Secretion of gamma-glutamyltranspeptidase by the pancreas: evidence for a membrane shedding process during exocytosis

Gamma Glutamyltranspeptidase (GGT) is a membrane-bound enzyme involved in glutathione metabolism. It is present in rat exocrine pancreas at a level which is only exceeded by the kidney. It has been previously shown that most of the enzyme activity is located in the apical area of the acinar cell, more precisely at the level of zymogen granules and plasma membrane. The aim of the present study was to examine the secretory behavior of that enzyme. Under resting conditions, in vivo, high levels of GGT were found in pancreatic juice and its level was not related to protein concentration. Under secretin infusion, a relatively constant level of GGT was released, and again, there was no correlation between enzyme activity and protein concentration. Following a bolus injection of caerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. This observation is in agreement with in vitro determinations on isolated zymogen granules showing that GGT is totally associated with the ZG membrane and undetect-able in the content of these organelles. The present data show that 1 degree GGT is released from the rat pancreas acinar cells in a particulate form; 2 degree GGT release is elicited by hormonal stimulation coinciding with the exocytotic release of secretory proteins. Our observations lead us to propose that in rat pancreas, ZG membrane fragments are released along with secretory proteins during exocytosis.     

A Direct Screening Procedure for Gravitropism Mutants in Arabidopsis thaliana (L.) Heynh.

In order to isolate gravitropism mutants of Arabidopsis thaliana (L.) Heynh. var Estland for the genetic dissection of the gravitropism pathway, a direct screening procedure has been developed in which mutants are selected on the basis of their gravitropic response. Variability in hypocotyl curvature was dependent on the germination time of each seed stock, resulting in the incorrect identification of several lines as gravitropism mutants when a standard protocol for the potentiation of germination was used. When the protocol was adjusted to allow for differences in germination time, these lines were eliminated from the collection. Out of the 60,000 M2 seedlings screened, 0.3 to 0.4% exhibited altered gravitropism. In approximately 40% of these mutant lines, only gravitropism by the root or the hypocotyl was altered, while the response of the other organ was unaffected. These data support the hypothesis that root and hypocotyl gravitropism are genetically separable.     

cDNA cloning and localization of the mouse leukosialin gene (Ly48) to chromosome 7

Mouse leukosialin, previously known as the 3E8 antigen, is expressed primarily on cells of the hematopoietic and lymphoid lineages and is shown to be the mouse homologue to the human leukosialin/sialophorin and rat W3/13 molecules. A partial leukosialin cDNA clone was isolated via cross-species hybridization with a portion of a human leukosialin cDNA. This mouse cDNA clone was used to demonstrate that the leukosialin isoforms are encoded by a single mRNA species of approximately 4.2 kilobases (kb) and that the leukosialin gene is located on chromosome 7. Based on these results, mouse leukosialin is given the designation Ly48.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M30693.     

Organometallic derivatives of estradiol as bioligands: targetted binding of the estradiol receptor

The complexation of estrogens by transitional metal units e.g. (alkyne)Co2(CO)6 and (alkyne)Mo2Cp2(CO)4, at the 17 alpha-position brings about a dramatic change in the chemical behavior of these compounds with respect to that of the free ligands. The 17 beta-OH function becomes particularly labile, even in weakly acidic medium, giving rise to carbenium ion-like species, from which, depending on the metal and the nucleophile, substitution, elimination and rearrangement take place. This situation provides the basis for a new type of active site directed-reagent for estradiol receptor. The hypothesis of vicinal space positioning of an acidic and a nucleophilic group in the estradiol receptor cavity is examined in the light of the amino-acid composition of the steroid binding domain. The requirement of the sulfhydryl group of a cysteine residue is suspected in the first step of the receptor inactivation process.     

Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.     

Specific binding sites on human phagocytic blood cells for Gly-Leu-Phe and Val-Glu-Pro-Ile-Pro-Tyr, immunostimulating peptides from human milk proteins.

Two immunostimulating peptides were isolated from human milk proteins by enzymatic digestion, the tripeptide GLF and the hexapeptide VEPIPY. These peptides increased the phagocytosis of human and murine macrophages and protected mice against Klebsiella pneumoniae infection. The present study showed that this activity may be correlated to the presence of specific binding sites on human blood phagocytic cells. The receptor molecules implicated were different for the two peptides. [3H]GLF specifically bound to PMNL and monocytes, whereas [3H]VEPIPY only bound to monocytes. The leukemic promyelocytic cell line HL-60 differentiated into granulocytes or into macrophages (depending on inducer used) coroborated these results. Specific binding of [3H]GLF on plasma membrane preparations of human PMNL (20 degrees C) was saturable and Scatchard analysis indicated two classes of binding sites: high-affinity sites of Kd 2.3 +/- 1.0 nM and Bm 60 +/- 9 fmol/mg protein and low-affinity sites of Kd 26.0 +/- 3.5 nM and Bm 208 +/- 45 fmol/mg protein. [3H]GLF binding was inhibited in a concentration-dependent manner by various analogous peptides, such as LLF, GLY, LLY and RGDGLF, but not by RGD, RGDS, VEPIPY and the chemotactic peptide f-Met-Leu-Phe (f-MLF). Only at high concentrations the direct analog MLF competed with labeled GLF. An important inhibitory effect was also observed with C1q component of the complement whereas C3 and BSA were uneffective. Specific binding of [3H]VEPIPY on monocyte membranes (20 degrees C) was saturable and Scatchard analysis was consistent with one class of binding sites of Kd 3.7 +/- 0.3 nM and Bm 150 +/- 6 fmol/mg protein.