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Congenital diaphragmatic hernia (CDH) is a relatively common malformation of unknown cause with high mortality due to hypoplasia of the lungs and pulmonary hypertension. We studied a family in which two fetuses had CDH, and two pregnancies resulted in first trimester missed abortions. Both fetuses with CDH had an apparently normal karyotype. In a subsequent pregnancy, fluorescent in situ hybridization analysis of amniocytes showed a balanced translocation 46,XY, t(5;15) (p15.3;q24) also present in the mother and in a normal child, suggesting that the diaphragmatic hernia in the first two fetuses was caused by a cryptic unbalanced translocation. This hypothesis is supported by a previous observation of CDH in a distal deletion of 15q as part of a multiple congenital anomalies syndrome. It is suggested that a gene distal to 15q21 is important for the normal development of the diaphragm.

The effect of both diagnostic and therapeutic ultrasound on cellular RNA synthesis was investigated in amniocytes cultured in F-10 medium enriched with fetal calf serum. The rate of RNA synthesis was tested using incorporation of H3 uridine, which was added following the exposure to acid insoluble RNA. Cultured amniocytes which were not exposed to ultrasound served as the control. Different results were obtained following the two ranges of exposure. A significant decrease in RNA synthesis was demonstrated immediately following diagnostic ultrasound and a certain increase in RNA synthesis was demonstrated soon after exposure to therapeutic doses. These effects were transient and could not be shown 24 hours later. It is concluded that the routine use of ultrasound can not have any clinical implications on RNA synthesis.

Congenital diaphragmatic hernia (CDH) is a relatively common malformation of unknown cause with high mortality due to hypoplasia of the lungs and pulmonary hypertension. We studied a family in which two fetuses had CDH, and two pregnancies resulted in first trimester missed abortions. Both fetuses with CDH had an apparently normal karyotype. In a subsequent pregnancy, fluorescent in situ hybridization analysis of amniocytes showed a balanced translocation 46,XY, t(5;15) (p15.3;q24) also present in the mother and in a normal child, suggesting that the diaphragmatic hernia in the first two fetuses was caused by a cryptic unbalanced translocation. This hypothesis is supported by a previous observation of CDH in a distal deletion of 15q as part of a multiple congenital anomalies syndrome. It is suggested that a gene distal to 15q21 is important for the normal development of the diaphragm.

In order to clarify the role of mast cells in the aetiology of secretory otitis media (SOM), we compared the protein components of middle ear effusion (MEE) with human mast cells using acrylamide gradient gel electrophoresis and electrofocusing methods. This first direct comparison between the proteins of MEE and human mast cells has been made possible by a method developed in our laboratory for cultivation of human mast cells in tissue culture.On electrophoresis, we found that out of 12 bands of MEE proteins that were different from the serum, seven (58 per cent) had a similar electrophoretic migration rate (Rx) to mast cells. On electrofocusing, three of the four bands of MEE had a similar Rxto the mast cells. We have shown that proteins of mast cells and MEE had similar Rxs. Therefore, our study supports previous studies which suggests that mast cells play an important role in the aetiology of SOM.

Sera from 183 healthy, elderly subjects and 92 young, control subjects were tested by the Elisa method for antibodies against double-stranded (ds) and single-stranded (ss) DNA, and sera from the elderly were tested for antinuclear antibodies as well. Significantly higher levels of anti-ss-DNA were found in the elderly, but levels of anti-ds-DNA did not differ significantly; no sera positive for anti-ds-DNA were found. levels of antibodies to DNA were similar in men and women. In those 85 and over, mean levels of anti-ds-DNA were lower than in those 65-84 (63 vs. 44, p less than 0.05). 13 of those over 65 (7.1%) tested positive for antinuclear factor, 2 of whom had positive titers of anti-ss-DNA. The results of this study support the hypothesis that elevated levels of anti-ss-DNA are age-related and not disease-related.

