Studies in metachromatic leukodystrophy: XV. Purification of normal and mutant arylsulfatase A from human liver

In this report we describe a method to purify both normal and abnormal (inactive) arylsulfatase A. The abnormal enzyme protein was isolated both from cases of late infantile and early juvenile forms of metachromatic leukodystrophy. Conventional protein isolation methods reported earlier were followed by size exclusion high-performance liquid chromatography in the final purification stages. Both the mutant enzyme and the normal enzyme had the same HPLC elution behavior. They thus appeared to self-associate in a similar pH-dependent fashion. Both could be followed by their reaction to a rabbit antibody to normal human arylsulfatase A. The amount of homogenous protein obtained from about 500 grams of liver was 300-400 micrograms.     

Kinetic differences at low temperatures between R and T state carbon monoxide-carp hemoglobin.

We use the low-temperature recombination kinetics of carbon monoxide with carp hemoglobin to determine that the R and T states of hemoglobin exhibit different low-temperature geminate recombination kinetics. The peak of the fitted Gaussian activation energy spectrum is at 1.5 kcal/mol for R state and 1.8 kcal/mol for T state. The distribution in activation energies is fit well by the Agmon-Hopfield linear strain model. The T state is fit with a stronger elastic constant than R, and has a larger displacement of the protein conformation coordinate than does the R state, indicating that the T state does have a significantly greater rigidity and also stores more strain energy in its conformational states than does R hemoglobin. The pre-exponential in the activation energy spectrum is only a factor of two greater in the R than the T state and the low-temperature activation energy spectrum does not correctly predict the difference in the on rates for R and T states at 300 degrees K, indicating that processes removed from the binding site are important in cooperativity.     

Characterization of distinct tyrosine-specific protein kinases in B and T lymphocytes

Lymphocyte membrane fractions from both normal and neoplastic sources exhibit tyrosine-specific protein kinase activity. The molecular weights of the endogenous substrates phosphorylated on tyrosine residues differ in B and T cells. To further characterize membrane tyrosine phosphorylation in the two major classes of lymphocytes, the tryptic phosphopeptides of their endogenous substrates were compared and the sensitivity of the kinases to inhibition by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) was determined. The two major B cell substrates (61,000 and 55,000 daltons, p61 and p55) were gel purified after phosphorylation and exhaustively digested with trypsin. Separation by reverse phase high pressure liquid chromatography demonstrated that these two substrates had two identical phosphotyrosine containing tryptic phosphopeptides. p61 had an additional phosphotyrosine site. Parallel analysis of the two T cell substrates (64,000 and 58,000 daltons, p64 and p58) showed that they also contained two phosphotyrosine sites that were identical. However, the tryptic phosphopeptides from the B and T cell substrate pairs were clearly distinct suggesting that they arise from different gene products. When B and T cell membrane fractions were preincubated with TLCK (21 degrees C, 30 min) a dose-dependent decrease in p64 and p58 phosphorylation resulted. p61 and p55 phosphorylation was not affected at concentrations up to 10 mM TLCK. Tyrosine-specific kinase activity was also assessed by measuring phosphorylation of a tyrosine containing synthetic peptide. The kinase activity of T cell plasma membrane fractions was inhibited by TLCK; the B cell activity was unaffected. The results suggest that membrane fractions from normal and some neoplastic B and T cells have at least two different tyrosine-specific kinases.     

