Cytochemical examination of the compartments involved in the transcellular transport of horseradish peroxidase in rat hepatocytes

Horseradish peroxidase (HRP, 10 mg/100 g body weight) was intravenously injected into rats in order to investigate the nature of the compartments involved in the transcellular transport of the protein through hepatocytes into bile. Double cytochemistry for HRP and the marker enzymes for cytoplasmic organelles was used. HRP was shown to be taken up by hepatocytes via vesicles at the sinusoidal surface, some of which were positive for 5'-nucleotidase activity. HRP was then found in the smooth-surfaced vesicles and tubules which were negative in 5'-nucleotidase, glucose 6-phosphatase, thiamine pyrophosphatase and acid phosphatase activity, suggesting that the tubular structures are neither the endoplasmic reticulum, the Golgi apparatus nor lysosomes. Biochemical studies revealed that the lead procedures used for the double cytochemistry did not inhibit the peroxidatic activity of HRP, and conversely that HRP did not interfere with the marker enzyme activity. Such cytochemical observations seemed to be supported by the observation that administration of monensin (3.5 mg/100 g) and chloroquine (5 mg/100 g), which markedly altered the structure of the Golgi apparatus and lysosomes, respectively, slightly altered the biliary excretion of HRP but not to a significant extent.     

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.     

Maturation of Human Herpesvirus 6A Glycoprotein O Requires Coexpression of Glycoprotein H and Glycoprotein L

Glycoprotein O (gO) is conserved among betaherpesviruses, but little is known about the maturation process of gO in human herpesvirus 6 (HHV-6). We found that HHV-6 gO maturation was accompanied by cleavage of its carboxyl terminus and required coexpression of gH and gL, which promoted the export of gO out of the endoplasmic reticulum (ER). Finally, we also found that gO was not required for HHV-6A growth in T cells.     

Synthesis of 6-Sulfur Analogues of Oxanosine and Closely Related Derivatives Thereof

Abstract

Novel β-D-ribofuranosides having a 5-substituted imidazo [4,5-d] [1,3]thiazine ring, including the S⁶-congener 3 of oxanosine 2, were synthesized for screening their anticancer and antiviral activities.     

Conditions of the cervix for the increase of plasma levels of 13,14-dihydro-15-keto-prostaglandin F2 alpha after amniotomy at term

Amniotomy was performed in 12 multiparas at term but not in labor. In 6 of these patients (group I), the fetal head and cervix condition were favorable for amniotomy, and in the other 6 (group II), they were not favorable. In all group I patients, a sudden and progressive descent of the fetal head, and onset and progress of labor were noted within 5 hours. Plasma 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) levels increased significantly (P less than 0.05) in 4 of these cases with time. In group II patients, descent of the head was less than that in group I patients (P less than 0.05), and neither strong labor nor rise of PGFM levels was noted within 5 hours. These data support our view that amniotomy at an appropriate time results in the onset and progress of labor, and the rise of plasma PGFM in virtue of the sudden and exponential increase of the head to cervix force, but amniotomy at an inappropriate time does not, because this force is unchanged.     

Translocation of hepatocyte lysosomes following partial hepatectomy and its inhibition by colchicine

Hepatocyte microtubules were studied during the translocation of lysosomes which follows partial hepatectomy. In hepatocytes of normal rat liver the lysosomes are seen mainly along the bile canaliculi. After partial hepatectomy, these lysosomes lose their pericanalicular arrangement and move towards large inclusions (‘protein droplets’) which result from the marked pinocytosis induced by the removal of about two-thirds of the liver mass. The lysosomes fuse with the inclusions, and by 4 h after partial hepatectomy most of the inclusions demonstrate acid phosphatase activity histochemically. Many microtubules are found in close proximity to the lysosomes. When the rats are treated with colchicine prior to the partial hepatectomy, events are markedly different. The lysosomes change their location but do not hit the cytoplasmic inclusions, and the inclusions remain negative for acid phosphatase activity even 4 h post-hepatectomy. Quantitative ultrastructural study shows that the volume density of microtubules in the hepatocytes decreases to one-third of that in control hepatocytes. This strongly suggests an involvement of microtubules in giving orientation to the translocation of hepatocyte lysosomes under these conditions.     

UV resonance Raman studies on the activation mechanism of human hematopoietic prostaglandin D2 synthase by a divalent cation, Mg

The Mg²⁺ ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D₂ synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg²⁺ to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615 cm⁻¹ to 1600 cm⁻¹. This large shift to lower energy of the tyrosine Y8a vibrational mode is caused by the deprotonation of the tyrosine phenol group promoted by binding of Mg²⁺. Upon subsequent addition of glutathione (GSH), the Mg²⁺/H-PGDS solution showed the Tyr8 Raman band shifted to 1611 cm⁻¹, which is 11 cm⁻¹ higher than the frequency of the Mg²⁺ complex of H-PGDS, but 4 cm⁻¹ lower than the Mg²⁺ free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg²⁺ is re-protonated by the abstraction of H⁺ from the thiol group of GSH, and that the re-protonated Tyr8 species forms a hydrogen bond with the thiolate anion of GSH. Density functional theory calculations on several model complexes of p-cresol were also performed, which suggested that the pK_a and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue.