Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Increase in hepatic microsomal lipid peroxidation mediated by α-adrenergic system under cold stress and noradrenaline treatment

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.Abbreviations SqCHN Squamous Carcinoma of the Head and Neck - MTX Methotrexate - CDDP Cis-Platinum - DHFR Dihydrofolate Reductase     

Nitrogen fixation and indole acetic acid production by Azospi-rillum sp. as influenced by an insecticide, carbofuran

Both indole acetic acid (IAA) accumulation and nitrogen fixation were increased in Azospirillum cultures isolated from rice roots and soils by carbofuran (2, 3-dihydro-2, 2-dimethyl-7-benzofuranyl-N-methyl carbamate), an insecticide widely used in rice cultivation. Addition of carbofuran at 5 and 10 parts/10₆ significantly stimulated nitrogen fixation in Azospirillum. Indole acetic acid accumulation by Azospirillum cultures was more pronounced at a lower level (250 g/50 ml) of carbofuran. Evidence is provided for carbofuran degradation by Azospirillum cultures. The 7-benzofuranol (2, 3-dihydro-2–2-dimethyl-7 benzofuranol), a major degradation product of carbofuran, however, did not enhance the IAA accumulation. The higher accumulation of IAA in Azospirillum in the presence of carbofuran is probably related to the increased growth due to fixed N present in the insecticide. Results indicate the involvement of parent compound carbofuran and/or compounds other than the 7-benzofuranol in the higher accumulation of IAA by Azospirillum sp.     

ENDOSPERM DEVELOPMENT IN THE DATE PALM (PHOENIX DACTYLIFERA) (ARECACEAE)

Date seeds were sampled at regular intervals from pollination (March) to mature fruit (September) and processed for light microscopy and SDS-PAGE. Seed fresh weight rose until early June and then declined slightly through September due to a continuous decrease in water content. Cell wall formation started in May in the free nuclear endosperm and proceeded centripetally from the inner integument to the seed center. Wall thickening in each cell started in cell corners and showed a layered appearance with calcofluor white staining. It started in early June in the center of the seed and proceeded centrifugally such that the outer cells showed cell wall thickening in late June. Thickened cell walls were soft and PAS positive at inception, but staining disappeared and hardness increased during wall maturation. Cell elongation in the radial direction accompanied wall thickening. Protein body formation started after cell wall thickening and followed the same centrifugal developmental pattern. Mature protein bodies occurred in even the outermost cells by early July. No further structural changes occurred after this time. The high molecular weight storage proteins appeared in late June, which is when protein bodies had formed in all but the outer endosperm cells; however, these proteins did not appear simultaneously and minor changes in protein bands continued until maturation. α-Galactosidase activity was present in the developing endosperm and peaked at 13 wk after pollination. The data suggest that the thickened wall is deposited as a highly substituted galactomannan, but that most of the galactose side branches are clipped off presumably by α-galactosidase during cell wall polymerization.     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

Dimethyl fumarate protects thioacetamide‐induced liver damage in rats: Studies on Nrf2, NLRP3, and NF‐κB

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)‐induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA‐induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ‐glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA‐induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains‐containing protein 3; NLRP3, apoptosis‐associated speck like protein containing a caspase recruitment domain; ASC, caspase‐1, nuclear factor‐kappa B; NF‐κB, interleukin‐6), fibrogenic makers (α‐smooth muscle actin; ɑ‐SMA, transforming growth factor; TGF‐β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid‐derived 2)‐like factor 2; Nrf2, superoxide dismutase‐1; SOD‐1, catalase). The present findings concluded that DMF protects against TAA‐induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.