Cdk inhibitory nucleoside analogs prevent transcription from viral genomes.

Targeting viral proteins has lead to many successful antivirals. Yet, such antivirals rapidly select for resistance, tend to be active against only a few related viruses, and require previous characterization of the target proteins. Alternatively, antivirals may be targeted to cellular proteins. Replication of many viruses requires cellular CDKs and pharmacological CDK inhibitors (PCIs), such as the purine-based roscovitine (Rosco), are proving safe in clinical trials against cancer. Rosco inhibits replication of wild-type or (multi-)drug resistant HIV, HCMV, EBV, VZV, and HSV-1 and 2. However, the antiviral mechanisms of purine PCIs remain unknown. Our objective is to characterize these mechanisms using HSV as a model We have shown that Rosco prevents initiation of transcription from viral, but not cellular, genomes. This inhibition is promoter independent, but genome dependent, and requires no viral proteins. This is a novel antiviral mechanism and a previously unknown activity for purine PCIs.

Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.

Cluster of differentiation 1 (CD1) antigens are a family of non-MHC but Class I-like molecules that have been identified in humans and rodents. Although their function(s) remains unknown, it has been proposed that CD1 may present antigens to specific subsets of peripheral T-cells. We now provide evidence in support of this hypothesis through the demonstration by in situ hybridization that Paneth cells of the mouse intestine express CD1 mRNA. These cells are thought to be involved in the immunological regulation of intestinal flora and could accomplish this task through interactions with intestinal intraepithelial lymphocytes. The expression and localization of CD1 mRNA was confirmed by both autoradiographic and non-isotopic techniques. The relevance of these results to CD1 function as well as to Paneth cell biology is discussed.

Transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine during storage of human platelets for 5 days.

Human platelets are routinely stored for 5 days prior to transfusion, but they deteriorate during storage. Since very little information is available concerning the effect of storage on platelet phospholipid metabolism, the biosynthesis and remodelling of platelet phospholipids were studied. Platelets were incubated separately with [14C]glycerol, [14C]arachidonic acid, or a mixture of [14C]glycerol and [3H]arachidonic acid, and stored in a platelet storage medium at 22 degrees C. Maximum glycerol uptake (20%) was attained after 6 h. [14C]Glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, and to a much lesser extent phosphatidylserine, under storage conditions for 5 days. The distribution of the initial arachidonic acid uptake was not as would be expected based on the molar composition of endogenous phospholipids. The arachidonic acid (75%) which was taken up within 10 min of incubation distributed 55% into the phosphatidylcholine and only 14% into the phosphatidylethanolamine; the molar composition is actually 18% phosphatidylcholine and 47% phosphatidylethanolamine. During storage, there was a continuous transfer of the radiolabelled arachidonic from phosphatidylcholine to phosphatidylethanolamine until, after 5 days, the distribution of arachidonic acid was identical to the endogenous distribution. In contrast, no change in the glycerol incorporation pattern was detected during storage. This suggested that the mechanism for arachidonic acid redistribution was not through exchange of polar head groups, but through acyl transfer of arachidonic acid from phosphatidylcholine to phosphatidylethanolamine.

Cultured juxtaglomerular cells: production and localization of renin.

Trypsin- or collagenase-dispersed renal cortical cells from newborn mice were filtered and cultured. The cultures comprised 25% juxtaglomerular cells as identified by immunocytochemistry. Renin was measured by radioimmunoassay in the culture medium and in the cells at various time intervals. This in vitro system was responsive to isoproterenol, which stimulated renin release in a dose-dependent manner.

Immunocytochemical localization of renin in juxtaglomerular cells.

