Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

The amnesic shellfish poison domoic acid enhances neurotoxicity by excitatory amino acids in cultured neurons

Summary A recent episode of human intoxication by cultured mussels containing a rare excitatory amino acid named domoic acid, received particular attention for its neurological implications. The intoxication produced neurological problems, such as headache, confusion, and loss of memory, particularly severe at times. Neuronal damage was found in the hippocampus and amygdala of four patients. We now report that in neuronal cultures the neurotoxicity of a domoic acid-containing mussel extract is the result of domoic acid potentiation of the excitotoxic effect of glutamic acid and aspartic acid present in high amounts in mussel tissue. Moreover, we show that subtoxic concentrations of domoic acid are sufficient to potentiate glutamic acid and aspartic acid neurotoxicity. We present evidence suggesting that the neurotoxic synergism may be due to a reduction of Mg^{+ +} block at the NMDA receptor-associated channel, following activation of NON-NMDA receptors by domoic acid.     

Microvascular transposition of the arterial supply to the testis of the boar.

The origin of the arterial supply to a testis was changed in 7 boars with a microsurgical technique. A. testicularis was connected, end to side, to a branch of the femoral artery. The capillary blood flow was unchanged, indicating viable transplants. The temperature difference between testis and body disappeared after the surgery, indicating an interruption of the heat transfer in the pampiniform plexus. The lower values of the histological score and the possibly lower peripheral plasma concentration of testosterone after the arterial by-pass may indicate a physiological importance of the counter current exchange.     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

Cholesteryl hemisuccinate's inductive effect on membrane rigidization regarding both, its remodelling of the cells' surface receptor pattern and its decreasing the natural killer susceptibility of K-562 cells.

The relationship of changes in membrane fluidity to natural killer susceptibility of K-562 target cells was investigated. Membrane rigidization was performed by the chemical modulator cholesteryl hemisuccinate. Steady-state fluorescence polarization measurements of the diphenyl hexatriene labelled, modified K-562 cells revealed that cholesteryl hemisuccinate increased the structural order of the hydrophobic region of membranes in a dose dependent way. Investigation of natural killer susceptibility followed by 51Cr release assay indicated that modified cells are less sensitive to natural killer attack. To elucidate whether surface structures such as transferrin and lectin receptors are associated with the altered susceptibility, the surface density of these receptors was followed by (I-125)-transferrin binding assay and quantitative immunofluorescence. We found that the number of transferrin and concanavalin A receptors increased by a factor of 2.44 and 2.00, respectively, whereas that of the wheat germ agglutinin receptor failed to exhibit any changes upon rigidization. From the results we concluded that i the membrane structural order does influence the natural killer susceptibility, ii changes in membrane structural order result in alteration of the number of cell surface transferrin and lectin receptors, iii however, no direct relationship seems to exist between these two events.     

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.