Computed tomography and automated image analysis of prehistoric femora

Non-invasive characterization of limb bone cross-sectional geometry would be useful for biomechanical analyses of skeletal collections. Computed tomography (CT) is potentially the method of choice. Additionally, CT images are suitable for automated analysis. CT is here shown to be both accurate and precise in the analysis of cross-sectional geometry of prehistoric femora. Beam hardening artifacts can be reduced by using a water bath. As the availability of CT for research increases, both bone density and geometry could be determined simultaneously with this method.     

Enzymes of gluconate metabolism and glycolysis inPenicillium notatum

In addition to the ability of Penicillium notatum to grow on sucrose, glucose, fructose and gluconate, substantial growth occurred on 2-ketogluconate and 5-ketogluconate thereby indicating a diverse sugar metabolism. Cell-free extracts contained all the enzymes of the Embden-Meyerhof-Parnas pathway and for both oxidative and non-oxidative pentose phosphate metabolism. Despite inconsistencies in results between different assay methods for the conventional Entner-Doudoroff (ED) enzymes, the data indicated the route was enzymatically possible. Demonstrations of the activities of the enzymes of the non-phosphorylative equivalent of the ED pathway were achieved. No evidence was found of a phosphorylative linking enzyme between the two pathways. Both 2- and 5-ketogluconate reductases were detected along with gluconate dehydrogenase which suggested interconvertibility between the ketogluconates and gluconate. However, ketogluconokinase, responsible for the conversion of ketogluconate to 2-keto-6-phosphogluconate, was not detected. A scheme for the inter-relationships of routes of gluconate metabolism is discussed.     

Correlation of pulmonary ACE activity and capillary surface area during postnatal development

Although a considerable amount of information is available regarding the remodeling and growth of the pulmonary arterial circulation, relatively little is known regarding postnatal development of the pulmonary microcirculation. We hypothesized that the maximal velocity (Vmax) of pulmonary angiotensin-converting enzyme (ACE) activity, measured from indicator-dilution outflow curves using a synthetic substrate, 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), is directly related to the capillary endothelial cell surface area in the lungs of developing lambs. Accordingly we measured apparent kinetics of pulmonary ACE activity in 22 anesthetized ventilated lambs (2-171 days old) and compared our functional assessment to simultaneous in vivo determinations of CO diffusing capacity (DLCO) and postmortem structural assessment of alveolar septal dimensions using stereology and electron microscopy. There was a progressive increase in Vmax of ACE in this age group, with little change in apparent affinity for BPAP. Similar functional manifestation of growth was noted by an age-dependent increase in DLCO. Neither Vmax nor DLCO was significantly affected by an increase in left atrial pressure to 19 Torr (via inflation of a balloon in the left atrium), suggesting little recruitment of vessels under conditions of the present protocol. A close correlation was observed when either Vmax for ACE activity or DLCO was plotted vs. capillary endothelial cell surface area. Double logarithmic transformation of capillary endothelial cell surface area, Vmax-ACE and DLCO vs. lung volume revealed power functions with slopes all greater than that predicted from isotropic growth, suggesting selective differential postnatal development of the endothelium of the alveolar septum in lambs from 2-171 days of age.     

Lipolysis of serum-activated triacylglycerol at the surface of J774.1 macrophages

