Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

Histopathological studies ofSporothrix schenckii-inoculated mice

Defense mechanisms againstSporothrix schenckii were studied using mouse models. After an intracutaneous injection of the yeast form ofS. schenckii to the dorsal skin of the congenitally athymic nude and normal heterozygote littermate mice, nodules were formed. They regressed and disappeared in 10 weeks in the case of normal mice. On the other hand, nodules and then ulceration developed progressively in nude mice until all animals expired by dissemination of microorganisms at the 11th week of inoculation. Histopathologically the migrated cells were similar in both the normal and the nude mice, particularly during the early phase (within 24 h), with infiltration by PMNs being predominant. Fragmentation ofS. schenckii commenced early during the 12–24 h stage of inoculation in the normal mice, while such fragmentation was scarce in nude mice even though numerous PMNs accumulated. Microscopic observations in the early stages (within 24 h of inoculation) suggested that the lack of killing activity by PMNs in nude mice contributes more to the impaired defense than the lack of macrophage activation by T-cells.     

A robust comparison of biological shapes

Localized differences in the form of two related animal skeletons are more effectively determined when resistant fitting techniques are used rather than at least squares. The repeated median resistant fitting algorithm is introduced. The methods are tested by comparing hominid skulls with those of the least squares fit, in that the differences are more readily identified and agree more closely with the structural differences perceived on biological grounds.     

Les génotypes responsables de mucoviscidose ou d’absence bilatérale des canaux déférents ABCD

Over the last decade, the genetic basis for CBAVD has been identified by its association with CFTR gene mutations, and CBAVD is now generally considered to be a mild or incomplete form of CF. In this review, the author summarizes the main results of compilation of CFTR gene analysis conducted in French laboratories for 3,923 patients with CF and 800 males with CABVD. The degree of clinical expression can be affected by several variables, including the molecular mechanisms by which the various CFTR mutations impair or disrupt the function of the CFTR chloride channel. Phenotypic expression of CFTR mutational genotypes varies from severe, progressive pulmonary disease with pancreatic insufficiency (CF-PI), to mild pulmonary disease with pancreatic sufficiency (PS) or singleorgan forms of “CFTR-opathies”. In CF, a total of 310 different CFTR mutations accounting for 94% of 7,846 CF alleles have generated almost 500 different genotypes, comprising 2 severe mutations in 88% of cases (CF-PI), one severe mutation in trans to a mild mutation in 11% (CF-PS), and 2 mild mutations in 1% of identified genotypes. In CBAVD, 137 mutations scattered over the whole gene were identified in 60% of 1,600 CBAVD alleles during the study. Among the 150 characterized mutational CFTR genotypes, compound heterozygosity was the rule, and the most frequent CBAVD combinations were ΔF508/5T (35%), ΔF508/other mutation (30%, including ΔF508/R117H-7T: 5,6%), and 5T/other mutation (17%). No combination of two severe mutations was found in CBAVD (0%); by contrast with the CF population, 88% of genotypes identified in CBAVD comprised a severe mutation in trans to a mild mutation, and 12% consisted of 2 mild mutations. A total of 22 genotypes were shared by both CF and CBAVD. The role of the 5T allele as a splicing variant with variable, incomplete disease penetrance in CBAVD is reviewed. Other haplotype backgrounds, such as the TG12 sequence and the M470V polymorphism, may influence CFTR splicing and/or function. This study confirms the high molecular heterogeneity of CFTR mutations in CBAVD and emphasizes the importance of extensive CFTR analysis in these patients. Longterm follow-up studies of CBAVD patients are necessary in order to predict the phenotypic consequences of numerous CFTR mutational genotypes.     

Cerebrospinal fluid hypocretin (orexin) levels are elevated by play but are not raised by exercise and its associated heart rate, blood pressure, respiration or body temperature changes

Hypocretin (Hcrt) has been implicated in the control of motor activity and in respiration and cardiovascular changes. Loss of Hcrt in narcolepsy is linked to sleepiness and to cataplexy, a sudden loss of muscle tone which is triggered by sudden strong emotions. In the current study we have compared the effects of treadmill running, to yard play on cerebrospinal fluid (CSF) Hcrt level in normal dogs. We find that treadmill locomotion, at a wide range of speeds, does not increase Hcrt level beyond baseline, whereas yard play produces a substantial increase in Hcrt, even though both activities produce comparable increases in heart rate, respiration and body temperature. We conclude that motor and cardiovascular changes are not sufficient to elevate CSF levels of Hcrt and we hypothesize that the emotional aspects of yard play account for the observed increase in Hcrt.     

