrocytes. Emerging evidence shows the importance of cellular metabolism in the molecular control of the cartilage and its dysregulation in degenerative diseases like osteoarthritis (OA). Compared to most other tissues, chondrocytes are sparsely located in the extracellular matrix, lacking the typical proximity of neural, vascular, and lymphatic tissue. Making up under 5% of the total tissue weight of cartilage, chondrocytes have a relative deficiency of access to nutrients and oxygen, as well as limited pathways for metabolite removal. This makes cartilage a unique tissue with hypocellularity, prolonged metabolic rate, and tissue turnover. Studies in the past decade have shown that several pathways of central carbon metabolism are essential for cartilage homeostasis. Here, we summarised the literature findings on the role of cellular metabolism in determining the chondrocyte function and how this metabolic dysregulation led to cartilage aging in OA and provided an outlook on how the field may evolve in the coming years. Although the various energy metabolism pathways are inextricably linked with one another, for the purpose of this review, we initially endeavoured to examine them individually and in relative isolation. Subsequently, we comment on what is known regarding the integration and linked signalling pathways between these systems and the therapeutic opportunities for targeting OA metabolism.

Abstract P500: Naltrindole And Naltrindole Derivatives Exhibit Potent Cardioprotection In Myocardial Ischemia Reperfusion Injury

Previously, naltrindole (NTI; selective delta opioid receptor antagonist) was shown to improve post-reperfused cardiac function and reduced infarct size when given prior to ischemia (I)/ reperfusion (R) in ex-vivo rat hearts. Conversely, naloxone (NX, broad-spectrum opioid antagonist) and nor-binaltrophine (BNI, selective kappa receptor antagonist) were similar to control hearts. In this study, the effects of NTI derivatives naltriben (NTB, delta receptor antagonist) and guanidonaltrindole (GNTI, kappa receptor antagonist) were compared to NTI, BNI, and NX. Isolated hearts from male SD rats (300g) were subjected to global I(30min)/R(45min). Treatments were given 5 min before I (preconditioning) and during the first 5 min of R. Left ventricular (LV) cardiac function was measured using a pressure transducer. At the end of reperfusion, infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA. As shown in Table 1, NTI, NTB, and GNTI significantly improved post-reperfused cardiac function and reduced infarct size compared to control hearts. NTI and NTB elicited direct effects on cardiac function when given during preconditioning in contrast to all other study groups and were the most robust at reducing infarct size and restoring post reperfusion cardiac function. The negative inotropic effects of NTI and NTB were correlated with a decrease in the rise of ischemic pressure. GNTI also elicited significant improvement in post-reperfused cardiac function and reduction of infarct size compared to BNI which suggests a separate cardioprotective mechanism that this NTI derivative may exert in contrast to kappa opioid receptor inhibition. Results suggest that NTI and derivatives, GNTI and NTB, are cardioprotective against I/R injury resulting in reduced ischemic peak pressure (NTI/NTB) and infarct size. In future studies, we will examine the mechanism of the protective effects of NTI and derivatives in hearts subjected to I/R injury.

Impact of Carotenoid Cleaving Enzymes on Lycopene Accumulation in Transgenic Mice

Abstract Objectives To evaluate the role of β-carotene oxygenase 1 (BCO1) and BCO2 on lycopene tissue distribution. Methods Three-week old C57BL/6 male and female mice (wild type [WT], Bco1−/−, Bco2−/−, Bco1−/− × Bco2−/− double knock out [DKO]) were divided into groups based on genotype (n = 16 per group split evenly by sex) and fed a powdered AIN 93G control diet for 2 weeks. After this period, mice were gavaged daily for 2 weeks with 1 mg of lycopene dissolved in cottonseed oil. 12 h-fasted mice were then sacrificed, and liver, serum, heart, kidney, intestine, gonadal adipose, prostate, spleen, and testes were harvested. Tissues were preserved in liquid nitrogen and stored at −80 until analyses. We measured lycopene levels in all samples by using high-performance liquid chromatography. Data analyses were performed using two-way ANOVA, followed by the Sidaks test with a statistical significance threshold of P < 0.05. Results Female mice showed higher lycopene levels in the intestine (P < 0.045) and liver (P < 0.007) irrespective of genotype, while male mice had higher lycopene levels in serum (P < 0.004). Intestine, serum, and kidneys exhibited higher lycopene levels in DKO mice compared to all other genotypes (P < .0001), while having higher lycopene levels in testes (P < 0.0001) compared to Bco2−/− and WT mice and adipose (P < 0.005) only in comparison to Bco2−/− mice. DKO exhibited higher lycopene levels in the spleen compared to Bco1−/− mice (P < 0.02). Lycopene levels in the liver (P < 0.0001) were higher in Bco2−/− mice compared to Bco1 −/− and DKO mice, while Bco1−/− mice had lower hepatic lycopene levels compared to all other genotypes. Conclusions Female mice accumulated higher lycopene levels in most tissues compared to males. These results were consistent when data were corrected by total tissue weight. The data suggest the absence of BCO2 favors carotenoid accumulation in many extrahepatic tissues, an effect that is enhanced in the absence of both carotenoid cleaving enzymes. Funding Sources Internal funding, University of Illinois Urbana Champaign.

Naltrindole Exhibits Robust Cardioprotection in Myocardial Ischemia Reperfusion Injury

The predominant opioid receptor subtypes in heart tissue are delta and kappa opioid receptors. Activation of delta and kappa opioid receptors during preconditioning have been shown to reduce infarct size in myocardial ischemia reperfusion (I/R) injury. This cardioprotective effect has been abolished with naloxone (NX), a broad-spectrum opioid receptor antagonist (e.g., delta, kappa, and mu). Previously, we demonstrated that a novel tripeptide, structurally similar to other delta or kappa opioid peptide receptor agonists, when given prior to global ischemia, significantly reduced infarct size and improved post-reperfused cardiac function using a Langendorf rat I/R heart ex vivo model. To identify the opioid receptor subtype responsible for tripeptide's cardioprotective effects, we used naltrindole (NTI), a selective delta opioid receptor antagonist. The cardioprotective effects of tripeptide were blocked by NX, but not NTI. To determine whether the cardioprotective effects of the combination was due to tripeptide or NTI, the effects of NTI and NX were evaluated individually in the same model. Male Sprague-Dawley rats (~300g) were subjected to global I(30min)/R(50min). NX (10 μM, n=6) or NTI (5 μM, n=8) or H2O vehicle (control n= 10) were given to hearts 5 min prior to ischemia and during the first 5 min of reperfusion in a Langendorf perfused model. Left ventricular (LV) cardiac function indices were measured using a pressure transducer. At the end of reperfusion (50min), hearts were frozen, sectioned (2 mm), and stained with 1% triphenyltetrazolium chloride (TTC). Infarcted heart tissue was compared to total tissue weight. Data were evaluated using ANOVA Student-Newman-Keuls post-hoc analysis. Control hearts (n=10) exhibited increased final LV end diastolic pressure (LVEDP) of 60±5 mmHg from baseline values (~8mmHg for all groups) and a mean infarct size of 36 ± 3% at 50 min post-reperfusion. NX showed no significant cardioprotective effect, since final LVEDP (64 ± 5%) and infarct size (35 ± 5%) was similar to control hearts. Surprisingly, the selective delta opioid antagonist, NTI, remarkably improved final LVEDP to 17 ± 3mmHg to near baseline values and reduced infarct size to 7 ± 2% compared to control and NX hearts (both p<0.01). The results indicate that NTI can prevent ischemic injury and restore post-reperfused heart function to pre-ischemic levels. In future studies, we will examine the intracellular mechanisms of NTI responsible for preventing myocardial ischemic injury.

Abstract 449: Myristic Acid Conjugated Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Cardioprotective Effects in Rat Myocardial Ischemia-Reperfusion Injury

