Automorphosis of maize shoots under simulated microgravity on a three-dimensional clinostat

Seedlings of maize ( Zea mays L. cv. Golden Cross Bantam T-51) were grown under microgravity conditions simulated by a three-dimensional clinostat. On the clinostat, maize shoots exhibited curvatures in three different portions: (1) the basal transition zone connecting roots and mesocotyls, (2) the coleoptile node located between mesocotyls and coleoptiles, and (3) the elongating region of the coleoptiles. Even non-clinostatted control shoots showed some degree of curvature away from the caryopsis in the transition zone and bending toward the caryopsis in the coleoptile node. Clinostat rotation greatly stimulated these curvatures. Control coleoptiles elongated almost straightly, whereas coleoptiles on the clinostat bent either away from or toward the caryopsis depending on the timing of rotation. The curvature in all three portions became larger with time, both in control and clinostatted seedlings. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any portion. However, in coleoptile nodes and coleoptiles, the faster-expanding convex side exhibited a higher extensibility of the cell wall than the opposite side, and this appears to be a cause of the curvature. Thus, changes in the cell wall metabolism may be involved in automorphosis, which governs the life cycle of plants under a microgravity environment.     

Induction of hydrotropism in clinorotated seedling roots of Alaska pea,Pisum sativum L.

Roots of the agravitropic pea (Pisum sativum L.) mutantageotropum show positive hydrotropism, whereas roots of Alaska peas are hydrotropically almost non-responsive. When the gravitropic response was nullified by rotation on clinostats, however, roots of Alaska peas showed unequivocal positive hydrotropism in response to a water potential gradlent. These results suggest that roots of Alaska peas possess normal ability to respond hydrotropically and their weak hydrotropic response results from a counteracting effect of gravitropism.     

A Subtype of κ-Opioid Receptor Mediates Inhibition of High-Affinity GTPase Inherent in Gi1 in Guinea Pig Cerebellar Membranes

Abstract: The κ-opioid receptor agonists including U-50,488H and dynorphin A (1–17) in ranges of 0.1–100 n M inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 µ M . The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 µg/ml but not with preactivated cholera toxin (30 µg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 µ M ) of N -ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of G_i1.     

Genetic Mapping Using Microcell-Mediated Chromosome Transfer Suggests a Locus for Nijmegen Breakage Syndrome at Chromosome 8q21-24

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, short stature, immunodeficiency, and a high incidence of cancer. Cultured cells from NBS show chromosome instability, an increased sensitivity to radiation-induced cell killing, and an abnormal cell-cycle regulation after irradiation. Hitherto, patients with NBS have been divided into the two complementation groups V1 and V2, on the basis of restoration of radioresistant DNA synthesis, suggesting that each group arises from a different gene. However, the presence of genetic heterogeneity in NBS has been considered to be controversial. To localize the NBS gene, we have performed functional complementation assays using somatic cell fusion between NBS-V1 and NBS-V2 cells, on the basis of hyper-radiosensitivity, and then have performed a genomewide search for the NBS locus, using microcell-mediated chromosome transfer followed by complementation assays based on radiosensitivity. We found that radiation resistance was not restored in the fused NBS-V1 and NBS-V2 cells and that only human chromosome 8 complements the sensitivity to ionizing radiation, in NBS cell lines. In complementation assays performed after the transfer of a reduced chromosome, merely the long arm of chromosome 8 was sufficient for restoring the defect. Our results strongly suggest that NBS is a homogeneous disorder and that the gene for NBS is located at 8q21-24.     

Fructose 2,6-Bisphosphate Changes in Rat Brain During Ischemia

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.     

Adenosine triphosphate compartmentation in the rat heart: a 31P spin-lattice relaxation study

Longitudinal relaxation times (T1) of phosphorus compounds in the perfused rat heart and erythrocytes were measured using the 31P Driven-Equilibrium Single-Pulse Observation of T1 relaxation (DESPOT) method at 33 degrees C. Both creatine phosphate in the heart and the three phosphate groups of adenosine triphosphate (ATP) in erythrocytes showed single-exponential relaxation. The three phosphate groups of ATP in the heart, however, had two T1 components. The T1 values of the short and the long T1 components of the beta-phosphate of ATP were ca. 0.4 and 14 s, respectively. The fraction with the long T1 represented ca. 30% of the total ATP content. These results suggested that there were two major pools of intracellular ATP in the rat heart which could be determined by 31P NMR spectroscopy.     

