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Coated vesicles isolated from carrot suspension culture cells were immune-blotted against four antibodies to porcine brain clathrin. Positive cross-reaction was obtained with three antibodies. Two of these cross-reacted with both the heavy clathrin chain and the putative light chains. Three out of five antibodies immunofluorescently stained permeabilised carrot suspension culture cells.

The structure of plastids in the root cap of cress and maize was studied by low- and high-voltage electron microscopy after staining their membranes with a mixture of zinc iodide and osmium tetroxide. In plastids of both species electron-opaque membranes were found in the plastid interior while membranes of lesser electron-opacity comprised the outer envelope and vesicles and cisternae underlying it. Electron-opaque tubules, often in groups attached to the inner membrane of the amyloplast envelope, were found in cress but not in maize. The internal, less-opaque membranes were often found associated with the starch grains. No specific association could be seen between amyloplasts and endoplasmic reticulum (ER); their surfaces showed no regular contact or connexion, though the amyloplasts clearly indented the underlying ER. The ER in statocytes was predominantly tubular in cress but predominantly cisternal in maize.

The immunological interaction between related maternal and neonatal human lymphocytes was investigated by assaying monocyte chemotactic factor production in mixed cell culture. Lymphokine was detected in a significantly lower proportion of maternal-neonatal cell cultures compared to mixed cultures of cells from unrelated adults and/or neonates. It is possible that a reduced inflammatory mediator production at the feto-maternal interface may contribute to the survival of the fetal allograft.

High mortality rates (20% to 60%) have been reported in the repair of coarctation of the aorta in infancy. During a 4 year period, 34 infants less than 6 months of age had coarctation repair (two prior to 1976). Eleven were less than 2 weeks of age, nine were 2 weeks to 1 month, eight were 1 to 2 months, and six were 2 to 6 months. Associated lesions were patent ductus arteriosus (PDA) (82%), ventricular septal defect (VSD) (53%), and other intracardiac lesions (35%). Twenty-three patients (67%) had emergency operations; the other procedures were semielective. The indications for operation included congestive cardiac failure (91%), acidosis (32%), hypertension (29%), cardiogenic shock (26%), and cardiac arrest (18%). There was one operative death (2.9%) in a patient with severe pulmonary valve insufficiency and multiple VSDs. There was one late death a 4 months (Taussig-Bing complex). Primary repair was used in 15, patch-graft angioplasty in 19 (left subclavian artery in nine, left common carotid in one, and Dacron or pericardial patch in nine). Two (6%) required reoperation for recurrent coarctation (follow-up 3 to 36 months with a mean of 25.8). Of 15 patients with a large VSD, six had pulmonary artery banding with two deaths (one operative and one late), two had debanding plus VSD repair, and two are awaiting operation. The remaining nine patients did not have banding (no operative or late deaths), four patients required late VSD closure, two VSDs closed spontaneously, two VSDs became smaller, and one patient is awaiting VSD closure. The infrequent need for pulmonary artery banding may be partly due to "physiological banding" seen at Denver's high altitude. The VSD spontaneously closed or became smaller in 44% of nonbanded patients. The low operative mortality can be ascribed to (1) aggressive medical therapy, (2) emergency catheterization and repair, (3) avoidance of hypothermia, and (4) adequate relief of the coarctation.

The lymphocytes of human breast milk have been previously shown to be immunologically competent while the monocytes have been described as actively motile in some reports but not in another. Cells were separated from milk samples collected by lactating women 2 to 9 days postpartum. Milk cell migration and production of the lymphokine, monocyte chemotactic factor, by milk cells were assayed using membrane filters. Milk cells were poorly motile although prior culturing improved their migratory ability. Cell-free milk was chemotactic for blood monocytes. Chemotactic factor production was detected in only two of 16 phytohemagglutinin-stimulated milk cell cultures.

During the past 5 years, patent ductus arteriosus (PDA) presented in 734 preterm infants (less than 2.5 kg and 37 weeks gestation) of 2,532 admissions (29%). The ductus presented with murmur, bounding pulses, and often congestive heart failure. Medical treatment consisted of the following: fluid restriction, furosemide, respiratory support, and rarely digoxin. The patients who were unresponsive to medical treatment had surgical ligation (306 of 734 or 42%). The patients who had ductal ligation were smaller, i .e., 82% of the surgical patients weighed less than 1.5 kg as compared to 38% of the medical patients. Of those patients weighing less than 1.5 kg, the surgical and medical groups were compared and the following observations made: The incidence of respiratory distress syndrome was greater in the surgical group (86% or 216 of 252 patients versus 69% or 111 of 161 medical patients, p less than 0.001) and the long-term survival was better (89% or 224 of 252 surgical patients versus 77% or 124 of 161 medical patients, p less than 0.005). In addition, the average duration of intubation was shorter in this surgical subgroup (8.9 versus 13.6 days). Significant left atrial enlargement and echocardiographic left atrial/aortic (LA/Ao) ratios of greater than 1.5:1 occurred in 58% or 171 of 290 surgical patients versus 32% or 59 of 190 medical patients (p less than 0.001). During the first 3 years of this study (medical treatment averaged 5 days), the duration of intubation in the surgical patients averaged 15.2 days; by comparison, in the last 2 years of this study (medical treatment averaged 1 to 2 days), the duration of intubation was 6.5 days (p less than 0.001). Necrotizing enterocolitis (NEC) occurred in 11% or 46 of 428 medical patients versus 0.3% (one of 305 patients) in the ligated group postoperatively (p less than 0.001). Late deaths were related to lung disease, central nervous system problems, NEC, and so on. From this study, it was determined that ligation of a significant PDA is associated with (1) zero surgical risk, (2) a reduced incidence of NEC, (3) reduced duration of intubation, especially with early ligation, and (4) improvement in late survival. Thus the surgical approach is our treatment of choice for a refractory PDA.