Summary Cultured mouse (J774.1) macrophages accumulated triacylglycerol, but no cholesteryl ester or cholesterol, when incubated in albumin-poor medium with serum-activated lipid particles containing 84 mol% trioleoylglycerol and 9 mol% cholesteryl oleate. Accumulation of triacylglycerol by cells was associated with hydrolysis of particulate triacylglycerol to fatty acid and glycerol. Both acyl and glyceryl moieties of particulate triacylglycerol were recovered in cellular triacylglycerol with a molar ratio of 3.6. The cells also accumulated fatty acid and monoacylglycerol. Whether acylglycerol was taken up as a single molecular species, such as monoacylglycerol, or as several species can not be determined by the present findings. Macrophages incubated with lipid particles for 24 h had many lipid particles attached to cell surfaces and numerous intracellular lipid droplets. The surface film of attached particles was continuous with the outer leaflet of plasma membrane of the cells. Particles partially depleted of core triacylglycerol and collapsed surface films were found attached to surfaces of macrophages. There was no morphological evidence that lipid particles were taken up intact by cells, through endocytosis or phagocytosis. Macrophages incubated with lipid particles also contained intracellular lamellar structures. They varied in size and shape, and were located in the periphery of cells, sometimes near lipid droplets and endoplasmic reticulum. Only 3% of the lamellar structures were associated with lysosomes, indicating they probably were not of lysosomal origin. Lipid particles attached to cells decreased in size and number, and lamellar structures developed at the surface of particles, or replaced the particles, when glutaraldehyde-fixed specimens were incubated at 25° C, demonstrating lipolytic activity at the surface of macrophages. Our findings suggest that particulate triacylglycerol was hydrolyzed by lipoprotein lipase at the surface of macrophages, and that fatty acid and monoacylglycerol formed by lipolysis were transported directly into the cells to be reesterified. When lipolytic products were taken up faster than they could be utilized, they accumulated as lamellar structures in the cells.Abbreviations MEM Eagle's alpha modification of minimum essential medium     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Electrostatic potentials in membrane systems

A membrane with an arbitrary distribution of fixed charges inside and on its surfaces is considered. A procedure for calculating the local electrostatic potential at an arbitrary point of the system is described and its validity discussed. This procedure is based on the linearization of the 3-dimensional Poisson-Boltzmann equation around an exact 1-dimensional solution.     

LIPOFUSCIN (AGING) PIGMENT GRANULES OF THE NEWBORN HUMAN LIVER

We have observed pigmented cytoplasmic granules, with the characteristic staining properties of lipofuscin (ceroid, "wear-and-tear") pigment, in newborn human liver. The pigment is found at the periphery of the lobule in hepatocytes and some bile ductular cells. It is acid-fast, PAS-positive after diastase digestion, slightly argyophilic and sudanophilic, and markedly Schmorl's- and peroxidase positive in paraffin sections. Difficult to see in sections stained with hematoxylin and eosin, the pigment can be detected in unstained sections. The granules also resemble lipofuscin found in adult tissues, in their ultra-structural and enzymatic properties. They are polymorphic, contain granular material of moderate and high electron opacity, and are delimited by a single membrane. Acid phosphatase and β-glucuronidase activities are visualized in the newborn granules, identifying them as lysosomes. The granules also contain copper and, to a much lesser extent, iron. The accumulation of lipofuscin pigment in lysosomes in many tissues correlates well with aging, and this process has been interpreted as a reflection of cellular degeneration or wear-and-tear. However, the presence of lipofuscin granules as a constant component of neonatal liver suggests that they are not a measure of cellular senescence.     

Microbodies (peroxisomes) in the toad,Bufo marinus

Summary Microbodies (peroxisomes), a group of cytoplasmic organelles enriched in catalase, are demonstrated in the toad, Bufo marinus, by light and electron microscopy by means of a cytochemical staining procedure that demonstrates the peroxidatic activity of catalase with diaminobenzidine (DAB). Amphibian microbodies are similar to those of other classes in their fine structure and localization in hepatocytes and kidney, where they are prominent in the proximal tubular cells. Nucleoids are present only in renal microbodies. In the proximal renal tubule an unusual group of large brown granules are identified as lysosomes by their acid phosphatase, -glucosaminidase and -glucuronidase activities.This work was supported by U.S. Public Health Service Grants Nos. NS-06856 and HD 00674. We wish to thank Dr. Richard M. Hays who generously supplied us with toads; Dr. Alex B. Novikoff for making available facilities for ultramicrotomy, Miss Betty De Prest for technical assistance; Miss Marianne Van Hooren for preparation of the photomicrographs.