Green algal microtubule-associated protein with a molecular weight of 90 kDa which bundles microtubules

Summary Cytoplasmic streaming in the freshwater, coencytic green alga,Dichotomosiphon tuberosus, is regulated by light. Conspicuous changes are observed in the number of microtubules cross-linked together in bundles when the cytoplasmic streaming is modulated by light. In an attempt to identify the cross-linker, we stainedD. tuberosus cells with antibodies specific for several different microtubules-associated proteins (MAPs) from vertebrates. Antibodies raised against bovine adrenal 190 kDa MAP stained the algal cells, and the pattern of staining was quite similar to that obtained with tubulin-specific antibodies. Examination by immunoelectron microscopy revealed that the antibodies specific for the 190 kDa microtubule-associated protein (MAP) were located along the microtubules. Western blotting demonstrated that the antibodies crossreacted with a peptide fromD. tuberosus with a molecular weight of about 90 kDa. This peptide was heat-stable, a property shared by the bovine 190 kDa MAP. Moreover, this 90 kDa peptide, crossreacted with antibodies raised against a synthetic peptide, identical to the tubulin-binding domain found in the 190 kDa MAP and in a tau protein. Partially purified 90 kDa protein fromD. tuberosus has the ability to bundle microtubules when mixed with a tubulin fraction fromD. tuberosus, in the presence of taxol. These results suggest that the 90 kDa protein fromD. tuberosus is a MAP that bundles microtubules.Abbreviations APMSF (p-amidinophenyl) methanesulfonyl fluoride - BSA bovine serum albumin - CBB Coomassie Brilliant Blue R - DEAE diethylaminoethyl - DMSO dimethyl sulfoxide - DOC deoxycholic acid - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MAP microtubule-associated protein - MES 2[N-morpholino] ethanesulfonic acid - PBS phosphate-buffered saline - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - TLCK N-p-tosyl-lysine chloromethyl ketone     

In vitro roles of Escbericbia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids

Summary Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins, DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.     

Hymenolepis nana: immunity against oncosphere challenge in mice previously given viable or non-viable oncospheres of H. nana, H. diminuta, H. microstoma and Taenia taeniaeformis

When mice, previously given oral inoculation with viable oncospheres of the heterologous cestode species (Hymenolepis diminuta, H. microstoma, Taenia taeniaeformis) and the homologous one (H. nana), were challenged with oncospheres of H. nana 4 days after the primary inoculation, they showed strong and complete resistance to H. nana challenge, respectively. However, the resistance was not evoked in mice given either infective eggs of Toxocara canis or non-viable oncospheres of all cestode species examined. Congenitally athymic nude mice given viable oncospheres did not show any resistance to H. nana either. Eosinophil infiltration around cysticercoids of H. nana in the intestinal villi appeared to be more prominent in mice previously given viable oncospheres of H. diminuta than in mice given non-viable oncospheres or PBS only. Some of the eosinophils in the villus harboring cysticercoid(s) of H. nana invaded the epithelia in the former, whereas all eosinophils remained in the lamina propria in the latter. There was almost no eosinophil infiltration in nude mice. Microscopic observations revealed that oncospheres of H. diminuta, which require beetles as the intermediate host like H. microstoma, could invade the mouse intestinal tissue. Therefore, it is strongly suggested that the strong cross resistance to H. nana in mice, induced by oncospheres of all heterologous cestode species, is thymus-dependent and due to oncospheral invasion into the intestinal tissue of mice.     

Intracellular processing of complement pro-C3 and proalbumin is inhibited by rat alpha 1-protease inhibitor variant (Met352----Arg) in transfected cells

Complement C3, when its cDNA was transfected into COS-1 cells, was synthesized as a precursor, pro-C3, which was intracellularly processed into the alpha and beta subunits, although not completely. A cDNA for rat alpha 1-protease inhibitor (alpha 1-PI) was mutated in vitro to encode its variant with the modified active site (Met352----Arg). In cells co-transfected with the mutant alpha 1-PI cDNA and the C3 cDNA, pro-C3 expressed was secreted without being processed into the subunits. Co-transfection of the mutant alpha 1-PI cDNA and the albumin cDNA also resulted in the inhibition of intracellular conversion of proalbumin into serum-type albumin. No inhibition of the processing of each preform was observed in cells co-transfected with the normal alpha 1-PI cDNA. Taken together, the results indicate that the alpha 1-PI variant (Met352----Arg) expressed inhibits specifically an intracellular enzyme which is involved in the proteolytic processing of both pro-C3 and proalbumin.