Protease-sensitive anchoring of microfilament bundles provides tracks for cytoplasmic streaming inVallisneria

Summary In mesophyll cells of the aquatic angiospermVallisneria gigantea Graebner, the endoplasm streams rotationally along the cell walls normal to the leaf surface in situ. Bundles of microfilaments anchored in the ectoplasm serve as tracks for the cytoplasmic streaming. In single mesophyll cells isolated by enzymatic digestion, hypertonic treatment induces abnormal streaming concomitant with plasmolysis, specifically at one or both of the shorter sides of an approximate rectangle. The disorderly arrangement of microfilaments in such cells has been confirmed by fluorescence microscopy of cells stained with FITC-phalloidin. While inhibitors of proteases added to the enzyme solution used for isolation of cells suppress the disturbance of rotational streaming, exogenously applied protease promotes it. The results suggest that bundles of microfilaments in the ectoplasm are stabilized by protease-sensitive factor(s) in the presence of the cell wall.     

Architecture of implanted bone matrix gelatin influences heterotopic calcification and new bone formation

Heterotopic bone formation in skeletal muscle induced by compacted demineralized bone matrix gelatin (BMG) was studied histologically and biochemically. BMG was obtained by dehydrating diaphyseal shafts of femora and tibiae of 4-week-old male Sprague-Dawley rats, cutting the bone into chips, and demineralizing and extracting the chips with various solutions. The BMG was treated with 4 M guanidine-HCl, and compacted BMG was prepared by centrifugation. The compacted BMG was implanted into the rectus abdominis muscle of 5-week-old male Sprague-Dawley rats. The resulting specimens were examined histologically, and their alkaline phosphatase activity and the calcium content of the tissues were measured 3, 5, 7, 10, and 15 days after implantation. The BMG (separated BMG) with 75- to 500-microns particle sizes were implanted into control rats. The results showed that calcification, alkaline phosphatase activity, and bone formation were suppressed by implantation of the compacted BMG and that scarcely any vascularization occurred. Calcification, vascularization, and alkaline phosphatase activity were related and were indispensable for bone formation. In the control group, bone formation was observed at sites of high activity of alkaline phosphatase and well-developed vascularization. These results suggested that compacting of BMG suppressed vascularization, decreased calcification, and consequently reduced the induction of bone formation.     

Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.     

Karyotypes and X chromosome inactivation in segregants of a murine X-autosome translocation, T(X;4)37H

Karyotypes and X chromosome inactivation were studied in embryos obtained from female mice carrying T(X;4)37H translocation on day 6 to 8 of gestation by a BrdU-acridine orange method. A total of 18 different karyotypes were found in 477 embryos examined: 90.0% embryos were products expected from 2:2 alternate or adjacent 1 disjunction. 3:1 and adjacent 2 disjunctions accounted for approximately 8.0% and 0.7% conceptuses, respectively. In the embryo proper of balanced T37H/ + conceptuses, inactivation was random with respect to the normal X and the larger translocation X (4x) chromosome. In all the cells with the 4x inactive, the late replication apparently did not spread to the attached autosomal portion, although black/brown coat variegation implies spreading of inactivation into the autosomal region. The X chromosome segment deprived of the inactivation center remained active in all the cells examined and it exerted deleterious effects on embryonic or fetal development. Observation in embryos having two maternally derived X chromosomes showed that they were indeed resistant to inactivation in early extraembryonic cell lineages, and two copies of active X chromosomes in the trophectoderm fatally affected embryonic development due to inability to form the extraembryonic ectoderm and ectoplacental cone from the polar trophectoderm. In unbalanced X aneuploids the X chromosomes with the deletion were preferentially inactivated due to strong selection against nullisomy X.     

Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl molecules from gamma-hexachlorocyclohexane.

Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Sequences of two tandem genes regulated by carbon sources, one being essential for n-alkane assimilation in Candida maltosa

Several n-alkane-inducible clones were isolated from the genomic library of an n-alkane-assimilation yeast, Candida maltosa, by the differential hybridization method. Among these, one of the most predominantly expressed clones was analyzed. The nucleotide sequence of the cloned DNA fragment showed that it contained two open reading frames, one encoding a protein of 127 amino acids (aa) and the other a protein of 276 aa. The former was named POX18Cm, because the sequence was highly homologous to that of the Candida tropicalis gene, POX18, which already had been identified as encoding a small oleate-inducible peroxisomal protein. The latter, named ALI1, had no homologous sequences in the EMBL database (1990 release). Northern-blot hybridization indicated that the expression of these two genes was regulated by carbon sources in the media. From gene-disruption experiments, it was concluded that ALI1 was essential for assimilation of n-alkane by C. maltosa.     

In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Gene responsible for protecting Escherichia coli from sodium dodecyl sulfate and toluidine blue plus light.

Escherichia coli cells were killed by visible light irradiation in the presence of the photosensitizing dye, toluidine blue. Two uvrB mutant strains of E. coli K-12 (AB1885 and N3-1) were much more sensitive than the isogenic uvrA and uvrC strains to treatment with toluidine blue plus light, suggesting that the uvrB+ gene product was involved in repair of DNA damage induced by the treatment. The uvrB+ gene cloned in a high- or low-copy-number plasmid was transformed into the uvrB strain (AB1885). Although all the transformants showed the same resistance as its wild-type strain (AB1157) to UV irradiation, they were as sensitive as AB1885 was to treatment with toluidine blue plus light. The two uvrB strains were more sensitive to sodium dodecyl sulfate than the other strains, suggesting that these strains had a defect in the cell surface. A sodium dodecyl sulfate-resistant revertant obtained from AB1885 was more resistant than AB1885 was to treatment with toluidine blue plus light. The two uvrB strains (AB1885 and N3-1) appear to have a defective gene (tentatively called dvl) different from uvrB. Its map position was around 7 min on the E. coli map.     

Relaxation of supercoiled plasmid DNA by oxidative stresses in Escherichia coli.

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157 harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidine blue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation in vivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNA relaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.     

Aplysia myoglobins with an unusual amino acid sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana, a species distributed world-wide, has been determined and compared with the sequence of the myoglobin of Aplysia limacina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one, the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference.