Ultrastructure of putative germ granules in the penaeid shrimp Marsupenaeus japonicus.

Knowledge about the specification of the germ line in penaeid shrimp would allow development of techniques to control germ cell formation and/or fate to produce reproductively sterile shrimp for genetic copyright purposes. Recent studies have traced the localization of an RNA-enriched intracellular body (ICB) in the putative germ line of four penaeid shrimp species. It is hypothesized that the ICB may serve as a putative germ granule and marker of germ line fate. In this study semi-thin and ultra-thin sections of Marsupenaeus japonicus embryos were prepared, and the dimensions and ultrastructure of the ICB was examined at different stages of embryogenesis. The ICB was an aggregation of electron dense granules, small vesicles and multi-vesicular bodies (MVBs), similar to germ granules from other species. Lamellar membranes and mitochondria were localized at the periphery of the ICB. Using fluorescence microscopy, microtubules were also observed between the centrosome and the ICB. The localization of the ICB in the D lineage and putative germ cell line, the enrichment of RNA in the ICB, and the ultrastructural similarities to other germ granules characterized in this study support the hypothesis that the ICB contains germ granules.

Metabolic dysfunction and depletion of mitochondria in hearts of septic rats.

Our previous studies indicate that hearts from septic rats have decreased work with oxygen wasting. The present studies test if there is energy deficit, changes in cardiac mitochondrial content and caspase activation during sepsis. Anesthetized, male Sprague-Dawley rats received no surgical treatment (control), laparotomy (sham), or laparotomy with cecal ligation and puncture (CLP) to induce polymicrobial septic shock. Hearts were isolated 12-14 h later. Cardiac work, oxygen consumption, substrate oxidation and energy stores were measured in perfused hearts. Normalized density of mitochondria was determined in ventricles without perfusion by morphometric analysis with electron microscopy. Citrate synthase activity was assessed in homogenates and isolated mitochondria. Cardiac work decreased significantly in CLP (47%), while oxygen consumption and glucose oxidation were unchanged compared with control or sham hearts (oxygen and substrate wasting). Tissue adenosine triphosphate, creatine phosphate and glycogen were lower in CLP hearts (energy deficit). Mitochondrial grid intersects decreased significantly from 151 +/- 8 sham to 130 +/- 4 CLP out of 361 possible intersects and autophagy was observed in CLP hearts. Total activity of citrate synthase decreased in homogenates (99 +/- 8 micromol/min/g wet weight sham vs. 62 +/- 7 CLP, P < 0.05) and in the mitochondrial fraction (27 +/- 1 micromol/min/g wet weight sham to 22 +/- 1 CLP, P < 0.05). Calculated mitochondrial content decreased from 63 +/- 4 mg protein/g wet weight sham to 46 +/- 5 CLP, P < 0.05 (mitochondrial depletion). Caspase-3 activity doubled and tumor necrosis factor alpha content tripled in CLP hearts. CONCLUSIONS. - Oxygen and substrate wasting in CLP occurs with fewer mitochondria and energy deficit, processes that are coincident with caspase-3 activation.

Activation of poly(ADP-ribose) polymerase in severe hemorrhagic shock and resuscitation.

This study examines activation of poly(ADP-ribose) polymerase (PARP) in the ileum during hemorrhage and resuscitation and determines if inhibition of PARP reduces organ dysfunction and metabolic acidosis. Awake, nonheparinized rats were hemorrhaged (40 mmHg, 60 min). Resuscitation used Ringer's solution (2 1/3 x shed volume) and packed red blood cells (2/3 shed volume). Ileal PARP activity was elevated at the end of hemorrhage (3.6-fold) and 10 min of resuscitation (5-fold). The subsequent decline in PARP activity observed after 60 min of resuscitation was not due to cleavage by caspase-3. Ileum permeability increased 10-fold and circulating liver enzymes increased 4- to 6-fold following 60 min of resuscitation in animals pretreated with 3-aminobenzoic acid, a structural analog that does not inhibit PARP. Pretreatment with 3-aminobenzamide (3-AB), a PARP inhibitor, reduced these changes, whereas posttreatment with a bolus of 3-AB was ineffective. Metabolic acidosis, accumulation of lactate, and base deficit was reduced by pretreatment with 3-AB. PARP is activated in the ileum by hemorrhage and by resuscitation. Activation of PARP contributes to organ dysfunction in the ileum and liver and appears to be central to the development of metabolic acidosis.

