Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.     

The influence of magnesium on calcium-activated, vascular smooth muscle actomyosin ATPase activity

Histamine activation of adenylyl cyclase activity in sonicated enriched rat gastric parietal cells showed a time, temperature, and concentration dependence upon guanine diphosphoimide (Gpp(NH)p). Enzyme activation was first order with Gpp(NH)p alone or Gpp(NH)p plus histamine. The K_a for Gpp(NH)p was ~2 μm and was not influenced by histamine. GTP and GDP were inactive alone or with histamine and were competitive with Gpp(NH)p, showing apparent K_i's of near 0.4 and 0.3 μm, respectively. In the presence of Gpp(NH)p, parietal cell adenylyl cyclase was activated by histamine with an EC₅₀ of 24 μm, the most potent in a series of histamine analogs, further substantiating an H₂-receptor classification for this response. H₂-Receptor antagonists were competitive inhibitors with submicromolar K_i's. Preincubation of parietal cells with histamine and Gpp(NH)p resulted in adenylyl cyclase activity up to 15 times the basal level. The activated state was retained after washing the cells free of histamine and Gpp(NH)p and was not reversed by the subsequent addition of either histamine, cimetidine, or GTP. The other gastric acid secretagogues, pentagastrin and carbamylcholine, were without effect upon histamine activation or the activated state of adenylyl cyclase. These results describe a level of control of histamine-sensitive adenylyl cyclase that requires consideration in the activation of the parietal cell H₂-receptor system by histamine to modulate acid secretion.     

A systems-theory approach to the analysis of multiexponential fluorescence decay.

A mathematical model of the fluorescence decay experiment based on linear systems theory is presented. The model suggests an experimental technique that increases the probability of correctly determining the decay constants of a multicomponent system. The use of moment methods for data analysis improves accuracy by combining information obtained from several discrete experiments. Examples are presented to show that the analysis of a three component system composed of known standards is improved as the number of experimental determinations is increased from one to four. The discrete measurements are made by changing the excitation and emission wavelengths.     

The migration of lymphocytes across specialized vascular endothelium. I. The entry of lymphocytes into the isolated mesenteric lymph-node of the rat.

The chain of lymph-nodes in the rat mesentery was isolated and the preparation was perfused via cannulae in the superior mesenteric vessels. The perfusate consisted of serum to which labelled lymphocytes had usually been added. The entry of radioactively labelled lymphocytes from the blood vessels into the lymph-nodes was studied by scintillation counting and autoradiography. It was observed that: (1) In the perfused node labelled lymphocytes localized in and around post-capillary venules in the paracortex as they do early after i.v. injection. (2) The number of lymphocytes which entered the node was directly proportional to the concentration in the perfusate over the range studied. The proportion of cells retained in the node varied considerably around a mean of 11% of lymphocytes reaching it. (3) The isolated lymph-node released few if any lymphocytes into the effluent (venous) perfusate. (4) Large lymphocytes migrated into isolated lymph-nodes slightly more readliy than did small lymphocytes. (5) Unlike intact cells isolated lymphocyte membranes did not adhere to specialized vascular endothelium.     

THE MIGRATION OF LYMPHOCYTES ACROSS SPECIALIZED VASCULAR ENDOTHELIUM

Lymphocytes were exposed in vitro to either trypsin or neuraminidase. The ability of the treated cells to migrate into tissues was measured (a) by i.v. injection into intact recipients and (b) by vascular perfusion through an isolated lymph-node preparation. The localization of trypsinized cells in the lymph-nodes of recipients was deficient when compared to untreated lymphocytes and there was a surplus of trypsinized cells in the blood. Trypsinized cells migrated into the isolated nodes in reduced numbers. By contrast, neuraminidase treated lymphocytes were markedly deficient in the blood of recipients early after injection; their localization in the spleen and lymph-nodes was also deficient but they were in surplus in the liver. Moreover they migrated into the isolated nodes in slightly increased numbers. By 24 hr after injection the perturbed localization pattern produced by either enzyme was partly restored to normal. In conclusion, trypsin interfered with the capacity of lymphocytes to migrate into lymph-nodes but neuraminidase did not; the latter promoted the hepatic sequestration of cells and the reduced localization in the blood and tissues was a consequence of this. The hypothesis that lymphocytes adhere to specialized endothelia in lymph-nodes because of specific glycoside sequences on their surface lacks experimental support.     

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes

Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M₄ lactate dehydrogenase structures. One of these is the 2.0 Å resolution apoenzyme structure; the other is a 3.0 Å resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase²: NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.     

KINETIC-MICROARCHITECTURAL CORRELATIONS IN THE BONE MARROW OF THE MOUSE

Transverse histologic sections of bone marrow obtained from mice that were sacrificed by perfusion fixation at intervals following tritiated thymidine injection were studied by means of radioautography. A kinetic gradient was demonstrated across the marrow section, with the highest proliferative rate in the subendosteal region. Megakaryocytes were shown to originate from the rapidly proliferating subendosteal cells. The immediate proliferating precursors of mature granulocytes were slowly proliferating cells found predominantly in the central region of the marrow. It was concluded that in the steady state there must be a migration of cells from the subendosteal region to the central region with concomitant growth retardation of the migrating cells.     

Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex

Human leukemic cells corresponding to the earliest identifiable stages of intrathymic T cell differentiation lack cell surface expression of the T cell receptor(TCR alpha/beta)-T3 complex but transcribe TCR beta mRNA from either germ-line configuration (1/13) or partially (DJ) or fully (VDJ) rearranged (12/13) genes. These cells do not produce TCR alpha mRNA, but do contain T3 delta and T3 epsilon mRNA and accumulate T3 polypeptides, primarily in the perinuclear envelope. Equivalent normal T cells isolated from thymus have a predominantly germ-line configuration of TCR beta but contain intracellular T3 proteins. T3 gene expression is therefore a very early event in T cell differentiation. TCR alpha chain production appears to be the limiting maturation-linked event in the transport, assembly, and cell surface membrane insertion of the TCR alpha/beta-T3 complex.