Reactive oxygen species (ROS) induced ischemia-reperfusion (I/R) injury is a phenomenon causing paradoxical myocardial damage after cardio-angioplasty, coronary bypass and organ transplantation. Previous studies show that a cell-permeable myristic acid (myr-) conjugated PKCβII peptide inhibitor given at reperfusion prevents PKCβII translocation (N - myr-SLNPEWNET; myr-PKCβII-) and significantly attenuates ROS mediated I/R injury. We included a scrambled myr-PKCβII- (N-myr-WNPESLNTE; myr-PKCβII-scram) to examine the effects of myr separately. We hypothesize that myr-PKCβII- will improve and myr-PKCβII activator peptide (N-myr-SVEIWD; myr-PKCβII+) will exacerbate infarct size and post-reperfused cardiac function compared to myr-PKCβII-scram and untreated controls. Hearts isolated from male Sprague-Dawley rats (~300g) were perfused with Krebs’ buffer at a constant pressure of 80mmHg and subjected to 30 min of global ischemia and 50 min reperfusion. Myr-PKCβII-, myr-PKCβII+, myr-PKCβII-scram (all 20μM), or untreated control were given during the first five minutes of reperfusion. Left ventricular (LV) dP/dt max and dP/dt min (mmHg/s) were measured using a pressure transducer, and infarct size was determined using 1% triphenyltetrazolium chloride staining comparing infarcted tissue vs. total tissue weight. Data were evaluated using ANOVA with Student-Newman-Keuls post-hoc analysis. Myr-PKCβII- (n=17) significantly improved LV dP/dt max and dP/dt min to 1535±107 and 1063±83 at 50 min post-reperfusion compared to untreated control (815±107 and 722±89; n=15); myr-PKCβII-scram (513±78 and 433±66; n=12), and myr-PKCβII+ (860±118 and 694±81; n=14) (all p&lt;0.05). Myr-PKCβII- significantly reduced infarct size (%) to 13±2 compared to untreated control (24±4); myr-PKCβII-scram (22±2), and myr-PKCβII+ (21±3) (all p&lt;0.05). Unexpectedly, myr-PKCβII-scram significantly depressed post-reperfused LV dP/dt max and LV dP/dt min compared to untreated control and other treated groups (p&lt;0.05). Results suggest that myr-PKCβII- exerted significant cardioprotective effects compared to untreated controls, myr- PKCβII+ and myr-PKCβII-scram and would improve clinical outcomes after cardio-angioplasty or organ transplantation.

Myristic Acid Conjugation Itself is Not Responsible for Robust Cardioprotective Effects Observed with Protein Kinase C Beta II Peptide Inhibitor in Ischemia‐Reperfusion Injury

Ischemia‐reperfusion (I/R) injury mediated by excessive reactive oxygen species (ROS) is a well‐known phenomenon causing paradoxical myocardial damage after cardio‐angioplasty, coronary bypass, or organ transplantation following ischemic injury. Protein kinase C beta II isoform (PKCβII) inhibition using a cell‐permeable myristic acid (myr‐) conjugated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII−) given at reperfusion significantly attenuated ROS release in previous animal I/R studies. However, prior studies did not explore the possibility that myristic acid conjugation itself contributes to the attenuation of I/R injury. We hypothesize that myristic acid conjugation is not responsible for attenuation of ROS‐induced I/R damage and that myr‐PKCβII− will reduce infarct size and improve post‐reperfused cardiac function compared to scrambled myr‐PKCβII− peptide (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram), myr‐PKCβII activator peptide (N‐myr‐SVEIWD; myr‐PKCβII+), and plasma controls. Hearts isolated from male Sprague‐Dawley rats (~300g) were subjected to 30 min of global ischemia and myr‐PKCβII− (20μM), myr‐PKCβII+ (20μM), myr‐PKCβII‐scram (20μM), or plasma (control) was given at initial reperfusion during the first five minutes. Thereafter, Krebs’ buffer was reperfused into hearts at a constant pressure (80 mmHg) throughout the remainder of reperfusion (50 min). Left ventricular (LV) cardiac function indices were measured using a pressure transducer, and infarct size of frozen post‐reperfused hearts was determined using 1% triphenyltetrazolium chloride staining comparing infarcted tissue vs. total tissue weight. Data were evaluated using ANOVA with Bonferroni‐Dunn post‐hoc analysis. Myr‐PKCβII‐significantly improved both post‐reperfused cardiac relaxation function indices compared to all groups (p&lt;0.05). LV end diastolic pressure (LVEDP; mmHg) and the maximal rate of decline of LVEDP (mmHg/s) at 50 min post‐reperfusion significantly improved with myr‐PKCβII− (41±5 and 1088±84; n=16) compared to plasma‐control (61±4 and 731±95; n=14), myr‐PKCβII+ (58±4 and 716±84; n=13), or myr‐PKCβII‐scram (68±1 and 451±78; n=8) hearts. Additionally, myr‐PKCβII− significantly reduced infarct size (%) to 13±2 compared to either plasma‐control (24±4) or myr‐PKCβII‐scram (24±2; both p&lt;0.05), whereas myr‐PKCβII+ (21±3) did not differ significantly from myr‐PKCβII‐scram or plasma‐control. Results suggest that myr‐conjugation is not responsible for the cardioprotective effects observed with myr‐PKCβII− in I/R injury. Myr‐PKCβII− may be an effective therapeutic to improve clinical outcomes after coronary bypass, cardio‐angioplasty, or organ transplantation.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC. lindonyo@pcom.edu.

