Hemidesmosome formation in vitro

Intact, viable sheets of adult rabbit corneal epithelium, 9 mm in diameter, were prepared by the Dispase II method (Gipson, I. K., and S. M. Grill, 1982, Invest. Ophthalmol. Vis. Sci. 23:269-273). The sheets, freed of the basal lamina, retained their desmosomes and stratified epithelial characteristics, but lacked hemidesmosomes (HD). Epithelial sheets were placed on fresh segments of corneal stroma with denuded basal laminae and incubated in serum-free media for 1, 3, 6, 18, or 24 h. Tissue was processed for electron microscopy, and the number of HD/micron membrane, the number of HDs with anchoring fibrils directly across the lamina densa from them, and the number of anchoring fibrils not associated with HDs were counted. After 6 h in culture, the number of newly formed HD was 82% of controls (normal rabbit corneas), and by 24 h the number had reached 95% of controls. At all time periods studied, 80-86% of HDs had anchoring fibrils directly across the lamina densa from them. Anchoring fibrils not associated with HDs decreased with culture time. These data indicate that the sites where anchoring fibrils insert into the lamina densa may be nucleation sites for new HD formation. Corneal epithelial sheets placed on two other ocular basal laminae, Descemet's membrane and lens capsule, had not formed HDs after 24 h in culture. These two laminae do not have anchoring fibrils associated with them. Rabbit epithelial sheets placed on the denuded epithelial basal lamina of rat and human corneas formed new HDs. Thus, at least in these mammalian species, HD formation may involve some of the same molecular components.     

Irreversible Activation of the Opiate Receptor of Neuroblastoma × Glioma Hybrid Cells by an Alkylating Benzomorphan Derivative

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Structural alterations in fibronectin correlated with morphological changes in smooth muscle cells

Nontransformed cultures of vascular smooth muscle cells proliferate until they form a confluent sheet of cells. Subsequently, the cells become reorganized to form multicellular nodules that are loosely attached to the substrate. The formation of nodules is facilitated by the addition of medium conditioned by nodular cultures. Nodulation is inhibited by the addition of fibronectin. Fibronectins derived from monolayer culture conditioned medium or from plasma are maximally effective while fibronectin isolated from nodular cell conditioned medium is inactive. Analysis by NaDodSO4-polyacrylamide gel electrophoresis reveals that the nodular cell fibronectin has a molecular weight that is about 20-30 kd less than that of monolayer cell fibronectin. Further, nodular cell conditioned medium contains an activity that can convert both plasma fibronectin and monolayer cell fibronectin to the lower molecular weight correlated with the loss of biological activity.     

Immunological evidence for two distinct chondroitin sulfate proteoglycan core proteins: Differential expression in cartilage matrix deficient mice

The expression and core protein structure of two proteoglycans, the major cartilage proteoglycan isolated from a rat chondrosarcoma and a small molecular weight chondroitin sulfate proteoglycan isolated from a rat yolk sac tumor, have been compared. The cartilage proteoglycan was not detectable in the cartilage tissue of cartilage matrix deficient ($\text{cmd}\text{cmd}$) neonatal mice by immunofluorescence, but the cmd cartilage did react with antibodies against the core protein of the yolk sac tumor proteoglycan. Radioimmunoassays showed that the core proteins of these proteoglycans are not cross-reactive with each other. Analysis of the core proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after chondroitinase ABC treatment of the proteoglycan revealed a large difference in their sizes. The cartilage proteoglycan core protein had a molecular weight of about 200,000 while the yolk sac tumor proteoglycan core protein migrated with an apparent molecular weight of about 20,000. In addition, the cultured yolk sac tumor cells that make the small proteoglycan did not react with antiserum against the cartilage proteoglycan. These results indicate that the proteoglycan isolated from the yolk sac tumor is similar to the small chondroitin sulfate proteoglycan species found in cartilage and support the existence of at least two dissimilar and genetically independent chondroitin sulfate proteoglycan core proteins.     

Ribonucleic Acid Synthesis During the Differentiation of Sporangia in the Water Mold Achlya

The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.