Effect of anticoagulants in vitro on the viability of lymphocytes and content of free fatty acids in plasma

Summary These authors attempted to test the effect of anticoagulants on lymphocytes viability by reproducing the procedure used for lymphocyte isolation for various immunologic tests in which blood specimens are allowed to stay at room temperature for 2 h before lymphocytes are isolated. Blood was obtained with three different anticoagulants i.e. heparin, citrate, and CPDA (citrate, phosphate, dextrose, and adenine). Plasma was lyophilized and extracted with ethanol. Dried ethanol extracts were suspended in medium (RPMI 1640+10% fetal bovine serum) and incubated with a lymphocyte cell line (MOLT-4). After 24 h of incubation the viability of cells was examined. The following death rates of the cells were observed: heparin −63±4.6% (mean±SEM), citrate −27±6.7%, and CPDA 6.2±0.6% (P<0.0005). A significant correlation was found between these results and changes in the concentrations of free fatty acids in the extracts. These results emphasize the importance of choosing the right anticoagulant when the viability of lymphocytes is obligatory.     

Timing of Expression of r and Its Encoding mRNAs in the Developing Cerebral Neocortex and Cerebellum of the Mouse

The expression of tau mRNA and of the corresponding encoded protein variants was studied during postnatal development in two brain regions differing in their timing of differentiation: the cerebral neocortex and the cerebellum. (a) The expression of tau mRNA was different in the two regions. Maximal contents were found at early stages in the cerebral neocortex, with a 10-fold decrease at later stages. In the cerebellum, two peaks of tau mRNA were observed soon after birth and in adulthood, with minimal values at postnatal day 6. (b) The expression of total tau proteins was similar to that of their encoding mRNAs in the cerebral neocortex, i.e., high concentrations after birth and low contents at later stages. In contrast, two peaks of tau proteins were observed in the cerebellum: the first perinatally and the second with a maximum at postnatal day 15. (c) Both in the cerebral neocortex and especially in the cerebellum, increasing concentrations of mature tau variants were expressed at late developmental stages, i.e., when total tau protein contents were decreased. In conclusion, the fluctuations in expression of tau and of its encoding mRNA seen in the cerebellum seem to reflect differences in the timing of differentiation of the various cell types, i.e., the macroneurons and the interneurons, present in this brain region. The adult tau variants appear in both the neocortex and the cerebellum only at late developmental stages, i.e., when most of the circuitry has been established, although these two regions markedly differ in their timing of differentiation.     

Effect of alpha-fetoprotein on isolated mouse oocytes

Data are presented which indicate a possible action of alpha-fetoprotein (AFP) on female germinal cells. The in vitro maturation of mature mice oocytes was significantly inhibited when mouse AFP replaced albumin in the culture medium. In addition, the degenerative aspect of oocytes cultured with AFP seemed to indicate that this me?otic inhibition was caused by a premature degeneration of oocytes rather than by a blockage at a specific stage of maturation. Thus AFP, perhaps through its ligands, may play a role in the reduction of germinal cells during fetal and immediate post-natal life rather than in the arrest of me?osis at the diplotene stage.     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

A type VII secretion system of Streptococcus gallolyticus subsp. gallolyticus contributes to gut colonization and the development of colon tumors

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SS^T05). We showed that core genes within the SggT7SS^T05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SS^T05 is functional. Deletion of SggT7SS^T05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SS^T05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SS^T05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SS^T05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SS^T05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.