Synthesis and characterization of poly-L-lysine with controlled low molecular weight

N-Carboxy-(N^?-benzyloxycarbonyl)-L -lysine anhydride (Z-L -lysine NCA) was polymerized in dimethylformamide with triethylamine, diethylamine or hexylamine as initiator, at varying molar ratios of NCA to initiator (M/I ratio). After removal of the protecting Z-group the resulting poly-L -lysine was characterized with ¹H NMR and MALDI TOF MS. Both diethylamine- and hexylamine-initiated polymerization yielded poly-L -lysine in which the initiators were incorporated at the carboxylic end of the polymer. This indicates that the NCA polymerization occurred exclusively via nucleophilic attack of the initiator on the monomer. On the other, hand, when triethylamine was used as the initiator, poly-L -lysine was obtained in which no triethylamine-derived end group could be detected by MS. These polymer chains are most likely end-capped with an N-acyl-2,5-dioxopiperazine group at the carboxylic end of the polymer. Incorporation of diethylamine and hexylamine allowed determination of the degree of polymerization (DP) of the obtained polymers by ¹H NMR. The DP depended linearly on the M/I ratio, for both diethylamine and hexylamine, with higher DPs for the diethylamine-initiated poly-L -lysine at equal M/I ratio.     

The early phase of experimental acute renal failure

Experiments were conducted to establish whether diminished solute reabsorption in the loop of Henle during acute renal failure could explain the loss of urinary concentration and participate in generating a tubuloglomerular feedback-mediated reduction in filtration rate. The electrolyte content of the fluid in the ascending limb of the loop of Henle was determined in situ by monitoring its electrical conductivity after propulsion into the distal tubule with a sudden burst perfusion. The value of the minimum electrolyte concentration decreased exponentially with increasing equilibration time, reaching a steady-state value equivalent to 27±9 mM NaCl in normal kidneys, 34±15 mM in mercuric chloride kidneys and 53±22 mM following ischaemia. A mathematical model was derived to describe the process of sodium chloride dilution from which it was possible to calculate both the permeability and transport velocity of the cortical thick ascending limb. In the normal kidney, the transport velocity was calculated to be 4.65±0.92 ·10^–5 cm/s, a value not significantly different from that of the mercuric chloride or ischaemic kidneys, and the estimated permeability was 1.13±0.52·10^–5 cm/s, not different from that of the mercuric chloride kidneys but significantly lower than that calculated for the ischaemic kidneys. It is concluded that for the more severely damaged ischaemic model, the loss of urinary concentrating ability was accompanied by a reduction in diluting ability of the ascending limb of the short loop of Henle, which appears to be due, at least in part, to an elevation of the passive permeability to sodium chloride in this segment.     

A proposed autacoid mechanism controlling mastocyte behaviour

Evidence is provided here supporting the existence of a novel autacoid mechanism negatively modulating mast cell behaviour in response to noxious stimuliin vivo; hence, the denomination “autacoid local inflammation antagonism” (ALIA). In particular, as lipid amides of theN-acylethanolamine type have been reported to accumulate in tissues in degenerative inflammatory conditions, we examined whether theseN-acylated lipids could exert regulatory effects on mast cell activationin vivo. The results reported show that both long- and short-chainN-acylethanolamines, when systemically administered, are effective in reducing mast cell degranulation induced by local injection of substance P in the earpinna of developing rats. These and other data suggest that the endogenous production ofN-acylethanolamines may constitute a local autocrine/paracrine response for the negative feedback control of mast cell responses to various activating signals. Such a process may be of physio-pathological relevance in the regulation of functional neuroimmune-mast cell interactions.

     

Cell-surface sialoglycoconjugate structures in wild-type and mutant Crithidia fasciculata

The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFR^R1 drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac₂) structures mediate influenza C virus cell-binding. The SAα2,3Gal and SAα2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these param- eters the TFR^R1 mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac₂ surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to d-galactose. The bond involves SAα2,6Gal and SAα2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAα2,3Gal and SAα2,6Gal sequences were preferentially expressed by the TFR^R1 mutant. The SAα2,6 linkage markedly predominated. In the TFR^R1 mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAα2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata. Received: 17 September 1998 / Accepted: 22 October 1998     

