Alicyclobacilli from an unexplored geothermal soil in Antarctica: Mount Rittmann

The isolation of three species from soil samples from an unexplored site in Antarctica, belonging to the Alicyclobacillus genus, is reported. The isolates, named MR1, MR2 and MR4, were from 30 samples of geothermal soil from Mount Rittmann. All the isolates had lipids based mainly on fatty acids possessing a terminal cyclohexane; hopanoid and quinones of MK-7 were also present. All isolates were characterized by phenotypic features and the isolate MR1 was subjected to genetic analyses. The isolates were thermoacidophilic, showing an optimum temperature of 63°C and an optimum pH of 3.5–4.0. They were able to grow in the temperature range 45–70°C. The percentage C+G of DNA of MR1 was 64.9% and showed a 16S rDNA similarity of 99.3% with Alicyclobacillus acidocaldarius ATCC 43034 and a DNA-DNA homology of 69.7% with A. acidocaldarius DSM 446. The strain MR1 is unable to produce amylolytic activity under different growth conditions. It is proposed to accommodate MR1 in the genus Alicyclobacillus as subspecies rittmannii of A. acidocaldarius. Received: 29 May 1997 / Accepted: 26 September 1997     

Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours,with a characterization of the tumour phenotype

Summary Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histo-type (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for tumour-specific lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5–346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor (TNF) or interferon , and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3⁺ but 2 cases of osteosarcoma showed a predominance of CD3⁺TcR / cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16⁺ and CD25⁺ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogeneic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor (TGF). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.     

Purification of β-N-acetyl-d-glucosaminidase fromAspergillus niger using thermostabilization and heat as the initial step

A β-N-acetyl-d-glucosaminidase (EC 3.2.1.30) produced byAspergillus niger 419, was completely inactivated after heating 15 min at 65°C in 100 mM sodium phosphate buffer pH 7. The presence of 10% of polypropyleneglycol 1025 induced the thermal stability of the enzyme, the activity remaining unchanged after heating 60 min at 65°C. When this thermal treatment was used as the initial step of purification, the protein content of the crude extract was reduced by 98% without loss of the total initial enzymatic activity of the sample and a purification factor of 61. As the second and third step of purification DEAE-Sephacel, and Sephadex-G150 column chromatography were used, respectively. The final purification factor was 230 with a yield of 76%.     

Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca²⁺ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca²⁺ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca²⁺- and Pi-rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5–2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca²⁺-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca²⁺ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca²⁺ and Pi is directly involved in endochondral calcification.     

Differences between male,female and juvenile haddock (Melanogrammus aeglefinus L.) sounds

Female and juvenile haddock make sounds, as well as males. Examination of the sounds from different sexes indicates that the sound waveform is a function of fish maturity and it is gender-specific. Immature fish sounds were found to be made up of two pulses with similar frequencies and opposite polarities. Females produced two pulses with the same polarity, the first pulse having a higher frequency than the second. The acoustic characteristics of juvenile, female and male haddock sounds are compared. Sexual dimorphism in the mass of the drumming muscle mass has also been investigated. Female haddock possess less well-developed drumming muscles than males throughout the whole year. A significant difference in drumming muscle mass was observed not only in males but also in females at different seasons. A positive relation between drumming muscle mass and fish size has been highlighted in both male and female fish. The physical parameters of the sound units emitted by juveniles, females and males, which are likely affected by physiological condition and maturity stage, are discussed in relation to the sound-producing mechanism.     

Influence of Environmental Conditions on Hyphal Morphology in Pellets of Aspergillus niger: Role of β-N-Acetyl-D-Glucosaminidase

The influence of modifications of the environmental conditions of growth on β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) activity and on hyphal morphological patterns in pellets of Aspergillus niger was studied. It was found that changes in the degree of branching and, to a lesser extent, in the number of bulbous cells were directly related to the activity of the enzyme. Nevertheless, since β-N-acetyl-D-glucosaminidase is not the only enzyme involved in the lytic potential of the fungus, these findings do not exclude the possibility that other enzymes may be involved. Received: 30 December 1998 / Accepted: 9 March 1999     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: Modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-β family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused ∼2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7–14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3–5-fold vs. 2–3-fold increase in ALP; ∼40% vs. ∼20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by ∼2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca²⁺ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo. J. Cell. Biochem. 67:498–513, 1997. © 1997 Wiley-Liss, Inc.     

Nutrigenomics: a case for the common soil between cardiovascular disease and cancer

The border between health and disease is often set by a complex equilibrium between two elements, genetics on one hand, lifestyle on the other, To know it better, means to give new weapons, often crucial, in the hands of the doctors and their patients. It also means to adjust therapies, to find out which drug is good for a patient and which prevention strategy will work better for him/her. Nutrigenomics is an approach to individualize or personalize food and nutrition, and ultimately health, by tailoring the food to the individual genotype. In this review, we present the interaction between certain genetic polymorphisms and diet and increased cardiovascular or cancer risk. It is, indeed, now clear that a large number of bioactive food components may provide risk or protection at several stages of both atherosclerosis and cancer formation processes. We are giving here few examples of gene-food interactions relevant for both the risk of cardiovascular disease and cancer, since a common soil could exist in the genesis of cardiovascular disease and of some types of cancer (mainly gastrointestinal tract and hormone-dependent).