Energy-dependent formation of free ATP in yeast submitochondrial particles, and its stimulation by oligomycin

Yeast submitochondrial particles, in a P_i- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5–10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

The cephalic glands of Brazilian reptilia and amphibia. IV. Histology of labial, premaxillary, and Duvernoy's glands in the colubrid snake Oxyrhopus trigeminus

A study of the morphology of the salivary glands of the colubrid snake Oxyrhopus trigeminus showed the following: The acini of supralabial, infralabial, and premaxillary glands are formed by mucous and mucoserous cells; the tubules of Duvernoy's gland are formed by seromucous cells; and mucous cells produce neutral and acid mucosubstances, mucoserous cells secrete neutral and acid mucosubstances and protein, and seromucous cells have neutral mucosubstance and protein secretions.     

The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Isolated and purified plasma and thylakoid membranes of the cyanobacteriumAnacystis nidulans contain immunologically cross-reactive aa3-type cytochrome oxidase

Cell-free extracts ofAnacystis nidulans were fractionated by discontinuous sucrose density gradient centrifugation resulting in the separation of two distinct types of membranes, the heavier one containing the chlorophyll and the lighter one devoid of chlorophyll. Identity of the latter with plasma membrane was confirmed by labeling of intact cells with impermeant marker,³⁵S-diazobenzenesulfonate, prior to cell disruption. Both membrane fractions were purified individually by repeated recentrifugation on identical gradients. Purified membranes were subjected to dissociating polyacrylamide gel electrophoresis, either type of membranes yielding a distinct polypeptide pattern. After transfer of the polypeptides to nitrocellulose by Western blotting, two of the proteins, with molecular weights of approximately 55,000 and 32,000, respectively, gave strong and specifically complementary cross-reactions with antibodies raised against subunits I and II of the aa₃-type cytochrome oxidase fromParacoccus denitrificans. The findings will be discussed in terms of the presence of aa₃-type cytochrome oxidase in both plasma and thylakoid membranes ofAnacystis nidulans.     

A complex balanced chromosomal rearrangement in repeated abortions

Summary A double balanced reciprocal translocation involving four chromosomes, t(1;19;6;14) (1p11; 19p11; 6q25; 14q21), was found in the phenotypically normal husband in a couple referred because of repeated abortions. Reciprocal translocations, t(6;14), had been transmitted by his mother, his father being apparently homozygous for a translocation comprising pairs 1 and 19-t(1;19)(1;19). The genetic consequences of this complex chromosomal rearrangement are analyzed.     

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat

Summary The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and -fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and -fetoprotein genomic subclones with ³²P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG·dT repeats by using ³²P-labeled poly-(dG·dT)(dC·dA) oligomer probe. They occurred in 5 and 3 extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element, 4D12. The intense repetitive sequence, localized in the 3 non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the -fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.