Role of fatty acids in the recovery of cardiac function during resuscitation from hemorrhagic shock.

This study tested the hypothesis that removal of fatty acids as a fuel source would improve cardiac efficiency at the expense of reduced cardiac contractile function in the isolated working heart after hemorrhage-retransfusion. Non-heparinized male Sprague-Dawley rats were anesthetized with ketamine-xylazine and were hemorrhaged to a mean arterial blood pressure of 40 mmHg for 1 h. Two-thirds volume of shed blood was reinfused together with 0.9% NaCl in a volume equal to 2.3 times the shed blood volume, followed by continuous infusion of 0.9% NaCl at 10 mL/kg per h for 3 h. Hearts were removed and perfused in closed, recirculating working mode for 60 min to measure hydraulic work and cardiac efficiency. Rates of glycolysis and glucose oxidation were assessed with [5-3H/U-14C] glucose (11 mM) in the absence or presence of 0.4 mM palmitate. Compared to baseline measurements, hemorrhage-retransfusion significantly reduced arterial blood glucose (228+/-7 versus 118+/-12 mg/dL) and non-esterified fatty acid concentrations (0.36+/-0.01 versus 0.30+/-0.02 mM), while elevating blood lactate (0.8+/-0.1 versus 2.5+/-0.4 mM). Perfusion of sham hearts with glucose-only did not alter cardiac work compared to shams perfused with glucose plus palmitate. However, shocked hearts perfused with glucose-only demonstrated a significant reduction in cardiac work compared to shocked hearts perfused with glucose plus palmitate and compared to sham hearts perfused with glucose only (P < 0.05, repeated measures ANOVA). Shocked hearts perfused with glucose plus palmitate showed no reduction in cardiac work compared to shams. Shocked hearts perfused with glucose-only had increased glucose oxidation rates compared to shams perfused with glucose plus palmitate. In sham hearts perfused with glucose-only, myocardial glycogen and triacylglycerol contents were significantly reduced compared to hearts freeze-clamped in situ. These endogenous fuels were not decreased in shocked hearts. These data indicate that hemorrhagic shock renders the heart unable to mobilize endogenous fuels, and suggest that withdrawal of fatty acid oxidation will impair myocardial energy metabolism during resuscitation.

Activation of pyruvate dehydrogenase improves heart function and metabolism after hemorrhagic shock.

This study was designed to test the hypothesis that activation of myocardial pyruvate dehydrogenase (PDH) would improve recovery of heart function after brief, severe hemorrhagic shock. Pentobarbital-anesthetized rats were instrumented to monitor arterial blood pressure and right ventricular pressures. Rats were hemorrhaged via femoral artery to 25-30 mmHg mean arterial pressure (MAP) for 60 min, followed by retransfusion of shed blood with either 1.0 cc saline with no dichloroacetate (-DCA) or 1.0 cc saline containing 150 mg/kg sodium dichloroacetate (+DCA). Rats were observed for 3 h after retransfusion. Hearts were freeze-clamped in situ for analysis of adenosine triphosphate (ATP), creatine phosphate (CrP), lactate and pyruvate content as well as PDH activity (PDHa) and total PDH activity (PDHt). Three h after retransfusion, the rate pressure product (RPP=HRxPSP) was 23 000+/-2733 with no DCA treatment v 36 2769 mmHg/min with DCA treatment (P<0.05, ANOVA). Treatment with DCA also increased myocardial tissue content of high energy phosphates (ATP=10.1+/-1.1 and CrP=5.8+/-1.0 micromol/g weight-DCA, v 15.1+/-0.9 and 14.7+/-1.0 micromol/g dry weight+DCA, P<0.05, both measurements). DCA administration also significantly reduced myocardial lactate contents (14.6+/-2.7 micromol/g dry weight-DCA v 5.9+/-1.0+DCA). Hemorrhagic shock did not change PDHa or PDHt compared to hearts obtained during the pre-hemorrhage period. Retransfusion with DCA significantly increased PDHa activity (6.8+/-1.1 micromol/g dry weight/min-DCA v 29.7+/-2.0 micromol/g dry weight/min+DCA). PDHt was not different between controls and DCA-treated groups. These data indicate that activation of myocardial PDH by adding DCA to retransfused blood improved heart function and metabolism after severe hemorrhagic shock.