Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin

A full-length cDNA for rat lung beta-galactoside lectin (subunit Mr approximately 14,000, lectin 14K) was cloned and the nucleotide sequence determined. The deduced amino acid sequence agrees with the amino acid composition and direct amino acid sequence analysis of purified rat lung lectin peptides. We found that the amino-terminal alanine is blocked with an acetyl group. Comparison of the amino acid sequence with other proteins shows a high degree of homology only with other vertebrate lectin sequences, supporting the suggestion that these lectins may constitute a unique class of vertebrate proteins. The amino acid composition and sequence of lectin peptides, the sequence of lectin cDNA, and isoelectric focusing of purified lectin indicate that rat lung lectin 14K is composed predominantly of a single protein. In addition, rat uterus lectin 14K was found to be the same protein as that present in lung. We characterized the secondary and tertiary structure of rat lung lectin 14K by circular dichroism, by analytical ultracentrifugation, and by computer analysis of its primary structure. Results of these experiments suggest that lectin 14K is primarily a hydrophilic protein with an asymmetric, elongated structure consisting of approximately equal amounts of alpha helix, beta sheet, beta turn, and random coil. We found that Cys-2 and Cys-130 react most rapidly with iodoacetamide; one or both of these residues may be primarily responsible for the thiol requirement of lectin activity.     

Adrenalectomy and surfactant in adult rats

We examined the effect of adrenalectomy (ADX) on aspects of the surfactant system of adult rats. Five days after bilateral ADX, ADX rats had about 20% less disaturated phosphatidylcholine (DSPC) in lung lavage returns (airway DSPC) than sham-operated rats, but the amount of tissue DSPC was not different between the groups; airway DSPC formed 12.8 +/- 0.5% of total DSPC (airway + tissue) in ADX and 15.9 +/- 0.7% in sham-ADX rats. An ultrastructural morphometric analysis of alveolar type 2 cells did not reveal an effect of ADX on lamellar body volume density or surface-to-volume ratio. ADX rats had heavier lungs (not as a result of edema) than sham-ADX rats. Treatment of ADX rats with hydrocortisone returned the amount of DSPC toward normal and eliminated the increase of lung weight. ADX did not alter the recoil of saline-filled lungs but did slightly increase the recoil of air-filled lungs. We conclude that corticosteroid hormones influence the in vivo functioning of the surfactant system of adult rats, but this effect seems to be slight.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Surfactant aggregation in rat lungs: influence of temperature and ventilation

We examined the effect of the ventilatory rate and the temperature of excised lungs and of increased body temperature of anesthetized spontaneously breathing rats on the centrifugal sedimentation of disaturated phosphatidylcholine (DSPC) present in lung lavage returns. More DSPC sedimented from lungs ventilated at low than at high rates, and sedimentation of DSPC and lung volume loss were temperature dependent, 41 greater than 37 greater than 4 degrees C. Most of the noncellular sedimented material was tubular and common myelin; these had diminished ability to lower surface tension rapidly compared with less-aggregated surfactant. More aggregated DSPC accumulated and lung volume decreased more in spontaneously breathing rats anesthetized for 30 min than in rats killed immediately after being anesthetized; these changes were greater after 30 min of anesthesia in hyperthermic rats (40.4 +/- 0.3 degrees C) than in normothermic rats (37.4 +/- 0.1 degrees C). These studies have shown a correlation between the increased accumulation of surfactant as large aggregates and the loss of alveolar stability; however, a cause and effect between these events has not yet been shown.     

Lymphocytes and Macrophages Are Infected by Theileria equi,but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.