Isolation and detailed characterization of human cell lines resistant to -threo-chloramphenicol

The isolation and characterization of chloramphenicol resistant derivatives of the human cell line HeLa B is described. Growth of resistant lines was unaffected in the presence of 100 μg/ml -threo-chloramphenicol, whereas growth of the parental cells was inhibited at 12.5 μg/ml. The incorporation of [³⁵S]methionine into mitochondrial protein of intact resistant cells continued normally in the presence of 100 μg/ml chloramphenicol (cytoplasmic protein synthesis was blocked by addition of 50 μg/ml emetine). Under these conditions the electrophoretic profile of labelled, presumptive mitochondrially-made proteins was similar to that of the parental cell line labelled in the absence of chloramphenicol. The cell lines selected in the presence of chloramphenicol also showed increased resistance to some other inhibitors of mitochondrial protein synthesis, e.g. carbomycin and mikamycin. [¹⁴C]Chloramphenicol was found to have normal access to the interior of resistant cells and it is therefore unlikely that resistance results from altered cell permeability. No modification of the drug by acetylation or glucuronide conjugation mechanisms was observed. The possibilities remain that resistance is mediated by altered permeability of the mitochondrial membrane, or from modification to a component of the mitochondrial protein synthetic system.     

Photosynthetic activity of developing leaves of Zea mays is less affected by heat stress than that of developed leaves

Various physiological and biochemical characters of a leaf change with stages of its ontogeny. It is likely that the photosynthetic functions of leaves of different ontogeny have different levels of heat tolerance. This study was initiated to analyze the photosynthetic heat tolerance of fully-developed, nearly-developed (more than 2/3 expanded) and developing (10–12 cm visible) leaves of two maize genotypes, F223 and F250. The results indicate that the photosynthetic CO₂ assimilation rate (P_n) of developing leaves was less affected by heat stress (42°C in the dark for 90 min) than that of developed leaves. The impaired P_n recovered within 24 h in the developing leaves, while the P_n of developed and nearly-developed leaves did not reach the non-stress level, even after 72 h. The P_n of the developed leaves of genotype F250 was less affected by heat stress than that of genotype F223. After heat stress, the slightly affected P_n of the developing leaf was associated with the almost unchanged photochemical efficiency of photosystem II (F_v/F_m) and the quantum yield of photosystem II electron transport. The chlorophylls a and b were degraded by heat stress; the degradation was pronounced in the developed leaves. As a result of heat stress, the antheraxanthin and zeaxanthin of the xanthophyll cycle accumulated in both the nearly-developed and developed leaves but not in the developing leaves. Injury to the plasma membrane due to heat stress was much less severe in developing leaves than that in the developed leaves. From the physiological characters which we determined it would appear that the P_n functions of the developing leaves are more resistant to heat stress than those of nearly-developed and developed leaves.     

Binding and complement activation by C-reactive protein via the collagen-like region of C1q and inhibition of these reactions by monoclonal antibodies to C-reactive protein and C1q

Ligand-complexed C-reactive protein (CRP), like aggregated or complexed IgG, can react with C1q and activate the classical C pathway. Whereas IgG is known to bind to the globular region and not to the collagen-like region (CLR) of C1q, the site of interaction of C1q with CRP has not been defined. CRP-trimers were prepared by cross-linking and found to bind to C1q and to activate the C system. Heat-aggregated IgG (Agg-IgG) did not block the binding of CRP-trimers to C1q, nor did CRP-trimers block binding of Agg-IgG to C1q, suggesting that CRP and IgG bind at different sites. ELISA and Western blot analysis showed that CRP-trimers bound to the CLR, whereas Agg-IgG bound only to the globular region; similarly, anti-CLR mAb inhibited binding of CRP-trimers to C1q whereas anti-globular region mAb did not. Reactivity with CRP-trimers as well as with Agg-IgG was retained after reduction/alkylation and SDS treatment of C1q. A group of 22 anti-CRP mAb directed against at least six distinct native-CRP epitopes and eight distinct neo-CRP epitopes was tested for ability to inhibit the CRP-CLR interaction; one mAb, anti-native CRP mAb 8D8, with strong inhibitory activity was identified. Fab' of 8D8 blocked binding of CRP-trimers to intact C1q as well as CLR, and also inhibited CRP (CRP-trimers and CRP-protamine complexes) induced C activation, but had no effect on C1q binding or C activation by Agg-IgG. These results indicate that a conformation-determined region on CRP binds to a sequence-determined region on the CLR of C1q in an interaction which leads to C activation. Anti-CRP and anti-C1q mAb that specifically inhibit this interaction are described.