Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1)

Summary A new mapping system, based on nonisotopic in situ hybridization combined with fluorescent staining of replicated prometaphase R-bands, is described. Replication of the bands is achieved by treatment of thymidinesynchronized cells with bromodeoxyuridine. The human COL2A1 gene was mapped to band 12q13.11–q13.12 in this manner, to illustrate the potential of the technique for improving the precision of chromosomal mapping and physical ordering of genes.     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Changes in choline acetyltransferase activity and high-affinity choline uptake, but not in acetylcholinesterase activity and muscarinic cholinergic receptors, in rat somatosensory cortex after sciatic nerve injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone γ-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat

New aspects of the distribution and developmental appearance of the 44-kDa bone phosphoprotein (44K BPP, also called sialoprotein I or osteopontin) and bone gamma-carboxyglutamic acid (Gla)-containing protein (BGP, also called osteocalcin) during osteogenesis and dentinogenesis were investigated with immunocytochemical techniques using monospecific, affinity-purified polyclonal antibodies. Sections from newborn rat incisors and from various bone anlagen of newborn animals and fetuses were processed for detection of 44K BPP or BGP antigenicity. In addition, histochemical reactions for detection of alkaline phosphatase or calcium salts were performed on a number of the sections. The 44K BPP appears to be synthesized and secreted by chondrocytes only in the areas of cartilage-to-bone transition; these cells could participate indirectly in the process of bone formation by providing a suitable scaffold onto which primary marrow osteoblasts attach and spread. During osteogenesis, 44K BPP is found in bone-forming cells almost concomitantly with the appearance of alkaline phosphatase and before osteoid deposition, whereas BGP is still absent during early stages of mineralization. We hypothesize that this dramatic difference between the developmental appearance of 44K BPP and BGP reflects the delayed expression of the BGP gene relative to that of 44K BPP. In long-term cultures of bone marrow from adult mice, some fibroblastic cells expressed the 44K BPP phenotype; these cells could represent early osteogenic progenitor cells. Some experiments also suggested that, as with BGP, 44K BPP or an immunologically related protein is synthesized by some odontoblasts and secreted into predentin, prior to the onset of mineralization.     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

Summary By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.     

Construction of transposons encoding genes for β-glucosidase,amylase and polygalacturonatetrans-eliminase fromKlebsiella oxytoca and their expression in a range of gram-negative bacteria

DNA fragments containing theKlebsiella oxytoca genes encoding -glucosidase and amylase were cloned into the kanamycin resistance transposon Tn5. Another DNA fragment containing two genes for polygalacturonatetrans-eliminase was cloned into Tn1721. These newly constructed transposons were then each transposed in vivo onto the broad-host-range plasmid pR751 and conjugally transferred to a variety of Gram-negative bacteria. These were then screened for the newly acquired phenotypes.     

The effect of prolonged hypobaric hypoxia on growth of fetal sheep

The effect of prolonged hypobaric hypoxia on fetal sheep was studied. Pregnant ewes were subjected to an atmospheric pressure of 429 torr from 30 days to 135 days gestation (long-term study). Average fetal weight for the hypoxaemic group (3.35 +/- 0.53 kg; n = 4; mean +/- SD) was significantly lower than for the controls (4.23 +/- 0.29 kg; n = 7; P less than 0.05). A short-term study was undertaken with fetuses (n = 8) which were catheterized at 110 days gestation and whose dams were subjected to hypobaric hypoxia from 120 to 141 days gestation. The mean carotid PO2 of fetuses in the hypoxic group was 12.7 +/- 0.7 torr compared to 22.7 +/- 0.7 torr for the control group (n = 9; P less than 0.001) throughout the period of treatment. Fetal arterial oxygen content fell from 6.5 +/- 1.7 to 4.9 +/- 0.4 ml/dl (P less than 0.05), but rose to control values after 7 days due to an increase in fetal haemoglobin concentration (9.6 +/- 1.1 to 13.0 +/- 1.9 g/dl, P less than 0.001) and packed cell volume (33 +/- 3 to 45 +/- 4%, P less than 0.001). In the hypoxaemic fetuses, pH fell initially from 7.34 +/- 0.02 to 7.28 +/- 0.03 (P less than 0.05) and then recovered to 7.32 +/- 0.03 within 24 h. Mean fetal weight of the short-term hypoxic group was 3.46 +/- 0.72 kg compared to 4.15 +/- 0.51 for the control group (P less than 0.05). Both long- and short-term hypoxia produced a similar reduction in fetal body weight. The adrenal glands were significantly heavier in the hypoxic fetuses than in controls. Placental weight was not effected by hypoxia, but exposure from 30 days gestation reduced the average size of cotyledons (P less than 0.05). It is concluded that the fetal sheep increases its ability to acquire and transport oxygen in response to chronic hypoxia, but this compensation is not sufficient to prevent growth retardation or changes to the pattern of tissue growth.     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50