Immunochemical purification of probe-labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.     

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

No difference in pubertal growth and final height between treated hypogonadal and non-hypogonadal thalassemic patients

BACKGROUND: Many factors can negatively affect growth in thalassemic patients, and hypogonadism has been considered as the main factor responsible for their pubertal growth failure. OBJECTIVE: To evaluate the influence of hypogonadism and its treatment on pubertal growth and final height in thalassemic patients. METHODS: We compared the growth of 28 hypogonadal thalassemic patients in whom puberty was induced to that of 25 patients in whom puberty occurred spontaneously. RESULTS: In both groups of patients we observed reduced peak height velocity (induced puberty: females 4.9 +/- 2.1, males 6.0 +/- 1.8 cm/year; spontaneous puberty: females 6.1 +/- 1.5, males 7.3 +/- 2.1 cm/year) and pubertal height gain (induced puberty: females 11.3 +/- 4.0, males 18.0 +/- 4.5 cm/year; spontaneous puberty: females 15.8 +/- 2.7, males 18.1 +/- 5.3 cm/year) and a short final height (induced puberty: females -1.8 +/- 0.7, males -2.1 +/- 1.0 SDS; spontaneous puberty: females -2.3 +/- 1.0, males -1.9 +/- 1.0 SDS). CONCLUSIONS: Poor pubertal growth is present in thalassemic patients regardless of hypogonadism. Other factors are responsible for the reduced growth spurt and the final short stature observed in these patients.     

A case of granulomatous skin lesions caused by Mycobacterium marinum in the Campania region

The diagnosis of cutaneous Mycobacterium marinum infection is frequently presumptive, as detection by conventional methods is difficult. We describe a patient with granulomatous skin lesions on the right dorsal hand and forearm. Histological examinations were presumptive for mycobacterium lesions. We identified Mycobacterium marinum directly in the patient's lesional skin biopsy combining polymerase chain reaction (PCR) amplification using Mycobacterium genus-specific primers, and subsequent restriction enzyme analysis enabling identification to the species level. The symptoms were no longer present after specific therapy, thereby confirming the initial diagnosis.     

Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae

Twenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups.     

Innate defence functions of macrophages can be biased by nano-sized ceramic and metallic particles

Nano-sized particles of ceramic and metallic materials are generated by high-tech industrial activities, and can be generated from worn-out replacement and prosthetic implants. The interaction with the human body of such nanoparticles has been investigated, with a particular emphasis on innate defence mechanisms. Human macrophages (PMA-differentiated myelomonocytic U-937 cells) were exposed in vitro to non-toxic concentrations of TiO(2), SiO(2), ZrO(2), or Co nanoparticles, and their inflammatory response (expression of TLR receptors and co-receptors, and cytokine production) was examined. Expression of TLR receptors was generally unaffected by exposure to the different nanoparticles, except for some notable cases. Exposure to nanoparticles of ZrO(2) (and to a lesser extent TiO(2)), upregulated expression of viral TLR receptors TLR3 and TLR7. Expression of TLR10 was also increased by TiO(2) and ZrO(2) nanoparticles. On the other hand, TLR9 expression was decreased by SiO(2) nano-particles, and expression of the co-receptor CD14 was inhibited by Co nanoparticles. Basal and LPS-induced production of cytokines IL-1beta, TNF-alpha, and IL-1Ra was examined in macrophages exposed to nanoparticles. SiO(2) nanoparticles strongly biased naive macrophages towards inflammation (M1 polarisation), by selectively inducing production of inflammatory cytokines IL-1beta and TNF-alpha. SiO(2) nanoparticles also significantly amplified the inflammatory phenotype of LPS-polarised M1 macrophages. Other ceramic nanoparticles had little influence on cytokine production, either in resting macrophages, or in LPS-activated cells. Generally, Co nanoparticles had an overall pro-inflammatory effect on naive macrophages, by reducing anti-inflammatory IL-1Ra and inducing inflammatory TNF-alpha. However, Co nanoparticles reduced production of IL-1beta and IL-1Ra, but not TNF-alpha, in LPS-polarised M1 macrophages. Thus, exposure to different nanoparticles can modulate, in different ways, the defence/inflammatory capacities of macrophages. A thorough analysis of these biasing effects may shed light on the mechanisms of pathogenesis of several diseases based on dysregulation of the immune response (allergies, autoimmunity, tumours).     

Detection of DNA in Ancient Bones Using Histochemical Methods

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian “Casti Amanti” house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4' -' 6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.