Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABP(PM).

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP(PM)) may be a component of this system. To test the hypothesis that FABP(PM) is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP(PM). Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 +/- 1.2 pmol/mg protein/15 sec and 45.6 +/- 2.9 nmol/mg protein/15 min and Km values of 12.8 +/- 3.8 and 18.4 +/- 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP(PM). Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP(PM). Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP(PM) is a component of this process in muscle.

cDNA cloning and characterization of Npap60: a novel rat nuclear pore-associated protein with an unusual subcellular localization during male germ cell differentiation.

We have cloned and characterized a cDNA, Npap60, encoding a rat nuclear pore-associated protein. The 3-kb cDNA was obtained by antibody screening of a rat testis expression library. The predicted NPAP60 contains 381 amino acids with a composition of 25.6% charged residues and is highly hydrophilic. The Npap60 gene appears to be conserved in mouse, rat, and human. Immunofluorescence studies with anti-NPAP60 fusion protein antibody show that the NPAP60 protein colocalizes with nuclear pore complexes in RAT1A cells. The expression of Npap60 is about 10-20 times higher in rat testis than in somatic tissues. The subcellular localization of NPAP60 protein changes dramatically during male germ cell differentiation, from nuclear pore complex-like staining in spermatocytes to whole nucleus staining in spermatids and finally to a nuclear surface staining in mature spermatozoa. These changes are temporally and spatially related to nuclear reorganization during male germ cell differentiation.

Involvement of Escherichia coli DNA polymerase II in response to oxidative damage and adaptive mutation.

DNA polymerase II (Pol II) is regulated as part of the SOS response to DNA damage in Escherichia coli. We examined the participation of Pol II in the response to oxidative damage, adaptive mutation, and recombination. Cells lacking Pol II activity (polB delta 1 mutants) exhibited 5- to 10-fold-greater sensitivity to mode 1 killing by H2O2 compared with isogenic polB+ cells. Survival decreased by about 15-fold when polB mutants containing defective superoxide dismutase genes, sodA and sodB, were compared with polB+ sodA sodB mutants. Resistance to peroxide killing was restored following P1 transduction of polB cells to polB+ or by conjugation of polB cells with an F' plasmid carrying a copy of polB+. The rate at which Lac+ mutations arose in Lac- cells subjected to selection for lactose utilization, a phenomenon known as adaptive mutation, was increased threefold in polB backgrounds and returned to wild-type rates when polB cells were transduced to polB+. Following multiple passages of polB cells or prolonged starvation, a progressive loss of sensitivity to killing by peroxide was observed, suggesting that second-site suppressor mutations may be occurring with relatively high frequencies. The presence of suppressor mutations may account for the apparent lack of a mutant phenotype in earlier studies. A well-established polB strain, a dinA Mu d(Apr lac) fusion (GW1010), exhibited wild-type (Pol II+) sensitivity to killing by peroxide, consistent with the accumulation of second-site suppressor mutations. A high titer anti-Pol II polyclonal antibody was used to screen for the presence of Pol II in other bacteria and in the yeast Saccharomyces cerevisiae. Cross-reacting material was found in all gram-negative strains tested but was not detected in gram-positive strains or in S. cerevisiae. Induction of Pol II by nalidixic acid was observed in E. coli K-12, B, and C, in Shigella flexneri, and in Salmonella typhimurium.

Preparation of mouse type I and type II insulins for immunologic studies.

Mouse pancreata contain comparatively meager amounts of two insulin species, types I and II. When these insulins are to be prepared for immunogenetic studies, it is desirable to obtain equivalent amounts of both in concentrations suitable for immunization. Standard methods, based on isolating single species, favor recovery of one type. Moreover, published methods for separation of type I from type II produce very dilute insulin solutions. Methods are suggested here to overcome these disadvantages.

Immune function in congenital osteopetrosis: defective lymphocyte function in microphthalmic mice.

Osteopetrosis is a prominent feature of a congenital mutation described in microphthalmic mice and is thought to be due to defective osteoclast function which causes a generalized lack of bone resorption. Reversal of defective bone resorption in osteopetrotic mutants has been achieved by hematopoietic cell transplantations; and conversely, defective bone resorption has been transferred to normals by hematopoietic cells from osteopetrotic littermates. This suggested that osteopetrotic mutants might also demonstrate defective immune functions which could in turn be related to the lack of normal bone resorption. To that end, aspects of in vitro lymphocyte function in microphthalmic mice were compared to those of their phenotypically normal littermates. There was a significant diminution in the proliferative response of splenocytes to mitogens in microphthalmic mice. Microphthalmic splenocytes also were less responsive in an in vitro assay which measured the capacity to form antibody forming cells.