Annual gametogenesis and reproductive effort of the limpet Cellana grata (Gould, 1859) (Gastropoda: Nacellidae) in a rocky intertidal beach at Ulleungdo Island off the east coast of Korea

Widely distributed from the northern coast of Vietnam to the northern Japan, the limpet Cellana grata (Gould, 1859) occurs commonly on the south and east coasts of Korea. Despite their wide distribution range, few studies have investigated the annual gametogenesis and reproductive effort of C. grata. In an attempt to understand the reproductive physiology of the limpet, we investigated the annual gametogenesis and reproductive effort of C. grata from Ulleungdo Island off the east coast of Korea. Histology revealed that the gonial mitosis commenced in January, as the female exhibited small oogonia (10–40 μm) in the follicle. From March to June, the oocyte size increased dramatically, and fully mature eggs (110–170 μm in diameter) appeared in early summer. First spawning males and females were observed in July, as the surface seawater temperature (SST) reached 22.1°C. The spawning male and females could be observed until the end of December. Gonad somatic index (GSI), a ratio of gonad mass to the total tissue weight, of the male ranged from 0.6 (April) to 17.9 (July), while the female GSI varied from 1.0 (February) to 18.3 (July). GSI of male and female declined rapidly from July to August, suggesting that the major purse of the spawning at the study site was between July and August. Our study suggested that the commercial catch of C. grata during July and August must be suspended at Ulleungdo Island, in order to protect the spawning limpets, which enhances C. grata recruitment and the population.

Evidence of Nonuniformity in Urothelium Barrier Function between the Upper Urinary Tract and Bladder

We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg ml(-1) mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 μg cm(-2), respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C.

Differential effects of developmental hypo- and hyperthyroidism on acetylcholinesterase and butyrylcholinesterase activity in the spinal cord of developing postnatal rat pups

The plasticity and vulnerability of the rat spinal cord (SC) during postnatal development has been less investigated compared to other CNS structures. In this study, we determined the effects of thyroid hormonal (TH) deficiency and excess on postnatal growth and neurochemical development of the rat SC. The growth as well as the specific and total activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes of the SC were determined in hypo- and hyperthyroid rat pups at postnatal (P) days P1, P5, P10 and P21 (weaning), and were compared to age-matched untreated normal controls. AChE is a cholinergic synaptic enzyme while BuChE is a metabolic enzyme mainly found in glial cells and neurovascular cells. The SC is rich in somatic motor, autonomic cholinergic neurons and associated interneurons. Daily subcutaneous injection of pups with thyroxine (T4) and administration of antithyroid goitrogen propylthiouracil (PTU) in the litter's drinking water were used to induce hyper- and hypothyroidism, respectively. Enzyme assays were carried out spectrophotometrically at the above-mentioned ages, using SC homogenates with acetylthiocholine-chloride as the substrate, together with specific cholinesterase inhibitors, which specifically target AChE and BuChE. SC weights were significantly lower at P10 and P21 in hypothyroid pups but unchanged in the hyperthyroid ones. Hypothyroidism significantly reduced both specific and total AChE activity in SC of P10 and P21 rat pups, while having no effects on the BuChE activity, although total BuChE activity was decreased due to reduced total tissue weight. In contrast both specific and total AChE activities were markedly and significantly increased (>100%) in the P10 and P21 hyperthyroid pups. However, BuChE specific activity was unaffected by this treatment. The results indicate that hypothyroid condition significantly reduces, while hyperthyroidism increases, the postnatal development of cholinergic synapses, thereby influencing the functional development of this major sensory and motor structure. However, the neurochemical development of glia and other non-neuronal cells, where BuChE is mainly localized, is comparatively unaffected in these abnormal developmental conditions.