The involvement of various organelles in the synthesis, transport, and packaging of renin in the juxtaglomerular cells of newborn mice has been investigated by immunocytochemistry with the protein A-gold technique. Highly specific rabbit antibodies against mouse submandibular renin were used. Mild fixation and embedding in glycol methacrylate allowed enough sensitivity to identify a steep gradient of labeling from rough endoplasmic reticulum to Golgi complex to secretory granules. Routine fixation and embedding in Epon produced labeling differentials that allowed delineation of hitherto ill-defined types of secretory granules and vacuoles. The classical pattern of synthesis, transport, and packaging of secretory proteins involves the rough endoplasmic reticulum and Golgi complex and seems to apply to renin secretion. Immunoreactive renin is packaged as rhomboid crystals at the trans face of the Golgi complex. The limiting membrane of these rhomboids fuses to form coalescing protogranules where the crystals eventually yield their individuality maturing into secretory granules. Vacuoles containing a flocculent material, with or without a dense core, show significant immunocytochemical labeling. These vacuoles are not associated with the Golgi complex but occupy cytoplasmic areas well endowed with rough endoplasmic reticulum. As judged from their morphological features and their immunoreactivity, the vacuoles do not seem to follow the sequence of events typical of protogranules and coalescing protogranules. They possibly represent a parallel pathway of renin synthesis and transport, involving the nuclear envelope and bypassing the Golgi complex.

Ultrastructural immunocytochemical localization of renin and angiotensin II in the juxtaglomerular cells of the ischemic kidney in experimental renal hypertension.

Partial ligation of the rat aorta between the renal arteries induces acute hypertension with atrophy of the left (ischemic) kidney, intense stimulation of juxtaglomerular cell (JGC) secretory activity, and significant increases in renal cortical renin activity, in plasma renin activity, and in the plasma levels of angiotensin I (AI) and angiotensin II (AII). With the unlabeled antibody technique at the light-microscopic level and various dilutions of renin antiserum, immunoreactive renin can be visualized in the JGC of sham-operated controls with high dilutions of antiserum that do not reveal renin in the JGC of ischemic kidney. The reverse is true with AII antisera; ie, high dilutions of AII antisera immunostain the JGCs of ischemic kidney but not those of control kidney. With the protein A-gold technique at the electron-microscopic level, using gold particles of small and large size and immunoreacting the two faces of a fine section, renin and AII can be localized in the same JGC secretory granules. With the same technique (immunoreacting only one face of a fine section with small gold particles), quantitative analysis reveals a lower concentration of renin and a higher concentration of AII in the secretory granules of the ischemic kidney JGCs; these granules are also of smaller size than those of control kidney JGCs. AI cannot be visualized in these cells at either the light- or electron-microscopic level. These results indicate that AII co-localized with renin in JGC secretory granules and probably co-secreted, is not synthetized by these cells but is internalized following receptor binding.

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats. An ultrastructural radioautographic study.

Sections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine-3,5 3H or of L-fucose 3H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine 3H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4 h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4 h. After injection of L-fucose 3H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose 3H and tyrosine 3H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.

Ultrastructural cytochemistry of atrial and ventricular cardiocytes of the bullfrog (Rana catesbeiana). Relationship of specific granules with reninlike activity of the myocardium.

The ultrastructural cytochemical reactivity, renin activity, and cathepsin D activity of atria and ventricle of the bullfrog have been assessed. The specific granules (A, B, and D) were found to be argentaphobic when ultrathin sections of Araldite-embedded atria and ventricle were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of the specific granules was moderated positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate embedded atria and ventricle were stained by phosphotungstic acid at a low pH. A similar reaction was shown by the cell coat, intercalated discs, residual bodies (C granules), and Z discs as well as by a very small portion of the Golgi complex. Incubation of ultrathin sections of atria and ventricule fixed only in glutaraldehyde and embedded in glycol methacrylate with either pronase or trypsin resulted in selective digestion of specific granules and Z discs and, to a much lesser degree, of the cell coat. As cathepsin D activity and renin activity were present in both atria and ventricle, the generation of angiotensin I by these cardiocytes might have been due to either enzyme. Nevertheless, because of the glycoprotein nature of specific granules and of the endocrinelike ultrastructure of atrial and ventricular cardiocytes in the frog, the present results raise the possibility that specific granules may contain renin.

[Transhepatic percutaneous cholangiography: A comparative study]. / La cholangiographie transhépatique percutanée: une étude comparative.

A retrospective study of 75 cases (19 with the conventional method and 56 with the Chiba method) of percutaneous transhepatic cholangiography was done. The utilization of the Chiba method is associated with a higher success rate of opacification of the biliary tree: 97% versus 80% for dilated biliary ducts and 36% versus 0% for non-dilated biliary ducts. No serious complications were encountered with the Chiba method. With the utilization of the "skinny" Chiba needle, percutaneous transhepatic cholangiography has become a simple, safe, and efficient method in the investigation of hepatobiliary disorders.