An epitope with apparent specificity for the surface of human syncytiotrophoblast was defined by a murine monoclonal antibody, FDO46B (IgG1, kappa). The epitope was predominantly expressed during the first trimester of pregnancy. Binding was detected on frozen tissue sections and on cultured trophoblast by the immunoperoxidase technique. It was also detected on the surface of a small percentage (less than 10%) of cultured choriocarcinoma cells (JEG-3). A panel of human tissues was negative, as were normal and malignant human lymphocytes. The antigen bearing the FDO46B epitope was still expressed by trophoblast after culture in the presence of tunicamycin, indicating that it is possibly protein in nature. This antigen may have potential utility as a target for a contraceptive vaccine.

Murine monoclonal antibodies were raised to human first trimester trophoblast cells. Eleven antibodies reacted with first trimester trophoblast, tested by immunoperoxidase staining on frozen sections, but only one had apparent specificity for trophoblast after examining reactivities with a panel of other cells and tissues. This antibody, designated FD0161G, bound selectively to syncytiotrophoblast and non-villous trophoblast in first trimester and term placentae. Villous cytotrophoblast was negative. This was clearly demonstrated on freeze-dried, paraffin embedded tissue sections which have superior architecture to frozen sections. FD0161G reacted with extra-villous trophoblast cells in human decidua which are also delineated by a monoclonal anti-cytokeratin antibody. Unlike the latter, however, FD0161G did not react with decidual glands. Thus FD0161G could be used as a specific probe for extra-villous trophoblast in decidual tissue.

Two type of Z disc structure have been reported in insect supercontracting muscle fibres: (i) a perforated Z disc where Z material forms a reticulum and (ii) a fragmented Z disc composed of separate, discrete Z bodies. The use of thick (1 micron) sections in conjunction with high voltage electron microscopy can distinguish between these two types while conventional thin sections may lead to misinterpretation of structure. It is shown that in one insect, the crane-fly Tipula, the larval body-wall muscles, for which a fragmented Z disc has been proposed, do in fact have a perforated disc. In the wax moth Galleria, homologous muscle fibres have a similar type of Z disc, a finding which indicates the need for re-examination of other lepidopteran muscles claimed to have fragmented discs. A redefinition of supercontraction is proposed which includes reference to the perforated type of Z disc.

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 μm thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.

The standard Blalock-Taussig (B-T) shunt is preferred over other shunts in the treatment of infants with cyanotic heart disease and reduced pulmonary blood flow. The B-T shunt is not, however, without problems such as phrenic nerve injury and excessive bleeding. It is a time-consuming procedure owing to the degree of lateral mediastinal dissection required (the latter is especially important in critically hypoxic infants). An alternative procedure is described in which an intrapericardial approach is used. This approach requires minimal dissection of the subclavian artery and the anastomosis is performed to the pulmonary artery, medial to the superior vena cava (SVC) and lateral to the aorta. The procedure was performed on 17 infants from 1 day to 2 years of age (average age of 9.4 months, with seven of the 17 infants being less than 6 months of age). Follow-up ranged from 1 to 44 months (average of 17 months). In 94%, the shunt was functioning well. The potential advantages of this approach include: elimination of phrenic nerve injury, easy dissection with reduced operating time, reduced intraoperative hemorrhage, and perhaps a larger anastomosis with improved patency rate.

Studies of cell-mediated immunity, comprising delayed hypersensitivity skin testing with four antigens, PHA and antigen-induced lymphocyte transformation and detection of the lymphokine, monocyte chemotactic factor, were undertaken in 14 women during pregnancy and in the post-natal period. Skin test reaction sizes to SKSD, PPD, Candida albicans extract and tetanus toxoid were not significantly different during pregnancy. In vitro lymphocyte transformation induced by PHA, SKSD and Candida albicans was enhanced during pregnancy. Monocyte chemotactic factor production was not significantly different during pregnancy. These in vitro tests were performed in the absence of autologous sera. These studies indicate that cell-mediated immune responses are normal during human pregnancy.