Mouse Skeletal Muscle Fiber-Type-Specific Macroautophagy and Muscle Wasting Are Regulated by a Fyn/STAT3/Vps34 Signaling Pathway

Skeletal muscle atrophy induced by aging (sarcopenia), inactivity, and prolonged fasting states (starvation) is predominantly restricted to glycolytic type II muscle fibers and typical spares oxidative type I fibers. However, the mechanisms accounting for muscle fiber-type specificity of atrophy have remained enigmatic. In the current study, although the Fyn tyrosine kinase activated the mTORC1 signaling complex, it also induced marked atrophy of glycolytic fibers with relatively less effect on oxidative muscle fibers. This was due to inhibition of macroautophagy via an mTORC1-independent but STAT3-dependent reduction in Vps34 protein levels and decreased Vps34/p150/Beclin1/Atg14 complex 1. Physiologically, in the fed state endogenous Fyn kinase activity was increased in glycolytic but not oxidative skeletal muscle. In parallel, Y705-STAT3 phosphorylation increased with decreased Vps34 protein levels. Moreover, fed/starved regulation of Y705-STAT3 phosphorylation and Vps34 protein levels was prevented in skeletal muscle of Fyn null mice. These data demonstrate a Fyn/STAT3/Vps34 pathway that is responsible for fiber-type-specific regulation of macroautophagy and skeletal muscle atrophy.

Comparison of a New Reusable Gynecologic Laparoscopic Electric Morcellator with a Disposable Morcellator: a Preliminary Trial

We compared the efficiency and safety of a newly developed reusable morcellator (Olympus VarioCarve) with that of a disposable morcellator (GyneCare Morcellex) for gynecologic laparoscopic surgery. In group 1 (n = 15 patients; 11 myomectomies and 4 supracervical hysterectomies), the laparoscopic disposable morcellator was used, and in group 2 (n = 14; 11 myomectomies and 3 supracervical hysterectomies), the reusable morcellator was used. There were no statistical differences in total tissue weight. Morcellating time was significantly shorter in group 2, and the rate of morcellation was significantly greater in group 2. Significantly fewer and longer pieces of tissue were removed with the reusable morcellator. No iatrogenic organ damage was observed.

Reproductive cycle and conditioning of translocated scallops ( Pecten maximus) from five broodstock populations in Norway

In Norway sea ranching depends on deployment of animals of local origin, and growers demand large spat outside the natural production season. To develop stock-specific production of juveniles, knowledge on local scallop reproductive cycle and performance in hatchery was needed. Broodstock sized scallops ( Pecten maximus) were collected from 5 locations along the coast of Norway from 59°N to 65°N during 2004 and 2005, and translocated to natural sea location at 61°N. The reproductive cycles were characterised and the possibility of performing successful hatchery conditioning and spawning outside the natural reproduction cycle was investigated. Results showed that scallops from south-western Norway (3 locations) all had distinct season summer peaks in the reproductive cycles. Scallops from the northern part (2 locations) quickly rebuilt the gonad after spawning and seemed to have full gonads through most of the year. Gonad indices in the southern group varied between 5 and 15%, while in the northern group it was 10 to 25% of total tissue weight. Both in December and March most individuals (> 75%) in the southern group had empty gonads, while most individuals in the northern group were characterised as being in gonad maturation stages 3–6. There was no complete synchronous gonad development through the year, and most developmental stages were present at all times. Scallops from the southern locations showed small changes in gonad maturation after translocation to the new environment. Scallops from northern locations kept the original cycle, but seemed to spawn later in summer. Scallops from the 3 southern locations responded to conditioning methods in the hatchery from late autumn to spring, as did one of the northern populations, while the other northern population could not be conditioned in winter. Based on reproductive cycle and hatchery performance we suggest that scallops from the 5 locations belonged to 3 distinct different populations. The differences in timing of gametogenesis and response to conditioning environment have implications for spat production in the hatchery and must be accounted for in order to fulfil the requirements of site-specific spat for scallop sea ranching in Norway.