Transient neonatal hypothyroidism has been observed in three successive offspring of a mother with autoimmune thyroiditis. Thyroxine replacement therapy was initiated in a 23-year-old woman with overt clinical and laboratory findings of non-goitrous primary hypothyroidism. While on such treatment, she gave birth to three infants manifesting hypothyroidism immediately after birth. The neonates were treated with thyroxine replacement therapy which was discontinued in the three siblings at ages 2½ years, 3½ years, and 13 months. Continuous observation following cessation of therapy revealed clinical and biochemical euthyroidism in the children. Thyroid scanning during the neonatal period in the first child failed to identify functional thyroid tissue, suggesting thyroid agenesis, whereas thyroid scan performed on subsequent follow-up revealed a normal gland. Sequential serum measurements of autoantibodies directed towards the thyrotropin receptor were made in the mother and third child by a cAMP bioassay. High titres (five-six fold above normal) of blocking antibodies (tested by measuring the inhibition of TSH-stimulated cAMP production of cultured human thyroid cells by serum immunoglobulin preparations) were present in the mother and newborn 10 days after birth. The levels remained persistently high in the mother, whereas they declined and were undetectable in the child at four months. Thyroid-stimulating immunoglobulin was absent in both mother and child. The data are compatible with transient neonatal hypothyroidism caused by transplacental transfer of antibodies which block thyroid response to TSH. The half-life of the maternally-derived blocking antibody in the infant was estimated as 1-2 months. This is the first report on sequential serum measurements and estimate of half-life of the blocking antibodies performed by a cAMP bioassay (using thyroid cells of human origin). Unlike the radioreceptor assay employed so far in such cases, this assay can distinguish between stimulating and blocking TSH receptor antibodies.

Summary. Congenital dysgranulopoietic neutropenia is a severe life-threatening disease characterized by specific dysmorphic granulocytes. Prenatal diagnosis of congenital neutropenia was reported first in 1983. In 1989 we reported a family with congenital dysgranulopoietic neutropenia in two siblings (one male and one female): in 1992 the mother became pregnant and prenatal diagnosis was performed by cordocentesis. The results we obtained from the fetus at risk enabled us to suggest that it was not affected, and we advised the parents that the pregnancy could continue: a healthy female was born. The neutrophil count at 2 and 4 months was normal.

A convenient procedure for the efficient plating of cells and protoplasts at low densities (as low as 200/ml) was developed. Feeder cells from exponentially growing Petunia suspension cell cultures are embedded in solid medium and overlaid with a cellophane membrane on which the nursed cells are plated. This method allows for the rapid transfer of cells from one set of experimental conditions to another, without the need to wash the cells. Being transparent, the membrane allows for an easy macro- as well as microscopic follow-up of both feeder and nursed cells. The method was successfully utilized for the isolation and rescue of Petunia hybrida cells resistant to 6-aza-uridine (6-aza-U), methotrexate (MTX) or the aminoglycoside G-418. Finally, the possibility of utilizing feeders from distant species has been demonstrated for tobacco mesophyll protoplasts using petunia feeder cells, suggesting that this technique could also be useful in other plant cell culture systems.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymocyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferate in response to mitogens.

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.

Trypsin increases intracellular levels of cyclic AMP (cAMP) in lymphocytes. The trypsin-induced increase in cAMP is blocked by specific trypsin inhibitors and by high concentrations of different proteins. Several proteolytic enzymes from various sources, including other pancreatic proteases, do not cause an increase in cAMP under the same experimental conditions. Immobilized trypsin induces the same increase in cAMP as does free trypsin. The trypsin-induced rise in cAMP is not due to inhibition of cAMP phosphodiesterase, but consistent activation of adenylate cyclase by trypsin could not be demonstrated. The extent of the trypsin-induced increase in intracellular cAMP correlates with the type of the lymphocyte and with the state of maturity attained by the cells. Transformed lymphocytes and nonlymphoid cells do not react at all.

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.

1. 1,5-Diphenylcarbazide (DPC) was shown to compete with water as an electron donor to photosystem II in untreated chloroplasts. 2. 1,5-Diphenylcarbazone (DPCN), a product of the above photooxidation, was shown to be disproportionated in a Photosystem I-dependent reaction. The products of the disproportionation reaction were DPC and a more oxidized form, probably diphenylcarbodiazone. 3. The disproportionation reaction is insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, does not require cytochrome 552 in Euglena chloroplasts, but shows a requirement for plastocyanin. 4. The disproportionation reaction required intensities about 100-fold lower than those required for a typical Hill reaction. The quantum requirement was determined as 0.07 quantum per molecule of DPCN at 715 nm. It is therefore suggested that the disproportionation reaction is catalyzed by a product of Photosystem I. 5. It is suggested that the reaction requires a smaller number of components around Photosystem I, than any other described to date.

This communication reports on studies carried out with a mutant of Euglena gracilis unable to use water as an electron donor. It was found that: (1) DPC is a donor which acts very close to the reducing side of photosystem II (2) contrary to results published in the literature electron donation by DPC is not inhibited by PMA.

We describe here a case of common variable immunodeficiency with depression of both humoral and cellular immunity, manifested primarily by chronic toxoplasmosis. The presence of a lymphoma as the underlying etiology of the immunodeficiency was excluded. The clinical, histological, and immunological interrelations between immunodeficiency, toxoplasmosis and lymphoma are discussed.

Alterations in cyclic-AMP levels before and after trypsin treatment as compared with cyclic-AMP levels of normal individuals are found in peripheral blood lymphocytes of patients suffering from various clinical conditions in which a defect in cell mediated immunity is suspected. Such conditions include infectious mononucleosis and acute lymphatic leukemia. It is concluded that cyclic-AMP levels in lymphocytes correlate with the clinical and immunological state of the patients and might be of diagnostic value.

The effect of exposure of nuclei at different stages of meiosis to hydroxyurea (HU) was tested by examination of its effect on the frequency of auxotrophic mutations and on intergenic recombination. The results indicate that recombination is increased most significantly if the nuclei are exposed to the drug during the premeiotic-S phase and to a lesser extent if exposed during prophase. Similarly, nuclei exposed to HU during premeiotic-S phase and prophase mutate at a frequency 10 to 50 times higher than untreated meiotic nuclei. The degree of response of the mutagenic events and recombination events is not similar and it is suggested that the mutagenic events occur at stages prior to recombination. The temporal relations of the two events in response to HU suggest that the mutagenic events precede recombination and the results obtained with HU only amplify the mutagenic events that normally occur in meiotic nuclei and are related to the meiotic effect.

A strain with an elevated level of spontaneous mutations and an especially high rate of reversion at a specific locus ( pab − ) was identified. The mutator trait is recessive. UV sensitivity and the absence of a UV-specific endonucleolytic activity were associated with the enhancement of the mutation rate in mutator strains. The endonuclease associated with the regulation of the mutation rate also acted on single-stranded DNA. The molecular weight of this enzyme is about 38 000 daltons.

Supernatants of adherent mouse peritoneal exudate cells or human mononuclear cells were used as the source of lymphocyte activation factor (LAF). LAF was found to potentiate the effect of mitogens such as PHA and Con A on DNA synthesis by mouse thymocytes. However, LAF also was capable of reducing vigorous thymosyte reactions to Con A. Thus, LAF usually enhanced the effect of PHA on DNA synthesis by BALB/c thymocytes to a relatively greater degree than that of Con A. This change in the ratio of Con A to PHA response of thymocytes suggests that LAF can serve as a regulator of thymocyte DNA synthesis. Moreover, in the presence of LAF, allogeneic thymocytes developed the ability to have bidirectional mixed thymocyte reactions. Exposure to LAF not only improved the ability of parental thymocytes to act as responder cells, but, in addition, led to increased stimulatory activity of F1 thymocytes, presumably by promoting the differentiation of stimulator cells. These indications that LAF affected differentiation were investigated further by studying its effect on the cAMP content of thymocytes. LAF stimulated significant immediate but transient elevations of intracellular cAMP and adenylate cyclase activity in thymocyte membranes. In contrast, the mitogens themselves failed to elevate or to influence the effect of LAF on the content of intracellular cAMP of thymocytes. Furthermore, the potentiating effect of LAF on mitogen-induced thymocyte DNA synthesis at times was enhanced by exogenous cGMP, carbachol, or imidazole. These findings suggest that LAF, through its stimulation of cAMP levels in thymocytes may in turn promote thymocytes to differentiate sufficiently to become competent to proliferative in response to mitogens.

SUMMARY: Electron microscopic observations of an indigotin-producing dome mutant of Schizophyllum commune Fr. have shown that large wall ingrowths occur within the hyphae. These ingrowths are coupled with morphological abnormalities produced by the dome mutation. The pigment indigotin appears to be produced by progressive condensation within vacuoles and to a lesser extent within the wall ingrowths. Cytochemical techniques have shown that the wall ingrowths are similar in structure to the hyphal walls. There was no evidence for the passage of condensed indigotin into the medium; the pigment granules found in the medium must therefore form outside the hyphae.

The immune competency of peripheral blood (cord) lymphocytes of newborn infants has been investigated by a battery of assays in vitro. Number of T cells was enumerated by E-rosette formation and a cytotoxicity assay by using an anti-human-thymocyte antiserum. Lymphokine production as an indicator of T cell function was evaluated by leukocyte migration inhibition factor production following stimulation with various mitogens, and "viral" and "immune" interferon production in response to stimulation with polyriboinosinic -cytidilic acid and phytohemagglutinin, respectively. Ability to respond to mitogens was also tested by means of 3H-thymidine uptake into DNA, and by measuring the early synthesis of protein by lymphocytes by means of 3H-leucine uptake. Lymphocyte cyclic adenosine monophosphate levels in resting cells and after trypsin treatment was also used as a test of cellular competency. Total B cells was evaluated by EAC-rosette formation, and class specific surface membrane immunoglobulin-bearing cells were assayed by the peroxidase immunoenzymatic method. Normal children and adults served as controls. The results indicated that no significant differences in immune competency could be shown between newborns and older people except for lymphocyte cyclic adenosine monophosphate levels which were lower in the newborns, both in resting and in stimulated cells. The relationship between this latter finding and the immune status is as yet not clear. It is concluded that neonatal lymphocytes are essentially immunocompetent, with the expression of their immune capabilities in vivo becoming apparent only after encounter with environmental antigenic stimuli.

