Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)     

Expression of lipoprotein lipase gene in combined lipase deficiency

The expression of the gene for lipoprotein lipase (LPL) was studied in brown adipose tissue and the liver of combined lipase deficient (cld/cld) and unaffected mice. The mRNA specific for LPL was detected in both animals. Although the size of LPL mRNA in cld mice was similar to that of unaffected mice, the mRNA concentration in affected animals was higher than in unaffected animals. We also studied the LPL gene mutation in cld mice by Southern blot analysis. No restriction fragment length polymorphisms were observed after digestion with 16 endonucleases. These data indicate that there is no gene insertion or deletion, but do not exclude the possibility of point mutation in the LPL structural gene. However, the present results agree with the hypothesis that the genetic defect in cld is not due to a mutation in the LPL structural gene, but instead involves the defective post-translational processing of LPL or defective cellular function affecting transport and secretion of this enzyme group.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

A novel IgA protease from Clostridium sp. capable of cleaving IgA1 and IgA2 A2m(1) but not IgA2 A2m(2) allotype paraproteins

Three bacterial strains of Bifidobacterium and Clostridium sp. from patients with inflammatory bowel disease (I.B.D.) and Streptococcus pneumoniae from a patient with pneumonia were identified to produce extracellular proteases cleaving IgA into Fab and Fc fragments. Although the proteases from the Bifidobacterium and the Streptococcus pneumoniae showed the characteristics of typical IgA1 proteases, cleaving the IgA of only the IgA1 subclass, the protease from Clostridium sp. revealed a dual substrate specificity, in that it cleaved both IgA1 and IgA2 of the A2m(1) allotype. The latter protease, however, did not show any activity with respect to the IgA2 of the A2m(2) allotype. Fc fragments isolated from the IgA1 and the IgA2 A2m(1) by digestion with the Clostridium sp. protease were identified to have an identical amino terminal residue of valine. The site of cleavage in both the alpha 1 and the alpha 2 of A2m(1) by the protease was assumed to be an identical peptide bond at Pro(221)-Val(222), which is a common one present just before the hinge of both the alpha 1 and the alpha 2 of the A2m(1) but not of the alpha 2 of the A2m(2). The protease was sensitive to ethylene-diamino tetraacetic acid, a chelating agent, similar to other already reported IgA1 proteases.     

Clostridium ramosum, an IgA protease-producing species and its ecology in the human intestinal tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (propionate-rifampicin-gentamicin-colimycin-polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease-positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease-positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.     

Membrane Potentials of Heterotrophically Cultured Tobacco Cells

The electrophysiological properties of the membrane of Nicotianatabacum var. Sarnsun cultured cells were determined using amicroelectrode technique in standard medium containing 1 mMKC1, 1 mM NaCl and 1 mu CaCl₂ at pH 7. Tobacco callus was derivedfrom the pith (E_m=–104.4%16.2 mV). The membrane potentialsof the callus cells did not show a symmetrical Gaussian distributionbut were scattered over a wide range. The percentage of highmembrane potential cells increased as the subculture was continueduntil about 11 months and then decreased. The response of themembrane potential to electric stimulus, ionic composition,metabolic inhibitors, sugars and amino acids was characteristicof high (E_m=–{small tilde}–160 220 mV; H-cells)and low (E_m=–80{small tilde}–90 mV; L-cells) membranepotential cells. The membrane potential of H-cells was largelydepolarized by addition of CN^–, carbonium cyanide m-chlorophenylhydrazone,decyclohexylcarbodiimide, and triphenyltin chloride and transientlydepolarized by addition of glucose, galactose, mannose or sucrose,and D-alanine, L-alanine or Llysine, but the membrane potentialof L-cells was not. (Received December 3, 1982; Accepted March 16, 1983)     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.     

Infrared analysis of pea stem cell walls and oriented structure of matrix polysaccharides in them

Infrared absorption spectra of film specimens of the epidermaland parenchyma cell walls of the third internode of pea stem,before and after protease treatment and after treatment forremoval of lipid materials, pectic substances and hemicellulose,were recorded, and characteristic bands in the spectrum of thewall were assigned. Polarization spectrum measurements of thewall provided evidence indicating that the non-cellulosic polysaccharidematrix as well as cellulose microfibrils has an oriented structurein the wall which changes during extension growth as well asupon mechanical extension of the walls. (Received March 9, 1978; )