MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle.

The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.

Betaine-homocysteine methyltransferase expression in porcine and human tissues and chromosomal localization of the human gene.

We have prepared antibodies against porcine liver betaine-homocysteine methyltransferase (BHMT; EC 2.1.1.5) and recently cloned cDNAs encoding the porcine and human liver enzymes. Porcine tissues were evaluated for BHMT expression by measuring catalytic activity and Western analysis. Liver and kidney were the only organs tested that had immunodetectable levels of BHMT, and these organs expressed high levels of enzyme activity. BHMT was expressed in the kidney cortex and not the medulla. Porcine pancreas, brain, heart, lung, and spleen were devoid of BHMT activity and immunodetectable protein. Human tissues were tested for BHMT expression by Northern analysis. Human liver and kidney were the only organs tested that expressed BHMT mRNA. Human pancreas, brain, heart, skeletal muscle, spleen, and placenta were devoid of BHMT mRNA. The human BHMT gene has been mapped to chromosome 5q13.1-q15.

Antibody networks and imaging: elicitation of anti-fluorescein antibodies in response to the metatypic state of fluorescein-specific monoclonal antibodies.

Studies are described regarding generation of anti-hapten antibodies starting with a monoclonal Ig immunogen in the ligand-induced conformation or metatypic state. Liganded monoclonal Ab1 antibodies represent the unique feature of the study since previous reports investigating internal imaging in the original Idiotype Network Hypothesis [Jerne, 1974 (Ann. Immun. 125C, 373-389)] were based on the non-liganded or idiotypic state [as reviewed in: Rodkey, 1980 (Microbiol. Rev. 44, 631-659); Kohler et al., 1979 (In: Methods in Enzymology: Antibodies, Antigens and Molecular Mimicry, pp. 3-35); Greenspan and Bona, 1993 (FASEB J. 7,437-444)]. Affinity-labeled liganded murine monoclonal anti-fluorescein antibodies served as immunogens administered both in the syngenic and xenogenic modes to determine if the metatypic state elicited anti-hapten antibodies through imaging-like mechanisms. Polyclonal and monoclonal anti-Ab1 reagents in various hosts were assayed for anti-fluorescein and/or anti-metatype specificity. Significant anti-fluorescein responses were measured indicating that the metatypic state directly or indirectly stimulates an anti-hapten antibody population.

Determination of hydrolysed fumonisin B1 (HFB1) in corn by competitive direct enzyme-linked immunosorbent assay.

Fumonisin B1, a mycotoxin produced by certain Fusarium moulds, consists of two tricarballyic acid groups esterified to a 20-carbon backbone. Under alkaline conditions, or through metabolism, the aminopentol backbone, also known as hydrolysed Fumonisin B1 (HFB1) can be formed and is itself cytotoxic. Although the occurrence of HFB1 in corn-based foods is suspected, because of the ubiquitous nature of FB1 in corn, analytical methods for its detection are difficult. In the present report we describe a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for the rapid analysis of HFB1 in corn. The concentration required to inhibit enzyme conjugate binding by 50% (IC50) was 36 ng/ml. The limit of detection of HFB, by the CD-ELISA was 2ng/ml, when HFB1 was added in bovine serum albumin-phosphate buffered saline. The antibody also cross-reacted with the hydrolysis products of FB2, FB3, and FB4, having IC50 values of 331, 174, and 1700 ng/ml respectively. The antibody did not react with the intact fumonisins, sphingosine, sphinganine, or tricarballylic acid. Samples of corn spiked with HFB1 over the range of 5-1000 ng/g indicated the CD-ELISA has a limit of detection of 5 ng/g and an IC50 of 41 ng/g in the matrix. The CD-ELISA provides a sensitive and rapid tool for the analysis of corn-based foods for HFB1.

High-affinity rat anti-fluorescein monoclonal antibody with unique fine specificity properties including differential recognition of dynamic ligand analogues.

The ability of antibodies to specifically select and stabilize through binding one or more isomers of highly dynamic ligands remains a relatively unexplored immunochemical problem. The experimental strategy employed in this study was to elicit homogeneous antibodies to polyaromatic fluorescein which exists in one isomeric form. The binding properties of a monoclonal rat antifluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune spleenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Cell line P2A12-1-C8 was selected for further characterization from the original 23 stable rat hybrids, since it produced a monoclonal antibody with a binding affinity 2.0 x 10(10)/M for fluorescein based on dissociation rate measurements. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red. Computer-based molecular modeling and energy minimization studies of fluorescein, HPF, phenol red, and phenolphthalein showed that in terms of the most energetically favorable orientation of the three aromatic rings, phenol red more closely simulated fluorescein than phenolphthalein. The results were analyzed in terms of the mechanisms of dynamic ligand stabilization and binding involving accommodation of specific ligand isomers by energetically permissible conformational states exhibited by an antibody active site. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities.

Elicitation of distinct populations of monoclonal antibodies specific for the variable domains of monoclonal anti-fluorescein antibody 4-4-20.

