Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis.

Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.     

Molecular basis of organ-specific selection of viral variants during chronic infection.

Viral variants of different phenotypes are present in the central nervous system (CNS) and lymphoid tissues of carrier mice infected at birth with the Armstrong strain of lymphocytic choriomeningitis virus. The CNS isolates are similar to the parental virus and cause acute infections in adult mice, whereas the lymphoid isolates cause chronic infections associated with suppressed T-cell responses. In this study, we provide a molecular basis for this organ-specific selection and identify a single amino acid change in the viral glycoprotein that correlates with the tissue specific selection and the persistent and immunosuppressive phenotype of the variants. This phenylalanine (F)-to-leucine (L) change at position 260 of the viral glycoprotein was seen in the vast majority (43 of 47) of the lymphoid isolates, and variants with L at this residue were selected in spleens of persistently infected mice. In striking contrast, isolates with the parental sequence (F at residue 260) predominated (48 of 59 isolates) in the CNS of the same carrier mice. Complete nucleotide sequence analysis of the major structural genes of several independently derived (from different mice) spleen isolates showed that these variants were greater than 99.8% identical to the parental virus. In fact, the only common change among these spleen isolates was the F----L mutation at residue 260 of the glycoprotein. These results show that an RNA virus can exhibit minimal genetic drift during chronic infection in its natural host, and yet a single or few mutations can result in the organ-specific selection of variants that are markedly different from the parental virus.     

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Interaction of Co,Mn, Mg and Al with d(GCGTACGC): a spectroscopic study

Spectroscopic study on the interactions of trace elements Co, Mn, Mg and Al with d(GCGTACGC) indicated the following: Al and Mg did not alter T_m values. Mn enhanced T_m at lower concentration and decreased it at higher concentrations. Interestingly Co at higher concentration elevated the T_m. These studies also showed lower concentrations of Mn displaced EtBr, whereas Al could displace it at higher ionic strength. Mg and Co displaced EtBr fluorescence at moderate concentrations. The binding constant values and CD spectra clearly indicated strong binding of these elements to DNA.     

Anti-H-2 hybridoma antibodies as immunoselective agents

Using immunoselection with an H-2K^k-specific monoclonal antibody following mutagenesis on an (H-2 ^k/H-2^d) F₁ cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonseactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertantly got mixed into the resistant mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybrid oma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2K^k-gene product.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An H-2 antigen variant, referred to as ?4 + 31 clone 1, was selected by its resistance to an anti-H-2D^d antiserum (BALB.G anti-BALB/c.H-2 ^g antiH-2 ^d ). When tested by cell-mediated cytolysis, this variant was found to be sensitive to cytolytic T lymphocytes raised in the same donor-host combination as that used in raising the antiserum. Further CML characterization of this variant, reported here, indicates that the cell line is in fact resistant to anti-H-2D^d killer cells raised in a more restricted immunization, viz. BALB.G anti-BALB/cH-2 ^db ,H-2 ^g anti-H-2 ^db . It is, however, sensitive to cytolytic cells raised in (BALB.B xBALB/c-H-2db) F¹ H-2 ^b /H-2 ^db ) against the BALB/c strain. These results suggest that the variant does not express H-2D^d itself, but probably expresses CML target antigens that are missing in theH-2 ^db mutation. This in vitro-isolated variant might thus be the complementary mutation to the in vivoobtainedH-2 ^db mutation.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.