Modulation of polymorphonuclear leukocytes function by incubation with human serum from oxidant-challenged individuals

Polymorphonuclear leukocytes (PMN) from healthy donors were tested for stimulated release of superoxide anions after being incubated with serum of welders and of a group of unexposed individuals. These two groups were further subdivided either according to age or to smoking habits. The experiments showed that stimulated superoxide production from PMN was inhibited (P < 0.05) by serum from young smokers as compared to that of young nonsmokers, both from the unexposed group. Incubation of PMN with serum from elderly nonsmoking individuals decreased superoxide production as compared to incubation with serum from young nonsmoking individuals, both from the unexposed group. A decrease in superoxide production by incubation with serum of welders as compared to that of unexposed individuals was significant only when the comparison was carried out between the young, non-smoking subgroups. These findings suggest that age, smoking, and exposure to oxidants induce appearance in serum of factors that affect the PMN function     

Red blood cell rouleaux formation in dextran solution: dependence on polymer conformation

The velocity of rouleaux formation (RF), as previously shown, increases with increasing dextran concentration up to a critical concentration (C_a), beyond which the addition of dextran reduces the RF velocity (RFV). de Gennes' model for polymer solutions suggests that dextrans exist in two conformations: a coil structure at low concentrations, which changes to a network beyond a critical concentration (C^*). In the present study we examined the relation between C_a and C^* for dextrans of different molecular weight, and found that they coincide. This suggests that the change in dextran behavior, from increasing to decreasing RFV, occurs when their conformation changes from coil to network. In addition, it has been reported that in dilute dextran solutions the intercellular distance (D) between RBC in rouleaux increases with the molecular weight of the dextran. We found that D correlates with R_f, the end-to-end distance of the polymer molecule, and for all dextrans D ≤ 1.5 R_f. In accord with de Gennes' Model for polymers between surfaces, this corresponds to intercellular interaction with two overlapping surface-associated polymer layers, which may extend “tails” to interact with the opposing cells. Received: 8 August 1997 / Accepted: 28 November 1997     

Post-fertilization changes in the zona pellucida glycoproteins of rat eggs

The zona pellucida (ZP) is the extracellular coat surrounding the mammalian egg. Numerious evidence supports the role of ZP carbohydrate residues as the specific sperm receptors. In this study we used lectins to study different distribution patterns of carbohydrate residues in the rat ZP, and to follow changes at fertilization. ZP were collected from follicular, ovulated, and fertilized eggs, incubated with one of 11 different biotin-labeled lectins, followed by avidin-fluorescein isothiocyanate (FITC) complex, and visualized by epifluorescent microscopy. For electron microscope (EM) histochemistry, eggs were embedded in LR white and ultrathin sections were stained with the complexRicinus communis lectin (RCA-1)-colloidal gold. Some lectins (RCA-I,Glycine max) bound to the entire ZP while others were restricted to the inner or outer zones [Griffonia simplicifolia, Concanovalia ensiformis, Triticum vulgaris (WGA), succinyl-WGA]. Other lectins (Lens culinaris, Ulex europhaeus) were totally excluded. The RCA-1 binding pattern changed following sperm penetration, from homogeneous in ZP of ovulated eggs (57%) to uneven in ZP of fertilized (71%) or activated (68%) eggs. Our results demonstrate an uneven distribution of different sugar residues in the rat ZP, and a post-fertilization change in the distribution of β-galactose, which is specifically recognized by RCA-I, presumably correlated with other changes in the ZP that lead to the block to polyspermy. This work is in partial fulfillment of the requirements for the PhD degree of Tamar Raz at the Sackler School of Medicine, Tel Aviv University     

5-Hydroxytryptophyl peptides: potent inhibitors of a storage compartment of serotonin.

Several analogues of 5-hydroxytryptophan were tested for their ability to inhibit the binding of serotonin to serotonin-binding protein (SBP), a protein found within serotonergic neurons which has a high affinity for serotonin. An N-substituted dipeptide, N-acetyl-5-hydroxytryptophan-5-hydroxytryptophan amide, was found to be an inhibitor of this binding. The inhibition (50% at 1.0 μM) was specific, since it did not affect other known sites of serotonin binding. The binding of serotonin to its membrane receptor was not affected by the dipeptide (up to 10 μM). Uptake of serotonin by synaptosomes was only slightly affected (9% at 10 μM), and aromatic-L-amino-acid carboxy-lyase(EC 4.1.1.28) and amine: oxygen oxidoreductase (deaminating) (flavin-containing) (EC 1.4.3.4) were not inhibited (10 μM and 5 mM respectively), The peptide was not hydrolyzed by honiogenates of brain or myenteric plexus. The ¹⁴C-labelled dipeptide was shown to be taken up by synaptosomes. However, the uptake of the peptide was not affected either by drugs that inhibit serotonin uptake or by serotonin itself although the uptake was abolished by excess 5-hydroxytryptophan. Intraventricular injection of N-acetyl dipeptide caused a biphasic effect depending on dose. Lower doses (10nmol) induced a decrease in serotonin brain levels (40%). Higher doses (300 nmol) caused a 95% increase in serotonin levels. It is suggested that 5-hydroxytryptophyl peptides may be used as potent specific inhibitors of SBP, a storage compartment of serotonin.     

Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.     

The effect of vitamin E administration to pregnant women, on the concentration of linoleic acid in adipose tissue of the newborns

The composition of various lipids were measured in ten healthy newborns of mothers who were treated with 100 mg vitamin E per day, starting in the 35th week of pregnancy. The maternal serum vitamin E levels following the treatment (1.10 +/- 0.49 mg/dl) showed no increase as compared to the pretreatment values (1.06 +/- 0.38 mg/dl). There was no significant difference in serum vitamin E levels in those newborns whose mothers were treated--(0.19 +/- 0.12 mg/dl) as compared to 10 newborns whose mothers were not treated with vitamin E (0.13 +/- 0.07 mg/dl). However, there was a striking increase in the relative concentration of linoleic acid in the adipose tissue to those newborns whose mothers were treated (9.4%--versus--4.1%).     

Identification, Purification, and Characterization of Two Forms of Serotonin Binding Protein from Rat Brain

Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K from of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by--SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants--56K form showed higher affinity for 5-HT (KD1 = 0.4 nM; KD2 = 32 nM), whereas the 45K form showed lower affinity (KD1 = 9.7 nM; KD2 = 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity--56K (19:1), 45K (10:1); (3) isoelectric point--the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)