Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01- 10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

The groin flap in reparative surgery of the hand

The historical literature of the use of axial vascular pattern flaps from the hypogastric and iliofemoral regions in reparative surgery of the hand is concisely reviewed. Thirty-six iliofemoral (groin) flaps were utilized for delayed primary resurfacing and secondary reconstruction of defects of the hand and forearm. Two flaps (6 percent) were complicated by partial necrosis. We caution against the immediate resurfacing (within 24 hours of injury) of acute crushed hand wounds by distant flaps. The immediate application of a healthy flap on a soiled or crushed wound invites complications of local tissue necrosis, infection, and subsequent loss of the flap. When distant flaps are indicated for coverage of acute hand wounds, delayed primary coverage following complete removal of all nonviable tissue is a safe and reliable regimen. It is advantageous to design the serviceable portion of the flap on the distal area of the vascular territory of the groin flap. Thoughtful yet "radical" defatting can be performed on the lateral portion of the groin flap territory. Constructed in this way, the long medial base of the groin flap allows freedom for movement at the wrist and metacarpophalangeal and interphalangeal joints, thus decreasing edema and stiffness. In the management of soft-tissue defects in the hand requiring distant flap coverage, we choose to utilize the conventional groin flap in preference to the microvascular free flap when both techniques will deliver equal results.     

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.     

Modification of susceptibility to Klebsiella pneumoniae during murine cytomegalovirus infection

The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 X 10(3) cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30-40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice.     

Long-acting contraceptive agents: Norethisterone esters of arylcarboxylic acids

The synthesis of esters of norethisterone (17α-ethynyl-17β-hydroxy-estr-4-en-3-one) with acids containing a benzene ring is described, two methods of esterification being compared in terms of yield and convenience. The activities of these esters as long-acting contraceptive agents have been evaluated.     

Identification and physical characterization of yeast maltase structural genes

Summary Each of at least five unlinked MAL loci (MAL1 through MAL4 and MAL6) on the yeast genome controls the ability to synthesize an inducible -D-glucosidase (maltase). A subcloned fragment of the coding sequence of the MAL6 maltase structural gene was used as a hybridization probe to investigate the physical structure of the family of MAL structural genes in the genomes of different Saccharomyces strains. Mal⁺ strains, each carrying a genetically defined MAL locus, were crossed with a Mal^- strain and the segregation behavior of the functional locus and of sequences complementary to the maltase structural gene at that locus analyzed. The maltase structural gene sequences of each MAL locus were detected by Southern blot hybridization using BamH1 digests of genomic DNA of the meiotic products. This restriction enzyme was previously shown to cleave outside the confines of the MAL6 locus.The results of such experiments indicate that each MAL locus encompasses at least one maltase structural gene sequence homologous to that of MAL6, that yeast strains that lack functional MAL loci may or may not contain the corresponding maltase structural gene sequence, that the MAL1 maltase structural gene sequence or one of its alleles can be detected in all laboratory yeast strains examined and that each MAL locus can be identified as a characteristic BamH1 fragment of genomic DNA which includes a maltase structural gene.Yeast strains vary in the number of maltase structural gene sequences that they carry. By using the approach described in this report, the ones corresponding to the different functional MAL loci and residing within a BamH1 generated restriction fragment can be identified.     

Purification and Properties of 4-Hydroxy-2-Ketopimelate Aldolase from Acinetobacter

The chemical synthesis of 4-hydroxy-2-ketopimelic acid is described. An aldolase that cleaves this compound to succinic semialdehyde and pyruvate has been purified from Acinetobacter grown at the expense of 4-hydroxyphenylacetic acid. The molecular weight of the enzyme was about 158,000 from sedimentation equilibrium data; other physical determinations gave values in reasonable agreement. The protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 25,700. The enzyme attacked both enantiomers of synthetic 4-hydroxy-2-ketopimelate and was stimulated by Mg(2+) and Mn(2+) ions.     

Mitotic chromosomes of species in the subgenusDrosophila (Diptera:Drosophilidae)

The karyotypes of 17 species in the subgenusDrosophila are compared according to their taxonomical relationships. Although closely related species often possess similar karyotypes, the karyotypes diverge considerably within the subgenus. Thus extensive chromosome rearrangements did occur during the speciation. Species with higher chromosome numbers do not necessarily have higher average of total chromosome length per cell.