Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation

Sialyltransferase (Galβ1,4GlcNAc α2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.     

Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands.

Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.     

Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E

We previouslyshowed that increased macrophage andPGE₂ production with age is due toenhanced cyclooxygenase (COX) activity and COX-2 expression. This studydetermined the effect of vitamin E supplementation on macrophagePGE₂ synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts permillion vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly morePGE₂ than those from young mice.Vitamin E supplementation reversed the increasedPGE₂ production in old mice buthad no effect on macrophage PGE₂production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in youngmice at all intervals. Vitamin E supplementation completely reversedthe increased COX activity in old mice to levels comparable to those ofyoung mice but had no effect on macrophage COX activity of young miceor on COX-1 and COX-2 protein or COX-2 mRNA expression in young or oldmice. Thus vitamin E reverses the age-associated increase in macrophagePGE₂ production and COX activity.Vitamin E exerts its effect posttranslationally, by inhibiting COXactivity.

     

Command-related distribution of regional cerebral blood flow during attempted handgrip.

To localize a central nervous feed-forward mechanism involved in cardiovascular regulation during exercise, brain activation patterns were measured in eight subjects by employing positron emission tomography and oxygen-15-labeled water. Scans were performed at rest and during rhythmic handgrip before and after axillary blockade with bupivacaine. After the blockade, handgrip strength was reduced to 25% (range 0-50%) of control values, whereas handgrip-induced heart rate and blood pressure increases were unaffected (13 +/- 3 beats/min and 12 +/- 5 mmHg, respectively; means +/- SE). Before regional anesthesia, handgrip caused increased activation in the contralateral sensory motor area, the supplementary motor area, and the ipsilateral cerebellum. We found no evidence for changes in the activation pattern due to an interaction between handgrip and regional anesthesia. This was true for both the blocked and unblocked arm. It remains unclear whether the activated areas are responsible for the increase in cardiovascular variables, but neural feedback from the contracting muscles was not necessary for the activation in the mentioned areas during rhythmic handgrip.     

The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.     

Changes in Muscle Volume and Composition After Treatment of Hip Dysplasia with Periacetabular Osteotomy

BackgroundPeriacetabular osteotomy (PAO) is a common treatment for pre-arthritic hip dysplasia in young adults. The purpose of this study was to better understand changes in muscle volume and composition after PAO visualized using magnetic resonance imaging (MRI).MethodsA prospectively collected series of individuals that underwent PAO for hip dysplasia were reviewed to identify subjects with pre- and postoperative MRI. In our practice, MRI was obtained preoperatively and greater than 6 months after PAO for persistent hip pain. MRI sequences were selected to optimize visualization of the muscle volume, fatty infiltration, and hip joint cartilage. MRI images were selected at predetermined bony landmarks and analyzed using 3D Slicer (©2021, www.slicer.org) software to measure muscle diameter and calculate muscle cross-sectional area (CSA) in 17 individual muscles surrounding the hip. Muscle atrophy was graded using the Goutallier classification for fatty infiltration and acetabular cartilage condition was graded using the Outerbridge classification. We compared pre- and postoperative muscle area and composition as well as cartilage for each case.ResultsA series of six female patients met our inclusion criteria. Mean age was 26 years at time of surgery. All cases had MRI sequences adequate for muscle volume measurements. Fatty infiltration and cartilage changes were recorded in four subjects with appropriate MRI sequences. Separating muscle groups, external rotators underwent the largest volume increase. Hip flexors demonstrated mild volume decrease. CSA change among external rotators averaged +12%, hip flexors -9.3%, and hip abductors -9.2% after PAO. All muscles had either the same or increased fatty infiltration after surgery, with gluteus medius and iliacus undergoing the most average increase. Similarly, cartilage condition worsened by a small margin in this series.ConclusionOur results provide preliminary indication that PAO may have noticeable effects on muscle characteristics and cartilage in the early postoperative period. This was a limited case series of subjects with adequate pre- and post-operative MRI imaging.Level of Evidence: IV