Endogenous lipid pneumonia in opossums from Louisiana

Endogenous lipid pneumonia was present in 19 of 27 opossums (Didelphis virginiana) trapped in the vicinity of Baton Rouge, Louisiana. The severity of lesions varied from small pleural and subpleural aggregates of foamy macrophages with minimal disruption of pulmonary architecture to large nodular accumulations of foam cells with cholesterol clefts and localized emphysema. The cause of the lipid pneumonia may be related to pulmonary nematode parasite infections, which were evident in 13 of the affected animals.     

Analgesia induced by N-acetylserotonin in the central nervous system

The relationship between N-acetylserotonin (NAS) in the central nervous system (CNS) and responses to pain was investigated. Using the rat tail-flick model, we initially replicated the work of others showing that intraventricular (IVC) injection of a dipeptide structurally similar to both NAS and serotonin was capable of inducing analgesia in the rat. We then showed that IVC-NAS, but not serotonin elicited analgesia in much the same manner as the dipeptide. This effect proved to be very specific as it required the presence of both an acetyl group on the terminal side chain amine as well as a hydroxyl group on the C-5 position of the indole ring. Substitution of the C-5 hydroxyl by a methoxyl group (melatonin) abolished the analgesic effect. Similarly, removing the N-acetyl substitution (serotonin) also eliminated the analgesia. IVC injection of highly specific antiserum to NAS induced hyperalgesia. Furthermore, an interaction was found between NAS and opiate systems. We demonstrated that while naloxone, the opiate antagonist, has no hyperalgesic properties of itself, it did counteract the analgesia induced by NAS. Similarly, NAS antiserum reversed the analgesia induced by the opiate morphine. This work provides evidence that NAS is an endogenously active substance within the CNS pain network.     

Taxol-induced reorganization of the microtubule system in murine splenic lymphocytes inhibits response to allogeneic cells but not to concanavalin A

The exposure of mouse splenic lymphocytes to the microtubule assembly-promoting drug taxol (10 microM for 4 h) results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules, which extend from the centrosome. Lymphocytes pretreated with taxol for 4 h, or cultured in the continued presence of taxol, respond normally to the mitogen concanavalin A up to, and including, the stage of DNA replication. In contrast, the induction of DNA synthesis during the alloactivation of lymphocytes is inhibited when taxol is present in the mixed leukocyte culture. If the stimulators are pretreated with this drug, the mixed leukocyte reaction occurs normally, but pretreatment of the responders inhibits the proliferative response markedly. Microscopic observations of nuclear morphologies in these populations and autoradiography indicate that taxol inhibition occurs early in alloactivation, prior to DNA replication. The responding ability of taxol-treated lymphocytes is not restored to control levels by the addition of interleukin 2, leading to the suggestion that interleukin 2 receptors do not emerge or function normally in these cells. We conclude that the capacity to respond to allogeneic cells, but not to a mitogen, is dependent on the presence of the normal submembranous organization of the microtubule system.     

The statistical analysis of circadian phase and amplitude in constant-routine core-temperature data.

Accurate estimation of the phases and amplitude of the endogenous circadian pacemaker from constant-routine core-temperature series is crucial for making inferences about the properties of the human biological clock from data collected under this protocol. This paper presents a set of statistical methods based on a harmonic-regression-plus-correlated-noise model for estimating the phases and the amplitude of the endogenous circadian pacemaker from constant-routine core-temperature data. The methods include a Bayesian Monte Carlo procedure for computing the uncertainty in these circadian functions. We illustrate the techniques with a detailed study of a single subject's core-temperature series and describe their relationship to other statistical methods for circadian data analysis. In our laboratory, these methods have been successfully used to analyze more than 300 constant routines and provide a highly reliable means of extracting phase and amplitude information from core-temperature data.     

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger.

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single KM(+)-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.     

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli

The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The bkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. aeruginosa. The relative molecular mass of the encoded protein was 45,134 (45,003 minus methionine) vs 47,000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1-E2 binding domains of E2b, E2p and E2o. There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched-chain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.     

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.     

Cloning and expression of a cDNA encoding a catalytically active fragment of calf thymus DNA polymerase alpha

A calf thymus cDNA expression library was constructed in the EcoRI site of lambda gt11 and probed with an antibody raised against calf thymus DNA polymerase alpha. Three classes of antibody-reactive clones were isolated. The largest class carried a 1.9 kilobase calf cDNA insert and expressed a 165-175 kilodalton beta-galactosidase:calf fusion protein which displayed DNA polymerase activity. The characteristic responses of the polymerase activity to alpha-specific inhibitors and antibodies identified the 1.9 kilobase cDNA as a sequence specifically derived from the structural gene encoding the pol alpha catalytic core.