The activity of neutral ribonucleases in nuclei of rat sympathetic ganglia and effects of nerve injury

Nuclei were isolated from homogenates of rat superior cervical ganglion by a conventional differential centrifugation technique with approximately 60% recovery. Ribonuclease activity at pH 7.1 (neutral ribonuclease) was associated with the nuclei fraction and represented 19% of the overall activity in normal ganglia. Ribonuclease in the nuclei fraction was stimulated variably by the sulfhydryl blocker N-ethylmaleimide indicating that a proportion was bound to the endogenous ribonuclease inhibitor present in these ganglia. The total activity of nuclear ribonuclease was increased 2–6 days after postaganglionic nerve injury, such that the inhibitor-bound form of the enzyme increased maximally by 600% at day 4. The percentage of the total ganglionic activity in the nuclei fraction decreased in injured ganglia as a result of a rise in the activity of non-nuclear components. The changes in nuclear ribonuclease activity were distinct from those in the 850g supernatant indicating that specific nuclear enzymes are being affected during regeneration.Special Issue dedicated to Dr. O. H. Lowry.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation-Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

DEVELOPMENT OF A BIMODAL HEART RATE PATTERN IN UNDISTURBED FETAL HARBOR SEALS

Fetal and maternal heart rates were studied in unrestrained, pregnant harbor seals during the last third of gestation. Heart rates were recorded while the mothers were resting on land or performing trained simulated dives of up to 2.25 min. Data from resting mothers showed the development of a bimodal or two-speed fetal heart rate pattern during late gestation. The mean faster and slower fetal heart rates at term were 125 ± 3.7 and 79 ± 3.1 (mean + SEM) respectively. The amount of fetal bradycardia observed increased steadily towards term, and fetal heart rate changes were not correlated with changes in maternal heart rate or maternal respiration. The bimodal fetal heart rate was also seen during the simulated dives, and no decrease in either the faster or slower heart rate was found. Heart rates from resting, unrestrained harbor seal pups were also studied. The pups displayed a bimodal heart rate similar to the fetuses' with the slower rate occurring during breath-holds. The bradycardia in the pups was equivalent to the slower fetal heart rate. These findings suggest that the regulatory mechanism that determines the apneic bradycardia in young harbor seals during non-stressful conditions develops in the last quarter of gestation.     

Convolvulus grandiflorus Jacq. (Convolvulaceae) re-examined

Convolvulus grandiflorus Jacq. has been treated as a synonym ofOperculina ventricosa andStictocardia tiliifolia. It is here accepted as identifiable withIpomoea (sect.Calonyction)macrantha which is the correct name for Jacquin’s taxon. Full synonymies ofIpomoea macrantha andOperculina ventricosa are given, along with a comment about a variant occurring in Micronesia.     

Seasonal variations in the density of a small West African lake

The seasonal variation in the vertical density profile of Lake Asejire waters was investigated from February 1974 to February 1976.The range of density was of the magnitude of 2.0 mg/cc, at the temperatures of Lake Asejire (21°C–32°C). Water densities were generally lower in the dry season when aquatic temperatures were high and generally higher in the rainy season when aquatic temperatures were low.During the dry season, the pycnocline was observed at 3m–7m in 1974 and 6 m–9 m in 1975. During periods of density stratification, the water body was clearly layered.Seasonal longitudinal differences in water density were such that during the floods of 1975 the inflow station waters had a higher density than the dam-site waters which however had a concomitantly higher conductivity. This suggests the insignificance of conductivity in water density changes in a lake with rather low conductivity values.     

Developmental dissociation of myelin synthesis and “myelin-associated” enzyme activities in the shiverer mouse

Developmental changes in three enzymes associated with myelin lipids were studied in the shiverer mouse, a murine mutant showing a severe deficiency of CNS myelin. Age-related changes in cerebroside sulfotransferase (measured in brain) and arylsulfatase A and cerebroside B-galactosidase (measured in brain and liver) were the same for shiverer and control mice. The shiverer mouse, therefore, demonstrates a dissociation between the genetic mechanisms regulating myelination in the CNS and developmental changes in enzyme activities thought to be closely related to the synthesis of myelin. In addition, we found no defect in the shiverer mouse in the incorporation of glycine-labeled basic protein into CNS myelin, indicating an important metabolic difference between the morphologically similar shiverer and quaking mutants.     

Numerical taxonomy and ecology of petroleum-degrading bacteria.

A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 of the petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.