A method was developed for the isolation of photosynthetic mutants of Euglena gracilis. It consists of the following steps. (a) Incubation of the cells under phototrophic conditions in the presence of 3 (3,4-dichlorophenyl)-1, 1-dimethylurea for 1 week. This step caused a drastic reduction in the number of chloroplasts per cell; (b) mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine; (c) phototrophic growth for a few days to allow for phenotype expression; (d) selection by incubation in the presence of arsenate under phototrophic conditions for 2 days; (e) plating and growth under photoorganotrophic conditions; (f) assay of green colonies for ability to evolve oxygen. About 10% of the green colonies were found to be deficient in their ability to evolve oxygen. In principle the method may prove suitable for the isolation of other types of mutants of Euglena.

Peripheral blood lymphocytes of asthmatic patients in the acute stage manifest low basal levels of cyclic AMP. These levels were higher in the lymphocytes of patients in remission than in controls. Trypsin treatment of the lymphocytes increased cyclic AMP content to almost the same additional activation site of the receptor in theophylline- and catecholamines-treated patients.

Positive titers of antibodies against double-stranded (ds) and single-stranded (ss) DNA were found in the sera of 4 and 6 patients, respectively, of 18 who had familial Mediterranean fever (FMF). While anti-dsDNA antibodies were found only in patients with active disease, there was no correlation between the presence of anti-ssDNA antibodies and disease activity. The antibody titers were lower than those found in patients with active systemic lupus erythematosus. This may be due in part to the fact that all the FMF patients were treated with colchicine.

Two patients with subacute sclerosing panencephalitis (SSPE) showed impairment of cell-mediated immunity, as indicated by a low T cell number, decreased intracellular cyclic AMP levels of peripheral blood lymphocytes, negative graft-vs.-host reaction in vivo, negative skin reactions to common antigens and, in one of the patients, abnormal reactions in migration inhibition factor tests. Since some of the impaired T cell functions in one of the patients were reconstituted in vitro by the administration of thymus humoral factor (THF), a thymic hormone shown in an earlier study to regulate maturation of T lymphocytes in in vitro and in vivo animal models, a course of THF administration was given to both patients in this study. In vitro and in vivo assays, which reflect T cell competence, were performed before and after a daily schedule of THF administration that lasted for 10 days in one patient and 21 days in the other. The results of this preliminary trial suggested that THF was capable of reconstituting the impaired T cell functions in both patients after a short term of treatment. These preliminary results should encourage additional long-term therapeutic trials with THF in SSPE patients with impaired cell-mediated immunity.

Adding plastocyanin to plastocyanin-depleted chloroplast particles, restored both their ability to catalyse the photoinduced electron transfer from ascorbate-DCIP to NADP, and to induce the photooxidation of cytochrome f . It is concluded, therefore, that plastocyanin mediates the photoinduced oxidation of cytochrome f , as previously suggested.

Euglena cells grown in an autotrophic medium with DCMU in the light, show a reduction in the number of chloroplasts per cell, and gross alterations in the structure of the remaining chloroplasts. The chloroplasts change both in dimensions and in the number of lamella per chloroplast. They tend to fuse with each other forming giant twisted chloroplasts. The intensity of the morphological changes reaches its maximum after the 5th day of incubation. All these changes are fully reversible and disappear 2–3 days after the removal of DCMU. Target analysis by ultraviolet irradiation indicates that the reduction in the number of chloroplasts per cell is associated with a reduction in the chloroplastic DNA.

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants. 2. An Arrhenius plot of the photoreduction of NADP + from water by chloroplasts isolated from tomato ( Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole −1 . Between 11 and 3 °C the activation energy increases to 22 kcal·mole −1 . Photoreduction of NADP + by chloroplasts from another chilling-sensitive plant, bean ( Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole −1 below about 12 °C. 3. The photoreduction of NADP + by chloroplasts isolated from two chilling-resistant plants, lettuce ( Lactuca sativa var. winter lake) and pea ( Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole −1 , respectively, over the temperature range 3–25 °C. 4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP + from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP + reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C. 5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible. 6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP + , would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.