Biology Reference
In-Depth Information
data on primary CTL and memory responses from ARC made of TPL from methano-
gens and hyperthermophiles, suggests that the headgroups are of crucial importance
in the induction of immune responses, as recently determined by employing synthetic
glycoarchaeols [45, 46].
To sum up, even though our results for IgG isotyping in a single strain of mice
(C3H/HeN) may not be extrapolated to the response in humans, it is promising to note
that, in preclinical evaluation in animal model, ARC-
BM
/
GC
-BSA would appear as
an effi cient adjuvant delivery system to promote both humoral and, probably, cell-
mediated immunity to the entrapped antigen.
MATERIALS AND METHODS
Sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro) ben-
zene sulfonic acid hydrate (XTT), BSA, antifoam 204 and cholesterol were provided
by Sigma-Aldrich (Argentina). Hydrogenated phosphatidylcholine from soybean
(HSPC) was obtained from Northern Lipids (Vancouver, Canada). The RPMI 1640
culture medium was purchased from Invitrogen Corporation. Endotoxin-free fetal bo-
vine serum (FBS) was bought from Hyclone. The L-Glutamine, Trypsin, EDTA and
penicillin/streptomycin were provided by PAA Laboratories GmbH (Austria). The flu-
orophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and the quencher p-xylene-
bis-pyridinium bromide (DPX) were purchased from Molecular Probes (Eugene, OR,
USA).
Halorubrum tebenquichense
strain ALT6-92 was purchased from Deutsche
Sammlung von Mikroorganismen and Zellkulturen (DSMZ). Tris buffer and all the
other analytical grade reagents were from Anedra (Argentina).
Culture Growth and Characterization
Soil samples were collected from Salina Chica, Península de Valdés, Chubut, Argen-
tina. Samples were classified according to their strata source as upper
GCs
and deeper
BM
. Isolation of microorganisms from each strata was done by seeding an aliquot on
basal growth medium [48] with 1.6% agar (solid basal medium) at 37°C. The resulting
colonies were grown on liquid medium at 40°C, at 160 rpm with chloramphenicol (30
mg/l) and then seeded on solid basal medium or brain-heart broth supplemented with
yeast extract, glucose or blood (enriched medium).
Optimal NaCl concentration, pH, temperature and Mg
+2
requirements for growth
were determined as described by Oren [49]. Gram staining as described by Dussault
[50], cell shape and pigmentation examined by optical microscopy, were performed on
colonies grown in basal medium and also in medium supplemented with blood or with
brain-heart broth. Standard biochemical test methods were assessed for each colony.
Salt requirement for maintaining stability of the cell envelope was determined as
described by Oren [49] by measuring the loss of turbidity of cell suspensions in me-
dium of decreasing concentrations of NaCl (8, 6, 4, 2, and 0% w/v).
Biomass was generated in 8 l batch cultures in basal medium supplemented with
yeast extract, glucose, and antifoam (20 μl/l). Cultures were monitored by absorbance
at 660 nm and harvested in late stationary phase for storage as frozen cell pastes.