Electron microscopy of the red pulp of human spleen

Terminating arterial vessels, the structure of sinuses and cords, and the passage of cells through the sinus wall in the red pulp of human spleen were studied. All terminating arterial capillaries arterial capillaries observed opened into cords. The distance between terminating arterial capillaries and sinuses varied. Macrophages were commonly present at arterial terminations. Arterial capillary endothelium contained filaments about 80 Å in diameter. Blood cells were frequently present in the capillary lumen or in passage through the capillary wall into cords. Endothelial cells of sinuses contained three distinctive structures: loosely organized cytoplasmic filaments, tightly organized finer filaments, and micropinocytotic vesicles. Many micropinocytotic vesicles about 0.1 μ in diameter were present just beneath the plasma membrane of the lateral and luminal sides of sinus endothelial cells and a few at the basal aspect. Loosely organized filaments about 80 Å in diameter ran parallel to the longitudinal axis of the sinus endothelium. The finer filaments about 30–50 Å in diameter were tightly organized as filamentous bands and present basally. The filaments of the bands appeared inserted upon the plasma membrane. They were also present in the cordal reticular cells and terminating arterial capillaries. Free cells were frequently present in passage through the slits of the sinus wall. There were no preformed or fixed apertures in the sinus wall. The basement membrane and reticular fibers were completely covered by the endothelial cells and/or cordal reticular cells. It is likely that those slits between endothelial cells in the sinus wall not covered by the basement membrane are potential passageways for cells moving from the cords into the sinus. The larger cytoplasmic filaments are likely contractile. The filamentous bands appear to maintain cell shape, stabilize the wall in relation to the basement membrane, and are probably operative in the control of cellular passage through the slits of sinus wall.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Novel missense mutations and a 288-bp exonic insertion in PAX9 in families with autosomal dominant hypodontia

We describe the molecular analysis of three families with hypodontia involving primarily molar teeth and report two novel mutational mechanisms. Linkage analysis of two large families revealed that the hypodontia was linked to the PAX9 locus. These two families revealed missense mutations consisting of a glutamic acid substitution for lysine and a proline substitution for leucine within the paired domain of PAX9. A pair of identical twins affected with hypodontia in a third family demonstrated a 288-bp insertion within exon 2 that resulted in a putative frameshift mutation and a premature stop codon. The insertion was associated with the loss of 7-bp from exon 2. A block of 256-bp of sequence within the insertion was completely identical to downstream sequence from the second intron of the PAX9 gene. These studies extend the spectrum of mutations in PAX9 associated with hypodontia to include heretofore undescribed categories, including missense mutations.     

RSV-induced bronchiolitis but not upper respiratory tract infection is accompanied by an increased nasal IL-18 response

The aim of this study was to investigate potential differences in the local nasal immune response between bronchiolitis and upper respiratory tract infection induced by respiratory syncytial virus (RSV). Nasal brush samples were obtained from 14 infants with RSV bronchiolitis and from 8 infants with RSV upper respiratory tract infection. The samples were taken during infection (acute phase) and 2-4 weeks later (convalescent phase). Cytospin preparations were stained immunohistochemically for T cells, macrophages, and eosinophils. Staining also took place for intercellular adhesion molecule-1 (ICAM-1), T-helper 1 (Th1)-like (interleukin-12 [IL-12], interferon-gamma [IFN-gamma]), Th2-like (IL-4, IL-10), and proinflammatory cytokines (IL-6, IL-8, IL-18). During both RSV-induced bronchiolitis and upper respiratory tract infection, cellular inflammation was observed. This was characterised by an increase in the numbers of nasal macrophages, which tended to be higher in bronchiolitis than in upper respiratory tract infection. Numbers of T lymphocytes and ICAM-1 positive cells increased during both bronchiolitis and upper respiratory tract infection. There were no differences between numbers in the groups. Interestingly, a distinct nasal proinflammatory cytokine response was observed in RSV-induced bronchiolitis. This is characterised by an increase in the number of IL-18 positive cells. This increase is specific for bronchiolitis, as a similar increase could not be detected in RSV-induced upper respiratory tract infection. Numbers of IL-6 and IL-12 positive cells were higher in both bronchiolitis and upper respiratory tract infection, and there were no differences between the groups. By contrast, the number of IL-8, IFN-gamma, IL-4, and IL-10-positive cells remained constant. In conclusion, clear differences were found in nasal immune responses of children with RSV-induced upper respiratory tract infection or bronchiolitis. The induction of a strong IL-18 response was typical for bronchiolitis, as this could not be observed in RSV-induced upper respiratory tract infection, and could explain the eosinophilia that is observed frequently during bronchiolitis.