Armenian hamsters were immunized with non-liganded, partially liganded or affinity-labeled anti-fluorescein Mab 4-4-20. Seventeen hybridoma producing monoclonal anti-4-4-20 antibodies were characterized from chemically-mediated fusions of immune hamster lymphocytes with murine Sp2/O-Ag14 myeloma cells. Distinct populations of anti-4-4-20 monoclonal antibodies were isolated from hamsters receiving immunizations with partially liganded Mab 4-4-20 relative to those receiving affinity-labeled 4-4-20. Two of the three monoclonal antibodies produced in response to partially liganded 4-4-20 were inhibited in their interaction with 4-4-20 by fluorescyl ligand. These two clones, 1F4 and 1B7, recognized unique epitopes on the 4-4-20 molecules, as demonstrated by non-reactivity with members of the 4-4-20 idiotype family. Additionally, 1F4 and 1B7 demonstrated the ability to delay the association of fluorescein with Mab 4-4-20. The 14 characterized non-ligand-inhibitable Mabs elicited to affinity-labeled 4-4-20 were classified into four separate groups based on various binding properties with members of the 4-4-20 idiotype family and binding to resolved H- and L-chains in a western blot. Members of three of the four groups showed strong reactivity with both 04-01 Ig and 04-01 SCA, which utilizes the same L-chain as Mab 4-4-20. Six non-ligand-inhibitable Mabs, 4A6, P1E11, 3A5-1, 2C3, 2C4, and 1A4, delayed the dissociation rate of ligand from Mab 4-4-20 and mutant 4-4-20 SCA L32phe.

Monoclonal antibodies against a dopamine-releasing protein (DARP) arrest fetal development, decrease brain catecholamines, and increase adrenal weight of neonatal rats.

Monoclonal antibodies (MAb) were produced against a partially purified dopamine-releasing protein (DARP) to examine the feasible physiological role of this factor during the development of the rat. These IgM isotype MAbs, and not other IgM antibodies (controls), induced a remarkable increase in fetal resorption and stillbirth rate and impaired the developmental increase of catecholamine concentration in the corpus striatum and hypothalamus of newborn rats. Neonatal injections of the anti-DARP MAb (a single injection of 200 mug, 24 h after birth or 40 mug on alternate days during the first 10 days) decreased the dopamine (DA) concentration of the corpus striatum by 30% on Day 10 and 15% on Day 25 and drastically impaired (by 43% on Day 25) the developmental increase in hypothalamic DA. Furthermore, in the hypothalamus, there was a marked decrease in epinephrine and an increase in norepinephrine (NE) concentration, suggesting an impairment in PNMT function. Neonatal anti-DARP injections also resulted in increased adrenal weight (45 and 44% on Days 10 and 25, respectively) and elevated NE content (anti-DARP, 315 +/- 12 ng, vs control, 223 +/- 16 ng, n = 6). Intrafetal injection of anti-DARP MAb (40 mug) on E 17 resulted in increased resorption and stillbirth accounting for 83% fetal loss. A 10-mug dose of the antibody produced 33% of fetal resorption or stillborn fetuses, whereas control injections resulted in only 4.4% of fetal loss. These data strongly suggest that DARP may be a neurotrophic factor involved in the growth and differentiation of central catecholaminergic neurons and probably necessary for the maturation of PNMT during the early development of rat brain.

Hemocytes contribute to both the formation and breakdown of the basal lamina in developing wings of Manduca sexta.

Monoclonal antibodies (MAbs) raised against wing tissues of Manduca sexta recognize epitopes shared by both hemocytes and basal laminae. During the last larval stadium, the basal lamina of moth wing epithelium forms after hemocytes have migrated into the space adjacent to basal surfaces of epithelial cells. As adult development commences, hemocytes participate in phagocytosis of the same basal lamina; and as dissolution of the basal lamina proceeds (day 2-day 5 post-pupation), wing epithelial cells send forth long basal processes and rearrange within the plane of the epithelium. During this period of cell rearrangement, the immunoreactivity of the basal lamina decreases in concert with an increase in immunoreactive vesicles within hemocytes; and at the ultrastructural level, hemocytes have been observed to engulf fragments of basal lamina. The distribution of immunolabel in the developing moth wing suggests that hemocytes contribute not only to the formation of the wing's basal lamina but also to its breakdown. Since basal laminae are probably important determinants of epithelial form and pattern, hemocytes also contribute to the shaping of epithelial populations.

Polyclonal antibodies specific for liganded active site (metatype) of a high affinity anti-hapten monoclonal antibody.

Syngeneic polyclonal antibodies were elicited to an affinity labeled high affinity (2-3 X 10(10) M-1) anti-fluorescein murine IgG2a monoclonal antibody. Hyperimmune ascites fluid was tested for reactivity with homologous liganded, affinity labeled and non-liganded Fab fragments derived from the high affinity antibody. Binding results demonstrated antibody specificity for the liganded or affinity labeled site, but no reactivity with either the non-liganded form or the fluorescyl ligand. Kinetic analysis showed that the rate of dissociation of the fluorescein ligand was slowed down significantly upon binding of the anti-affinity labeled reagent to the liganded antibody. Antibodies specific for the affinity labeled prototype were not reactive with the liganded form of an IgM monoclonal anti-fluorescyl antibody of the same affinity but idiotypically unrelated. Results of the immunological studies suggested that the antibody active site stabilized by bound ligand differed from the idiotype of the antibody. The term "metatype" was proposed for the immunological definition of the liganded active site to distinguish it from idiotype (non-liganded). The general nature of metatopes is discussed in terms of conformational or sequential epitopes.

Functional and structural implications of variable region immunoglobulin dynamic states.

The consequence of multiple conformational states in the immunoglobulin variable region are considered. Bound ligand is viewed as a stabilizer of one conformer from a series of non-liganded conformers. Kinetic, equilibrium, thermodynamic and crystal formation information are used as supporting evidence for a unique conformer which is the crystallizable liganded state. The multi-state model is discussed in terms of certain biological and biochemical properties exhibited by the immunoglobulin molecule.