Characterization of the phospholipid and fatty acid composition of Sendai virus

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.     

The thymic microenvironment. Characterization of in vitro differentiation of the IT26R21 rat thymic epithelial cell line

We have previously postulated an in vivo pathway of thymic epithelial (TE) cell maturation in pre- and postnatal thymus, whereby endocrine medullary TE cells terminally differentiate to form Hassall's bodies. Epithelial-cell differentiation has been well documented in vitro using epidermal keratinocytes. Therefore, to characterize TE-cell differentiation in vitro, we observed clones of the rat TE cell line, IT26R21, after 4 and 14 days in culture. We found alterations in cell morphology, the cessation of cell proliferation, and the acquisition of a differentiation antigen defined by monoclonal antibody TE-19 (a marker of terminally differentiated epithelial cells). At light and electron microscopy, we detected progressive TE-cell stratification and squamous-cell formation between 4 and 14 days of culture. Autoradiography on day 14 showed that squamous TE cells in stratified layers did not incorporate tritiated thymidine, while surrounding smaller cells adhering to the substratum continued to synthesize DNA. At indirect immunofluorescence, only 3% of cells reacted with monoclonal antibody TE-19 at day 4, while on day 14, 22% of the TE cells were TE-19 positive (P less than 0.02). Antibody-TE-19 reactivity was limited to stratified, squamous TE cells. Additionally, we isolated a clone of the IT26R21 cell line that did not undergo these changes characteristic of TE cell differentiation. We conclude that IT26R21 TE cells are capable of undergoing programs of both terminal differentiation and cell renewal in vitro.     

Membrane peptidases in the pig choroid plexus and on other cell surfaces in contact with the cerebrospinal fluid.

A comprehensive survey of 11 peptidases, all of which are markers for renal microvillar membranes, has been made in membrane fractions prepared from pig choroid plexus. Two fractionation schemes were explored, both depending on a MgCl2-precipitation step, the preferred one having advantages in speed and yield of the activities. The specific activities of the peptidases in the choroid-plexus membranes were, with the exception of carboxypeptidase M, lower than in renal microvillar membranes: those of aminopeptidase N, peptidyl dipeptidase A ('angiotensin-converting enzyme') and gamma-glutamyltransferase were 3-5-fold lower, those of aminopeptidase A and endopeptidase-24.11 were 12-15 fold lower, and those of dipeptidyl peptidase IV and aminopeptidase W were 50-70-fold lower. Carboxypeptidase M had a similar activity in both membranes. Alkaline phosphatase and (Na+ + K+)-activated ATPase were more active in the choroid-plexus membranes. No activity for microsomal dipeptidase, aminopeptidase P and carboxypeptidase P could be detected. Six of the peptidases and (Na+ + K+)-activated ATPase were also studied by immunoperoxidase histochemistry at light- and electron-microscopic levels. Endopeptidase-24.11 and (Na+ + K+)-activated ATPase were uniquely located on the brush border, and the other two peptidases appeared to be much more abundant on the endothelial lining of microvessels. Dipeptidyl peptidase IV and aminopeptidase W were also detected in microvasculature. Pial membranes associated with the brain and spinal cord also stained positively for endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A. The immunohistochemical studies indicated the subcellular fractionation did not discriminate between membranes derived from epithelial cells (i.e. microvilli) and those from endothelial cells. The possible significance of these studies in relation to neuropeptide metabolism and the control of cerebrospinal fluid production is discussed.     

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.     

Effect of winter high temperatures on reproduction and circannual rhythms in hibernating ground squirrels

We tested whether prevention of hibernation in ground squirrels by midwinter exposure to high ambient temperatures influenced timing of the spring phase of reproductive maturation and the phase and period of subsequent circannual rhythms of reproduction and body mass. Exposing hibernating adult male Spermophilus lateralis to 30 degrees C for 6 weeks beginning December 4 advanced the timing of testicular recrudescence by 4-5 weeks, compared to controls left at 4 degrees C. Males exposed to 30 degrees C for 6 weeks beginning at the average time of spontaneous end of hibernation (January 15) reached reproductive maturation at a time intermediate to those of controls and of the December 4 experimental group. However, neither the date of the subsequent fall's body mass peak, the date of the next year's reproductive maturation, nor the periods of circannual rhythms of body mass and reproduction differed among groups. Premature interruption of hibernation appears to allow early expression of reproduction, but does not affect the underlying timing mechanism.     

Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96h, and sometimes at 120h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.     

Biomechanical basis of optimal scoliosis surgical correction

For an optimal approach to surgical correction of scoliosis, it was deemed desirable to biomechanically simulate the set of corrective forces applied by alternative internal fixation systems, so as to determine and apply the internal fixation system producing the best correction under safe levels of forces applied by the fixation systems to the spinal structures. To this end, we have developed, and presented here, (1) a spinal finite-element model relating the applied corrective forces to the corrected spinal configurations, (2) a method for determining the stiffness of the patient's spine prior to surgery, (3) computerized finite-element analysis simulation of alternative internal correction-fixation systems, so as to determine the most efficacious system, (4) instrumentations for surgically implementing the recommendations of the surgical simulation analysis and (5) comparisons of the model-simulated and surgically-obtained corrected spinal configurations. These procedures together constitute the biomechanical foundations of scoliosis surgical correction.