Monoclonal antibody as a probe for structure and function of an Escherichia coli outer membrane protein

Eight independently derived monoclonal antibodies directed against the LamB protein were produced and characterized. By using these antibodies as probes, we identified four distinct topological and functional regions in the LamB molecule. Four monoclonal antibodies recognize antigenic determinants of the protein exposed on the outer side of the membrane. Two of these have their binding sites located in a region involved in maltose transport. One monoclonal antibody presumably binds to a determinant which is normally hidden in the membrane and three monoclonal antibodies recognize determinants facing the periplasmic space.     

Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter.

Little is known regarding the role of androgenic hormones in the maintenance of myosin heavy chain (MHC) composition of rodent masticatory muscles. Because the masseter is the principal jaw closer in rodents, we felt it was important to characterize the influence of androgenic hormones on the MHC composition of the masseter. To determine the extent of sexual dimorphism in the phenotype of masseter muscle fibers of adult (10-mo-old) C57 mice, we stained tissue sections with antibodies specific to type IIa and IIb MHC isoforms. Females contain twice as many fibers containing the IIa MHC as males, and males contain twice as many fibers containing the IIb MHC as females. There is a modest amount of regionalization of MHC phenotypes in the mouse masseter. The rostral portions of the masseter are composed mostly of type IIa fibers, whereas the midsuperficial and caudal regions contain mostly type IIb fibers. Using immunoblots, we showed that castration results in an increase in the expression of type IIa MHC fibers in males. Ovariectomy has no effect on the fiber type composition in females. We conclude that testosterone plays a role in the maintenance of MHC expression in the adult male mouse masseter.     

The phosphorylation of human link proteins

Three link proteins of 48,44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain 32P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO4/mole link protein.     

COMPARISON OF DNA RENEWAL IN GERM-FREE AND CONVENTIONAL MICE USING [125I]IODODEOXYURIDINE AND [3H]THYMIDINE

Germ-free (GF) and conventional (CV) C3H mice received a single injection of 1 μCi [³H]thymidine and 3 μCi [¹²⁵I]iododeoxyuridine to provide simultaneous labeling of DNA with the two precursors. Thymus, spleen, mesenteric lymph nodes, bone marrow (femora), small intestine, colon and skin were examined for total organ activity and rate of DNA renewal 1–8 days after injection. Precursor incorporation, assayed on day 1, was lower in the thymus, mesenteric lymph nodes and femora (and, to a lesser extent, in the spleen and colon) of GF mice as compared to CV animals. The opposite was observed in the small intestine and skin, i.e. total organ activity was higher in GF animals. Differences in precursor incorporation were partly due to differences in organ weights between the two groups of mice. In comparison to CV animals, DNA renewal rates were diminished in the mesenteric lymph nodes, bone marrow, colon (following a 3-day plateau) and spleen of GF mice. Little, if any, difference was observed between the two groups with respect to the rate of DNA turnover in the thymus and skin. Radioactivity of the small intestine remained constant for 2 days. Thereafter intestinal activity in GF mice declined at an initial slow rate between days 2 and 5 followed by a rapid decrease between days 5 and 8. In CV mice the first phase of activity loss was short with the rapid decline in intestinal activity beginning on day 3. From the slopes of the regression lines, the percentage thymidine reutilization was estimated. Reutilization varied from 0 to 63% in the various organs examined, with the greatest difference between GF and CV mice occurring in the mesenteric lymph nodes.     

The specificity of the syngeneic mixed leukocyte response, a primary anti-I region T cell proliferative response, is determined intrathymically

In previous studies, the syngeneic MLR of peripheral T cells was shown to be predominantly an I region-restricted function. In this report we show that adult thymocytes are also capable of responding to syngeneic irradiated stimulator cells in a syngeneic MLR, provided that TCGF is added to the culture system. Using this assay, it was possible for the first time to examine the pattern of I region restriction within the thymus itself. Analysis of the thymocyte syngeneic MLR in thymuses from radiation-induced bone marrow chimeras demonstrated that the MHC preference seen in the peripheral T cell population also existed in cells resident within the thymus. Experiments utilizing congenitally athymic mice transplanted with allogeneic thymic grafts demonstrated that both peripheral T cells and thymocytes from such animals displayed a strong preferential proliferation toward stimulator cells bearing thymic-type MHC determinants. The results in the nude model thus demonstrate that the thymus by itself is sufficient to impart such restriction specificity on a developing T cell repertoire. These results are consistent with the notion that the thymus exerts selective pressure on maturing T cell populations that results in a skewing of the T cell repertoire toward the recognition of thymic-type I region products, and that this MHC preference exists before expansion of T cells in the periphery.     

Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.