Aquaporin-1 and aquaporin-3 expressions in the temporo-mandibular joint condylar cartilage after an experimentally induced osteoarthritis

Over 70% of the total tissue weight in the cartilage matrix consists of water, and the early-stage osteoarthritic cartilage is characterized by swelling. Water transport in the cartilage matrix and across the membranes of chondrocytes may be important in normal and pathological conditions of cartilage. The purpose of this study was to identify aquaporin-1 (AQP1) and aquaporin-3 (AQP3) expressions in the mandibular condylar cartilage after experimentally induced osteoarthritis (OA) in rats. An experimental temporomandibular joint OA was induced by partial discectomy in rats. The pathological characteristics of the normal, early-stage, and late-stage osteoarthritic TMJ cartilages were verified by histological techniques. The AQP1 and AQP3 gene expressions in the normal and osteoarthritic cartilages were measured using quantitative real-time reverse-transcription PCR analysis. The cartilage sections were incubated in primary polyclonal antibodies to AQP3; immunofluorescent microscopy was used to examine the AQP3 expression shown by its protein level. The mRNA expression levels of AQP1 and AQP3, analyzed using quantitative PCR, revealed that AQP3 mRNA was highly up-regulated in the OA cartilage, which was considered significant. There was no notable difference in the expression of AQP1 mRNA between OA and normal controls. With the progressing of the OA, the localization of the AQP3 protein was quite different from that of the normal cartilage. Compared to the normal cartilage, the expressions of AQP3 protein were observed mainly in the proliferative zone and the upper mid-zone chondrocytes at the early-stage of OA, and were observed to appear frequently throughout the mid- and deep zone during the late-stage of OA. The high expression of AQP3 mRNA in the OA cartilage and the different localization of the AQP3 protein suggest that it may play a particular role in OA pathogenesis. Further study of AQP3 function may provide new insight into the understanding of the molecular mechanisms underlying OA.

Whole body distribution of deuterated linoleic and α-linolenic acids and their metabolites in the rat

Little is known about the uptake or metabolism of essential fatty acids (EFAs) in various mammalian organs. Thus, the distribution of deuterated alpha-linolenic acid (18:3n-3) and linoleic acid (18:2n-6) and their metabolites was studied using a stable isotope tracer technique. Rats were orally administered a single dose of a mixture (20 mg each) of ethyl D5-18:3n-3 and D5-18:2n-6, and 25 tissues per animal were analyzed for D5-labeled PUFAs at 4, 8, 24, 96, 168, 240, 360, and 600 h after dosing. Plasma, stomach, and spleen contained the highest concentrations of labeled precursors at the earliest time points, whereas other internal organs and red blood cells reached their maximal concentrations at 8 h. The time-course data were consistent with liver metabolism of EFAs, but local metabolism in other tissues could not be ruled out. Brain, spinal cord, heart, testis, and eye accumulated docosahexaenoic acid with time, whereas skin accumulated mainly 20:4n-6. On average, approximately 16-18% of the D5-18:3n-3 and D5-18:2n-6 initial dosage was eventually accumulated in tissues, principally in adipose, skin, and muscle. Approximately 6.0% of D5-18:3n-3 and 2.6% of D5-18:2n-6 were elongated/desaturated and stored, mainly in muscle, adipose, and the carcass. The remaining 78% of both precursors was apparently catabolized or excreted.

Comparative reproductive biology of Yezo scallop, Patinopecten yessoensis, under two different culture systems on the east coast of Korea

The annual gametogenesis of Yezo scallop, Patinopecten yessoensis, raised in suspended-hanging (SHCS) and bottom-sowing culture systems (BSCS) on the east coast of Korea was investigated over 2 years. Gametogenesis was initiated in October, when the surface water temperature reached 18 °C. The gametes matured fully and were ready for spawning in March and early April. Spawning occurred from late March to late May, when the water temperature rose from 7 °C to 11 °C. The gonado-somatic index (GSI), a ratio of gonad weight to total tissue weight, ranged from 3.05 (September) to 23.11 (April) for SHCS scallops and from 3.02 (August) to 20.30 (April) for BSCS scallops. The GSI of SHCS scallops was significantly higher ( P < 0.05) than that of BSCS scallops during the first reproductive season, but not the next spawning season. SHCS scallops also maintained a significantly higher condition factor (CF) than that of BSCS scallops ( P > 0.05) throughout most of the period, indicating that the SHCS may provide better growth and reproductive conditions. The higher GSI of SHCS scallops in the first spawning season may have resulted from different levels of food availability and the stocking size of the spats.

