Cells of origin of the hippocampal afferent projection from the nucleus reuniens thalami

Summary Neurons of the nucleus reuniens thalami stained with Golgi methods are compared to cells in this nucleus labelled in retrograde fashion after hippocampal injections of horseradish peroxidase. The cellular morphology ranges from fusiform to multiangular with most cells showing radiating processes characteristic of neurons in the reticular core. Dendrites are long and relatively smooth, with a few sparsely distributed spinous processes. These cells are comparable to the cholinergic cells of the median septal/diagonal band area which also project into the hippocampal formation.We would like to thank Mr. Al Cibivicious for his excellent technical assistance. This research was supported in part by general research funds awarded to R.H.B. by East Tennessee State University     

Refined structures of oxidized flavodoxin from Anacystis nidulans

Flavodoxin from Anacystis nidulans (Synechococcus PCC 7942) was the first member of the flavodoxin family to be characterized, and is the structural prototype for the "long-chain" flavodoxins that have molecular masses of approximately 20 kDa. Crystal structure analyses and refinements of three orthorhombic forms of oxidized A. nidulans flavodoxin are reported, and salient features of the fold and the FMN binding site are compared with other flavodoxins. The structure of form I (wild-type: P212121, a=57.08 A, b=69.24 A, c=45.55 A), determined initially by multiple isomorphous replacement, has been refined to R=0.183 and R(free)=0.211 for data from 10.0 to 1.7 A resolution. Structures of form II (wild-type: P212121, a=60.05 A, b=65.85 A, c=51.36 A) and form III (Asn58Gly: P212121, a=51.30 A, b=59.15 A, c=94.44 A) have been determined by molecular replacement and refined versus data to 2.0 A and 1.85 A, respectively; the R values for forms II and III are 0.147 and 0.150. Changes in the molecular contacts that produce the alternative packings in these crystalline forms are analyzed. Deletion of the Asn side-chain in the mutant Asn58Gly removes an intermolecular stacking interaction and allows the alternative packing found in form III crystals. The functionally important 50's loop of the FMN binding site is less restrained by intermolecular contacts in these crystals but maintains the same conformation as in oxidized wild type protein. The structures reported here provide the starting point for structure-function studies of the reduced states and of mutants, described in the accompanying paper.     

Alterations in cellular gene expression without changes in nuclear matrix protein content

Cell metabolism and function are modulated in part by cell and nuclear shape. Nuclear shape is controlled by the nuclear matrix, the RNA-protein skeleton the nucleus, and its interactions with cytoskeletal systems such as intermediate filaments and actin microfilaments. The nuclear matrix plays an important role in cell function and gene expression because active genes are bound to the nuclear matrix whereas inactive genes are not. It is unknown, however, how genes move on and off the matrix, and whether these events require compositional protein changes, i.e., alterations in protein content of the nuclear matrix, or other, more subtle alterations and/or modificatins. The purpose of this investigation was to begin to determine how nuclear matrix protein composition is related to gene expression. We demonstrate that gene expression can change without apparent changes in the protein composition of the nuclear matrix in MCF10A breast epithelial cells.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

High-resolution X-ray diffraction study of the complex between endothiapepsin and an oligopeptide inhibitor: the analysis of the inhibitor binding and description of the rigid body shift in the enzyme.

The conformation of the synthetic renin inhibitor CP-69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP-69,799 is an oligopeptide transition--state analogue inhibitor that contains a new dipeptide isostere at the P1-P1' position. This dipeptide isostere is a nitrogen analogue of the well-explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1' C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3' pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3'. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.     

In vitro correlation of ultrastructural morphology and creatine phosphokinase release in L6 skeletal muscle cells after exposure to parenteral antibiotics

Summary Morphologic changes in a rat skeletal muscle cell line (L6) exposed for 1 h to the parenteral antibiotics amphotericin B (AMP), tetracycline-HCl (TET), erythromycin lactobionate (ERY), and cephaloridine (CEP) were characterized by transmission and scanning electron microscopy and compared to cellular release of creatine phosphokinase (CRK). AMP (0.05, 0.1, 0.5 mg/ml) caused a concentration-related swelling of nuclei, endoplasmic reticulum, and mitochondria. Loss of membrane integrity associated with AMP exposure was evident at the middle concentration and extensive at the high concentration, which correlated well with the 43 and 90% depletion of CPK from the muscle cells, respectively. TET (0.25, 1.0, 2.5 mg/ml) caused dilation of endoplasmic reticulum and cytoplasmic blebbing at the low concentration but had no effect on the cytoplasmic membrane or CPK. Cells exposed to the high concentration of TET had extensive damage to the cytoplasmic membrane, and CPK was completely depleted. ERY (2.5, 5.0, 25 mg/ml) caused a pattern of morphologic changes and CPK depletion similar to TET. CEP (4.0, 20, 50 mg/ml) had no effect on membrane integrity or CPK; however, membranous whorls were prominent in the cytoplasm. A good correlation between CPK release and cytoplasmic membrane integrity was evident and the ability of these agents to release CPK from muscle cells in culture correlated with the known irritancy potential of these parenteral antibiotics. Furthermore, CPK depletion seems to be a reliable indicator of muscle cell damage after cytoplasmic membrane perturbation and is therefore an appropriate index of toxicity in this in vitro muscle irritation model.     

In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability. Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.