A monolayer depletion/adoptive immunization protocol that biased the immune response towards recognition of placental macrophage (pMO) antigens was established. BALB/c spleen cells immune to human pMO were adsorbed onto monolayers of the B-cell line QIMR-WIL. Monolayer-depleted or unfractionated cells were transferred to irradiated recipients, which subsequently were restimulated with pMO then killed for hybridoma production. Screening of hybridomas revealed an increased proportion of pMO-specific hybridomas following transfer and fusion of monolayer-depleted cells. Two monoclonal antibodies (mAb), L9 and L21, which were generated through application of this protocol, are described. L9 recognized an antigen on cells within the villi in sections of term placenta and freshly isolated pMO. With time in culture, expression of this antigen decreased markedly. Macrophages, but no other cell type, in placental cell suspensions expressed this antigen. L9 failed to react with any peripheral blood cells. Immunoprecipitation and SDS-PAGE analyses indicated that two proteins of molecular weight (MW) 40,000 and 43,000 were recognized by L9. Sections of term placenta and freshly isolated pMO failed to react with L21. After 2-3 days in culture, however, most macrophages expressed this antigen. L21 reacted weakly with peripheral monocytes and granulocytes but not other normal peripheral blood cells. Myeloid cell lines reacted strongly with this mAb only after activation with PMA. SDS-PAGE analyses of the L21 immunoprecipitate under non-reducing conditions revealed a single band of 61,000 MW, while two bands of 46,000 and 49,000 MW were detected under reducing conditions. Cellular distribution and molecular weight analyses indicated that the antigens recognized by these two mAb were apparently distinct from previously defined myeloid antigens.

A 20-year-old woman, admitted to a neurological ward with a diagnosis of benign intracranial hypertension, was found on specific protein electroimmunoassay to have a consistently decreased concentration of alpha 1-antichymotrypsin in her plasma. Serum from her father showed the same result. Further investigation of her family demonstrated that this partial deficiency was transmitted in an autosomal dominant fashion and was not associated with any obvious specific clinical abnormalities.

The structure and distribution of cytoplasmic membranes during mitosis and cytokinesis in maize root tip meristematic cells was investigated by low and high voltage electron microscopy. The electron opacity of the nuclear envelope and endoplasmic reticulum (ER) was enhanced by staining the tissue in a mixture of zinc iodide and osmium tetroxide. Thin sections show the nuclear envelope to disassemble at prophase and become indistinguishable from the surrounding ER and polar aggregations of ER. In thick sections under the high voltage electron microscope the spindle is seen to be surrounded by a mass of tubular (TER) and cisternal (CER) endoplasmic reticulum derived from both the nuclear envelope and ER, which persists through metaphase and anaphase. At anaphase strands of TER traverse the spindle between the arms of the chromosomes. The octagonal nuclear pore complexes disappear by metaphase, but irregular-shaped pores persist in the membranes during mitosis. It is suggested that these form a template for pore-complex reformation during telophase. Phragmoplast formation is preceded by an aggregation of TER across the spindle at anaphase. Evidence is presented to suggest that the formation of the desmotubule of a plasmodesma is by the squeezing of a strand of endoplasmic reticulum between the vesicles of the cell plate.

Incomplete Freund's adjuvant was used as a priming agent prior to the injection of hybridoma cells in mice to expand monoclonal antibody production. Two hybridoma cell lines, FDO28B (IgG1) and FDO31C (IgM), which produce monoclonal antibodies reactive towards human placenta, were used. Monoclonal antibody was detected in the ascites fluids by agarose gel electrophoresis. It was found that the time interval between adjuvant priming and cell injection could be reduced to 1 day, allowing collection of ascites fluid containing monoclonal antibody within 2 weeks of priming. In addition, as few as 1 X 10(5) hybridoma cells were needed to collect approximately 5-7 ml of ascites fluid containing antibody detectable by gel electrophoresis. Thus priming with incomplete Freund's adjuvant enables production of large amounts of monoclonal antibody in a short time using a low number of hybridoma cells.

Sixty children in this series underwent pulmonary artery banding (PAB); isolated ventricular septal defect (VSD) was present in 24, and 20 were corrected seven months to ten years and eight months after PAB. Other anomalies were present in 37, and 15 were eventually totally corrected. Survival after PAB was 90% (95.8% with isolated VSD), and 94.3% (95% with isolated VSD) after total correction. Mean age at banding was 12.9 months, and 52.6 months at debanding. All patients underwent catheterization before PAB; 39 underwent catheterization after PAB, and ten after total correction. Pulmonary artery blood pressure was reduced from 53.5 mm Hg to 25.3 mm Hg by banding. Most patients had only moderately increased pulmonary blood flow. Banding at the altitude of Denver (1,600 m) appears to be an effective means to convert high-risk infants into lower-risk children for total correction. The cumulative mortality for two-stage treatment of VSD was 8.3%.

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.