A test for overdominance at the phosphoglucomutase-2 locus in Pacific oysters ( Crassostrea gigas) from New Zealand

The biochemical basis of overdominance for specific activity at the phosphoglucomutase-2 (PGM-2) locus has been reported for Pacific oysters ( Crassostrea gigas) that are formed from heterozygous combinations of the allele at highest frequency and any other allele at this locus [Pogson, G.H., 1991. Expression of overdominance for specific activity at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas. Genetics 128, 133-141.]. It was suggested that this form of overdominance might explain soft tissue growth differences among adult wild Pacific oysters that are frequently reported in the literature. In the context of New Zealand's Pacific oyster aquaculture programme, we investigated the feasibility of using this overdominance as one of several different, but complementary, genetic approaches to achieve production gains for a hatchery-based breeding programme. There was no evidence of either a PGM-2 genotype-dependent effect or of overdominance for wet tissue weight among the wild juvenile oysters (9 months of age) or among the adults of this same batch of oysters 12 months later after they had been on grown. Among juveniles (5 months of age) from each of 8 full-sib hatchery reared families, there was no evidence of a PGM-2 genotype-dependent effect or of overdominance on dry shell weight, wet tissue weight, or total tissue weight within any of the families. When these juvenile oysters were pooled without regard to family, there was no evidence of overdominance for dry shell weight, wet tissue weight, or for total body weight (=dry shell weight plus wet tissue weight). Despite finding no evidence for an overdominant effect at the PGM-2 locus within either wild or hatchery-produced Pacific oysters from New Zealand, we suggest that further evaluation of the PGM-2 overdominance effect is warranted to determine if the overdominant growth advantage previously reported in the literature is present among adult oysters grown at aquaculture sites.

Application of enzyme-linked immunosorbent assay for studying of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia)I. Quantifying eggs

A polyclonal antibody specific to Ruditapes philippinarum egg protein (ME-ab) was developed to quantify clam eggs using an enzyme-linked immunosorbent assay (ELISA). Western blots revealed that ME-ab reacted with egg proteins of molecular masses 475, 84, and 40 kDa under nonreducing conditions and 330, 96, 64, 50, and 31 kDa under reducing conditions. With ELISA, ME-ab detected between 0.23 and 15 A gm l 1 of clam egg protein; the number of eggs per clam was quantified by dividing the weight of the total egg protein by the average weight per egg. Reproductive output, expressed as the gonadosomatic index (GSI), was calculated as the ratio of the egg weight to the total tissue weight. Seasonal changes in reproductive output were measured in clams collected on a monthly basis from Gomso Bay, Korea. Clam egg protein was detected during all months except January. The monthly mean GSI varied from 0 (January) to 0.25 (August), and the highest GSI (0.389) was recorded from a clam collected in late July. ELISA indicated that clams in Gomso Bay spawned when the gonad accounted for 20% of the total tissue weight. The fecundity estimated from individual clams before spawning ranged from 0.94 to 11.79 million eggs, with a mean of 4.15 million. In conclusion, the ELISA used in this study was a sensitive and rapid method

Application of enzyme-linked immunosorbent assay for studying of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia) : I. Quantifying eggs

A polyclonal antibody specific to Ruditapes philippinarum egg protein (ME-ab) was developed to quantify clam eggs using an enzyme-linked immunosorbent assay (ELISA). Western blots revealed that ME-ab reacted with egg proteins of molecular masses 475, 84, and 40 kDa under non-reducing conditions and 330, 96, 64, 50, and 31 kDa under reducing conditions. With ELISA, ME-ab detected between 0.23 and 15 μg ml −1 of clam egg protein; the number of eggs per clam was quantified by dividing the weight of the total egg protein by the average weight per egg. Reproductive output, expressed as the gonadosomatic index (GSI), was calculated as the ratio of the egg weight to the total tissue weight. Seasonal changes in reproductive output were measured in clams collected on a monthly basis from Gomso Bay, Korea. Clam egg protein was detected during all months except January. The monthly mean GSI varied from 0 (January) to 0.25 (August), and the highest GSI (0.389) was recorded from a clam collected in late July. ELISA indicated that clams in Gomso Bay spawned when the gonad accounted for 20% of the total tissue weight. The fecundity estimated from individual clams before spawning ranged from 0.94 to 11.79 million eggs, with a mean of 4.15 million. In conclusion, the ELISA used in this study was a sensitive and rapid method for quantifying eggs in individual clams and is considered the method of choice for quantifying the reproductive output.