Increased renal TXA2 synthesis in diabetes mellitus: simultaneous determination of urinary TXB2 and 2,3-dinor-TXB2

The present study was designed to determine urinary excretion of kallikrein(KAL)-kinin as well as prostaglandin (PG) E2, TXB2 and 2,3-dinor-TXB2, a major urinary metabolite of TXA2 synthesized in platelets, by specific RIAs in patients with diabetes mellitus (DM). KAL or kinin excretion in 26 type II DM did not differ from control values obtained in 18 age-matched healthy subjects (C), although DM with HbA1 greater than 11% excreted less KAL. Urinary PGE2 excretion (7.6 +/- 2.8 ng/mg creatinine, mean +/- SE) was significantly lower in DM compared to C (17.5 +/- 3.9, p less than 0.05), while DM excreted more TXB2 (0.57 +/- 0.09, p less than 0.01) and 2,3-dinor-TXB2 (0.56 +/- 0.12, N.S.) than C (0.19 +/- 0.02 or 0.33 +/- 0.01). DM with or without mild proteinuria demonstrated lower PGE2, but higher TXB2 and 2,3-dinor-TXB2 excretion. A positive correlation of TXB2/2,3-dinor-TXB2 with proteinuria was observed in this group. However, in DM with massive proteinuria over 500 micrograms/mg creatinine, TXB2 and 2,3-dinor-TXB2 excretion decreased to levels almost identical to C. As a whole, a ratio of TXB2 to PGE2 or 2,3-dinor-TXB2 in DM was significantly higher than in C. The results suggest that a relative preponderance of TXB2 to 2,3-dinor-TXB2 may indicate an augmented renal, in addition to platelet, TXA2 synthesis. An excessive vasoconstrictive and proaggregatory TXA2 renal synthesis, concomitant with a decrease in vasodilatory and antiaggregatory PGE2, may have profound effects on renal functions such as protein excretion in DM.     

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice

Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.     

Fluorescence resonance energy transfer between points on actin and the C-terminal region of tropomyosin in skeletal muscle thin filaments

Fluorescence resonance energy transfer between points on tropomyosin (positions 87 and 190) and actin (Gln-41, Lys-61, Cys-374, and the ATP-binding site) showed no positional change of tropomyosin relative to actin on the thin filament in response to changes in Ca2+ concentration (Miki et al. (1998) J. Biochem. 123, 1104-1111). This is consistent with recent electron cryo-microscopy analysis, which showed that the C-terminal one-third of tropomyosin shifted significantly towards the outer domain of actin, while the N-terminal half of tropomyosin shifted only a little (Narita et al. (2001) J. Mol. Biol. 308, 241-261). In order to detect any significant positional change of the C-terminal region of tropomyosin relative to actin, we generated mutant tropomyosin molecules with a unique cysteine residue at position 237, 245, 247, or 252 in the C-terminal region. The energy donor probe was attached to these positions on tropomyosin and the acceptor probe was attached to Cys-374 or Gln-41 of actin. These probe-labeled mutant tropomyosin molecules retain the ability to regulate the acto-S1 ATPase activity in conjunction with troponin and Ca2+. Fluorescence resonance energy transfer between these points of tropomyosin and actin showed a high transfer efficiency, which should be very sensitive to changes in distance between probes attached to actin and tropomyosin. However, the transfer efficiency did not change appreciably upon removal of Ca2+ ions, suggesting that the C-terminal region of tropomyosin did not shift significantly relative to actin on the reconstituted thin filament in response to the change of Ca2+ concentration.     

The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses

The proper intracellular distribution of mitochondria is assumed to be critical for normal physiology of neuronal cells, but direct evidence for this idea is lacking. Extension or movement of mitochondria into dendritic protrusions correlates with the development and morphological plasticity of spines. Molecular manipulations of dynamin-like GTPases Drp1 and OPA1 that reduce dendritic mitochondria content lead to loss of synapses and dendritic spines, whereas increasing dendritic mitochondrial content or mitochondrial activity enhances the number and plasticity of spines and synapses. Thus, the dendritic distribution of mitochondria is essential and limiting for the support of synapses. Reciprocally, synaptic activity modulates the motility and fusion/fission balance of mitochondria and controls mitochondrial distribution in dendrites.     

Variation in stress resistance patterns among stx genotypes and genetic lineages of shiga toxin-producing Escherichia coli O157

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.     

Reciprocal role of ERK and NF-kappaB pathways in survival and activation of osteoclasts

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-kappaB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (Ras(DN)), constitutively active MEK1 (MEK(CA)), dominant negative IkappaB kinase 2 (IKK(DN)), and constitutively active IKK2 (IKK(CA)). Inhibiting ERK activity by Ras(DN) overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEK(CA) remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-kappaB pathway with IKK(DN) virus suppressed the pit-forming activity of OCLs and NF-kappaB activation by IKK(CA) expression upregulated it without affecting their survival. Interleukin 1alpha (IL-1alpha) strongly induced ERK activation as well as NF-kappaB activation. Ras(DN) virus partially inhibited ERK activation, and OCL survival promoted by IL-1alpha. Inhibiting NF-kappaB activation by IKK(DN) virus significantly suppressed the pit-forming activity enhanced by IL-1alpha. These results indicate that ERK and NF-kappaB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-kappaB regulates osteoclast activation for bone resorption.     

Assessment of fresh and stored duck spermatozoa quality via in vitro sperm-egg interaction assay

An in vitro sperm-egg interaction assay was used to measue the quality of duck spermatozoa in fresh and stored semen. The inner perivitelline layer (IPVL), which had been separated from laid duck eggs, was incubated with spermatozoa in vitro. The number of points of sperm hydrolysis in the IPVL in vitro was logarithmically correlated with the fertility of the eggs laid by inseminated females, for both fresh semen (r = 0.85, P < 0.001) and stored semen at 5 degrees C for 24 h (r = 0.84, P < 0.001). After semen storage, the ability of spermatozoa to hydrolyze the IPVL decreased by 67.4% compared with the values for fresh semen, whereas egg fertility and sperm motility decreased by 47.8% and 15.2%, respectively. These results suggest that the in vitro sperm-egg interaction assay accurately reflects the fertilizing ability of fresh and stored duck spermatozoa and detects spermatozoal damage due to semen storage more sensitively than motility or fertility tests.     

H-ras gene is expressed at the G1 phase in primary cultures of hepatocytes

The expression of c-H-ras and proliferating cell nuclear antigen (PCNA) in primary cultures of rat hepatocytes was determined in order to elucidate the relationship between the c-H-ras gene and the S phase of the cell cycle. In cells treated with EGF, elevation of c-H-ras expression was detected at the 22nd, 34th, 44th, and 54th h after plating, PCNA expression and DNA synthesis were detected at the 44th and 54th h. In cells without EGF treatment, only c-H-ras expression was detected at the 44th and 54th h. In our previous report, we showed that c-myc expression increased within several hours after plating, suggesting that isolated hepatocytes traverse from G0 to G1 under culture conditions, regardless of EGF treatment. These results clearly showed that the c-H-ras gene of adult rat hepatocytes was expressed in the mid-to-late G1 phase of the cell cycle as well as in the early S phase in primary culture.