RiV Particles are Heat Stable

The “reaction pattern in vertebrate cells” (RiV) is mainly represented by characteristic particles with a diameter of 50 ± 15 nm and smaller RiV‐specific units probably as reaction products of the cells to specific stress. As vaccines they have been successfully applied against laryngeal papillomatosis and experimentally against foot‐and‐mouth disease of guinea pigs and mammary carcinoma of mice. Subsequently, the small‐scale treatment of AIDS, cancer and hepatitis C was started in a single doctor's practice. RiV samples of calf kidney cell culture contain identified proteins and non‐viral ribonucleic acid. Vaccine samples and samples for heat stability studies were produced from roller bottle cultivated cells after differential centrifugation and a short solvent treatment. The preparations were examined by electron microscopy, ELISA, protein determination, SDS‐PAGE, immunoblot and UV spectroscopy. Since the efficacy of heated RiV (10 min at 100 °C) against the growth of balb/c mouse myeloma was higher than that of the non‐heated sample, heated RiV samples were investigated by electron microscopy, immunoelectron microscopy (IEM) and ELISA to find a possible explanation for these results. The RiV particles were surprisingly heat‐stable, after 10 (up to 60) min of boiling no differences were observed in morphology and diameter in comparison to the untreated sample. Furthermore, no difference was seen by IEM in the reaction of these RiV particles with an anti‐RiV antiserum relative to the untreated sample. When two samples were compared by ELISA using the same antiserum, the reactivity of the heated samples decreased quickly during the first minutes of boiling, but the decrease became much slower later on. In conclusion, there seemed to be heat‐sensitive as well as heat‐stable reactive components within the RiV samples. The heat‐stable component(s) should be associated with the RiV particles. Further studies are necessary to find out whether the positive results of the mouse myeloma experiment could also be obtained following the treatment of other cancers, AIDS and hepatitis C and also to answer the question, which of the known proteins are important for the therapeutic activities of RiV samples.     

Profiles of Leaf Nitrogen and Light in Reproductive Canopies of Cotton (Gossypium hirsutum)

During vegetative growth, the vertical profile of leaf nitrogen(N) often parallels the profile of light distribution withinthe canopy. This is more advantageous in terms of canopy photosynthesisthan a uniform distribution of leaf N. We investigated the influenceof both reproductive growth and N supply on the profiles ofN and light in canopies of irrigated cotton crops (Gossypiumhirsutum L.). Regular samplings were made from soon after theonset of reproductive growth until crop maturity. Every 2 weeks,a 1 m²sample of the canopy was cut in four successive verticallayers of equal thickness. Leaf area and N concentration (%)in each layer were measured. The vertical N gradient becamemore marked with ongoing reproductive development. It is hypothesizedthat because of the high rate of growth after the onset of reproductivedevelopment and the long duration of this phase compared toother species, the whole canopy photosynthetic benefit thatwould accrue from maintaining the N gradient is likely to beaccentuated. The rate of decline in leaf N concentration ina layer was not related to either the initial concentrationin the leaves nor the boll load within the layer.Copyright 2001Annals of Botany Company Gossypium hirsutum, leaf nitrogen, light profile, nitrogen, nitrogen distribution, remobilization, reproductive growth     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Loss of rubidium and potassium from barley roots on sudden chilling

Sudden chilling of roots of barley pretreated in a labeled Rb⁺ solution at 25 C causes a transient loss of label from the nonfree space. The half-time of the release is about 10 minutes. Within the first few hours of uptake the loss increases proportionally to the root Rb⁺ content but varies between experiments as a fraction of this content: values from 4 to 20% were observed. The same phenomenon is found for K⁺ labeled with ⁸⁶Rb⁺     

Localization of inorganic ions by precipitative freeze dissolution

Summary A new method for localization of inorganic diffusible ions in tissue is introduced. It has been applied to localization of Tl⁺ and Rb⁺ in barley roots and is probably also suited for Cs⁺, Ca²⁺, Cl^–, Br^–, PO ₄ ^3– and perhaps K⁺. Its principle consists of dissolution of the ice from frozen tissue in a concentrated aqueous solution of a precipitating agent that is kept at a temperature just above its melting point.     

Diffusion of carbon dioxide through lipid bilayer membranes. Effects of carbonic anhydrase, bicarbonate, and unstirred layers

Diffusion of (14)C-labeled CO(2) was measured through lipid bilayer membranes composed of egg lecithin and cholesterol (1:1 mol ratio) dissolved in n-decane. The results indicate that CO(2), but not HCO(3-), crosses the membrane and that different steps in the transport process are rate limiting under different conditions. In one series of experiments we studied one-way fluxes between identical solutions at constant pCO(2) but differing [HCO(3-)] and pH. In the absence of carbonic anhydrase (CA) the diffusion of CO(2) through the aqueous unstirred layers is rate limiting because the uncatalyzed hydration-dehydration of CO(2) is too slow to permit the high [HCO(3-)] to facilitate tracer diffusion through the unstirred layers. Addition of CA (ca. 1 mg/ml) to both bathing solutions causes a 10-100-fold stimulation of the CO(2) flux, which is proportional to [HCO(3-)] over the pH range 7-8. In the presence of CA the hydration- dehydration reaction is so fast that CO(2) transport across the entire system is rate limited by diffusion of HCO(3-) through unstirred layers. However, in the presence of CA when the ratio [HCO(3-) + CO(3=)]:[CO(2)] more than 1,000 (pH 9-10) the CO(2) flux reaches a maximum value. Under these conditions the diffusion of CO(2) through the membrane becomes rate limiting, which allows us to estimate a permeability coefficient of the membrane to CO(2) of 0.35 cm s(-1). In a second series of experiments we studied the effects of CA and buffer concentration on the net flux of CO(2). CA stimulates the net CO(2) flux in well buffered, but no in unbuffered, solutions. The buffer provides a proton source on the upstream side of the membrane and proton sink on the downstream side, thus allowing HCO(3-) to facilitate the net transport of CO(2) through the unstirred layers.     

Bacillus subtilis Bactofilins Are Essential for Flagellar Hook- and Filament Assembly and Dynamically Localize into Structures of Less than 100 nm Diameter underneath the Cell Membrane

Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.     

Prevalence of methotrexate intolerance in rheumatoid arthritis and psoriatic arthritis

Introduction

The aim of this study was to determine the prevalence of gastrointestinal and behavioural symptoms occurring before (anticipatory/associative) and after methotrexate (MTX) administration, termed MTX intolerance, in rheumatoid (RA) and psoriatic arthritis (PsA).

Methods

Methotrexate Intolerance Severity Score (MISS), previously validated in juvenile idiopathic arthritis patients, was used to determine MTX intolerance prevalence in 291 RA/PsA patients. The MISS consisted of four domains: abdominal pain, nausea, vomiting and behavioural symptoms, occurring upon, prior to (anticipatory) and when thinking of MTX (associative). MTX intolerance was defined as ≥6 on the MISS with ≥1 point on anticipatory and/or associative and/or behavioural items.

Results

A total of 123 patients (42.3%) experienced at least one gastrointestinal adverse effect. The prevalence of MTX intolerance was 11%. MTX intolerance prevalence was higher in patients on parenteral (20.6%) than on oral MTX (6.2%) (p < 0.001).

Conclusion

Besides well-known gastrointestinal symptoms after MTX, RA and PsA patients experienced these symptoms also before MTX intake. RA and PsA patients on MTX should be closely monitored with the MISS for early detection of MTX intolerance, in order to intervene timely and avoid discontinuation of an effective treatment.     

Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.