Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.     

Insulin resistance in Cushing's syndrome

Insulin resistance is well established in Cushing's syndrome, but its mechanisms are not completely understood. We performed the euglycemic insulin clamp technique on four patients with Cushing's syndrome, five obese patients and five normal volunteers, in order to determine the role of impairments in insulin responsiveness and insulin clearance in hypercorticism and obesity. Insulin was infused at 0.3, 1, 3 and 10 mU/kg/min, and steady-state glucose-infusion rates required to maintain euglycemia were determined. Glucose disposal at maximal insulin levels was 11.9 +/- 0.4 mg/kg/min in normals, with a 29% decrease in obese and a 42% decrease in Cushing's syndrome patients. Half maximally effective insulin concentrations were increased in both abnormal groups compared to normals. Maximal insulin clearance rates were 1460 +/- 200 ml/min/m2 in normals, not significantly changed in obese and 40% decreased in Cushing's syndrome patients. These results indicate that the insulin resistance in Cushing's syndrome is distinct from that occurring in obesity and is characterized by both decreased insulin responsiveness and decreased insulin clearance. These impairments could be caused by a common defect which may be at or distal to the glucose transport level.     

Induction of oestrus in the camel (Camelus dromedarius) during seasonal anoestrus

Injection of 7000 i.u. PMSG induced oestrus in 7 camels during the last part of seasonal anoestrus. Mature follicles developed and a CL was formed after fertile mating. However, pregnancy was not maintained by Day 60 in the 3 females detected as pregnant by rectal palpation and increased progesterone concentrations at Day 50. A single male camel mated with 4 of the females 2-16 days after the PMSG injection, and 2 or 3 matings occurred. The failure of pregnancy after induction of oestrus and mating during seasonal anoestrus was probably due to inadequate luteal function.     

Deprenyl Protects Dopamine Neurons from the Neurotoxic Effect of 1-Methyl-4-Phenylpyridinium Ion

1-Methyl-4-phenylpyridinium ion (MPP+) is the product of the metabolic oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by monoamine oxidase (MAO). MPP+ is toxic to 3,4-dihydroxyphenylethylamine (dopamine, DA) neurons in explant cultures of rat embryonic midbrain. Addition of 2.5 microM MPP+ to the feeding medium for 6 days results in significant reduction of the DA levels in the cultures (to 19% of control) as well as in the uptake of [3H]DA (to 32% of control). When the cultures are treated with the MAO inhibitor deprenyl (10 microM) 24 h prior to and during exposure to MPP+, the DA neurons are protected from the toxicity of the drug. In the combined deprenyl plus MPP+ treatment, the levels of DA in the cultures remain at the control range and the [3H]DA uptake is reduced to only 73% of control. These results indicate that MAO is involved in the toxicity of MPP+ on DA neurons.     

Hybridization in the genus Lens by means of embryo culture

Summary The cultivated lentil L. culinaris and the wild lentil L. ervoides are reproductively isolated from one another due to their hybrid embryo breakdowns. Using embryo culture, vegetatively normal hybrids were obtained. One specific hybrid, heterozygous for a reciprocal translocation, had about 50% gamete viability and produced aborted and viable embryos in a 11 ratio. In the F₂, vegetatively normal and highly fertile plants were selected. With the aid of embryo culture techniques, L. ervoides can be included in the wild gene pool of the cultivated lentil.     

The integrated and free states of Streptomyces griseus plasmid pSG1

A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome.     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

The possible involvement of polyamines in the development of tomato fruits in vitro

The apparent involvement of ornithine decarboxylase (ODC) and putrescine in the early stages of fruit growth in tomato (Lycopersicon esculentum Mill.) has been previously described. Further evidence presented here supports the direct involvement of ODC and putrescine in the cell division process in tomato fruits. In tomato fruits grown in vitro, in which basic growth processes are inhibited, the activity of ODC and arginine decarboxylase (ADC) and the level of free polyamines were reduced. While ODC and ADC activity was correlated with the period of cell division in the tomato fruit, the free polyamine content was correlated with the DNA content, cell size, and fruit fresh weight. The addition of exogenous putrescine, however, did not restore the basic growth processes in the fruits grown in vitro.