Immunogen structure and cell selection. I. Cellular requirements for the in vitro response to moderately haptenated forms of the solid-phase immunogen DNP-O-Bio-Gel-P.

The cellular requirements for the in vitro response to DNP-O-Bio-Gel-P were determined. Results of these experiments indicate that: 1) splenic adherent cells are required for generation of anti-hapten PFC to the solid phase immunogen; and 2) cultured nude-mouse spleen cells have far less capacity to respond to the immunogen than do cells from wild type mice. These experiments suggest that both T and B cells are required for responsiveness to the moderately haptenated form of DNP-O-Bio-Gel-P.

A fetuin-like antigen from human nephroblastoma.

An antigen was detected in pooled human nephroblastomas using antiserum prepared in rabbits against an ethylemediaminetetra acetic acid (EDTA) extract of the tumors. This antigen was not found in normal human plasma or kidney extracts, and was not related to the ABO or Forssman blood groups. The antigen was detected in extracts of cultured nephroblastoma cells, but was not present in extracts of normal human fetal kidney cell cultures. The antigen is believed to be present at the cell surface, as cell viability was not significantly lowered during the extraction procedure. A reaction of complete identity was demonstrated by Ouchterlony double diffusion experiments with this antigen and purified bovine fetuin. The antigen was not found in extracts of human fetal spleen, thymus or kidney, nor in human fetal serum. Furthermore, the antigen does not possess determinants in common with the human alpha-fetoprotein of hepatomas, nor was it detected in human renal clear cell carcinoma. Initial characterization of the antigen showed it to be nondialysable, not sedimentable at 100,000 times g for 2 h, stable to repeated freeze-thawing and to incubation at 56 degrees C for 1 h, and water soluble over a wide pH range. The antigen was susceptible to digestion with pronase and trypsin and possibly hyaluronidase, but not to ribonuclease or neuraminidase. The protein portion is therefore of major importance to the structural integrity of this antigen. The relationship between this antigen and other abnormal materials reported previously in nephroblastoma patients is being studied.

Isolation of Wilms' tumor antigens by chelation.

A procedure for ethylenediaminetetraacetate extraction of minced Wilm's tumor was assessed as a method for isolating Wilm's tumor antigens. An antigen was detected by immunodiffusion using an adsorbed antiserum to this extract. This antigen was also found in ethylenediaminetetraacetate extracts of in vitro cultures of nephroblastoma cells.

In vitro response of mouse spleen cells to the solid phase immunogen DNP-O-Bio-Gel.

The in virto immunogenicity of the solid-phase hapten, dinitrophenyl-ornithine-Bio-Gel (DNP-O-Bio-Gel), was investigated in cultures of mouse spleen cells. Appropriate combinations of cells and immobilized hapten were determined. Large numbers of direct anti-hapten plaque-forming cells (PFC) were generated when 1 times 10-7 C57BL/6 or C57BL/10 spleen cells were cultured with 4 times 10-3 DNP-O-Bio-Gel beads. Specificity studies of the responses of cultured spleen cells to DNP-O-Bio-Gel yielded the following results: soluble DNP-ornithine or DNP-bovine gamma-globulin inhibited the induction of anti-hapten PFC by DNP-O-Bio-Gel; neither dinitrophenyl-Bio-gel (DNP-Bio-gel) nor ornithine-Bio-Gel (O-Bio-Gel) induced anti-hapten responsiveness; furthermore, neither DNP-Bio-Gel nor O-Bio-Gel inhibited the induction of PFC by DNP-O-Bio-Gel. It was concluded, from the results of these specificity experiments, that a spacer, ornithine, is required for immunogenicity of immobilized DNP; and that the Bio-Gel bead, itself, acts solely as a physical carrier for the hapten.

Electrophoretically homogeneous anti-DNP antibodies with restricted isoelectric points elicited in mice by immunization with the antigen papain-S-DNPL.

The primary immunization of outbred mice with the antigen Pap-S-DNPL results in the generation of low titers of anti-DNP antibody which in about one-third of the responding animals is as homogeneous as a myeloma protein by the combined criteria of (a) isoelectric focusing in gels and (b) gel electrophoresis of the antibody light chains. The electrophoretically homogeneous antibodies show a marked restriction of isoelectric points near pH 5.0. Such marked selectivity of the antihapten antibodies appears to result from the chemical and structural homogeneity of the Pap-S-DNPL antigen.