Depletion of florfenicol in lactating dairy cows after intramammary and subcutaneous administration.

Eighteen Holstein dairy cows ranging in body weight from 500-700 kg and with an average milk yield of 37 ± 6 kg/day were used to investigate the depletion of florfenicol (FFL) in milk and plasma of dairy cows. Three groups of six were administered FFL: Group A, intramammary (IMM) infusion of ~2.5 mg FFL/kg BW at three consecutive milking intervals (total amount of ~7.5 mg/kg BW); Group B, one IMM infusion (20 mg/kg BW) into one quarter and Group C, one subcutaneous (SC) treatment (40 mg/kg BW). IMM infusions were into the right front quarter. Cows were milked daily at 06:00 and 18:00 h. The highest concentrations (Cmax ) and time to Cmax (Tmax ) were: 1.6 ± 2.2 µg·FFL/mL milk at 22 h (Group A), 5.5 ± 3.6 µg·FFL/mL milk at 12 h (Group B), and 1.7 ± 0.4 µg·FFL/mL milk at 12 h (Group C). The half-lives (t1/2 ) were ~19, 5.5, and 60 h, for Groups A, B, and C, respectively. FFL was below the limit of detection (LOD) by 60 h in three Group B cows, but above the LOD at 72, 84, and 120 h in three cows. FFL was above the LOD in milk from Group C's cows for 432-588 h. Plasma values followed the same trends as milk. The results demonstrate that IMM-infused FFL is bioavailable and below the LOD within 72-120 h. The concentration of FFL was detectable in both plasma and milk over the course of 2-3 weeks after SC administration. The absence of residue depletion data presents problems in determining safe levels of FFL residues in milk and edible tissues. The data presented here must not be construed as approval for extra-label use in food animals.

Overcoming the low relaxivity of gadofosveset at high field with spin locking.

The contrast agent gadofosveset, which binds reversibly to serum albumin, has a high longitudinal relaxivity at lower magnetic fields (≤3.0 T) but a much lower relaxivity at high fields. Spin locking is sensitive to macromolecular content; it is hypothesized that combining this technique with the albumin-binding properties of gadofosveset may enable increased relaxivity at high fields. In vitro measurements at 4.7 T found significantly higher spin-lock relaxation rates, R1ρ (1/T1ρ), when gadofosveset was serum albumin-bound than when unbound. R1ρ values for a nonbinding contrast agent (gadopentetate dimeglumine) in serum albumin were similar to those for unbound gadofosveset. R2 (1/T2) values were also significantly higher at 4.7 T for serum albumin-bound gadofosveset than for unbound. Spin locking at high field generates significantly higher relaxation rates for gadofosveset than conventional contrast agents and may provide a method for differentiating free and bound molecules at these field strengths.

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Indirect induction of radiation lymphomas in mice. Evidence for a novel, transmissible leukemogen.

The transmission of a lymphomagenic agent(s) from the bone marrow of irradiated mice to thymic target cells has been demonstrated by: (a) the induction of T cell lymphomas in nonirradiated thymic grafts implanted in irradiated, Thy-l-congenic mice, (b) the induction of T cell lymphomas of host origin in mice infused with bone marrow from irradiated, Thy-l-congenic donors. The latter procedure also yields an appreciable number of pre-B cell lymphomas of uncertain origin. The results confirm Kaplan's theory that radiation induces thymic lymphomas in mice by an indirect mechanism. However, the previously described radiation leukemia virus is clearly not involved in the majority of transferred lymphomas. We propose that the mediating agent in radiation lymphomagenesis is a novel, transmissible agent induced in the bone marrow, but exerting its transforming activity on cells in the thymus. The nature and mode of action of the agent are under investigation.

Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs.

The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E(2) (PGE(2)) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE(2) occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B(2) (TXB(2)). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE(2) in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFalpha) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE(2) production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.

An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway.

The B cell activating factor BAFF (BlyS/TALL-1/zTNF4) is a tumor necrosis factor (TNF)-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI, and the recently described BAFF-R). Here we report an absolute requirement for BAFF in normal B cell development. Examination of secondary lymphoid organs from BAFF-deficient mice revealed an almost complete loss of follicular and marginal zone B lymphocytes. In contrast, mice lacking BCMA had normal-appearing B lymphocyte compartments. BAFF therefore plays a crucial role in B cell development and can function through receptors other than BCMA.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay.

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Essential roles for the Abl and Arg tyrosine kinases in neurulation.

The Abl and Arg tyrosine kinases play fundamental roles in the development and function of the central nervous system. Arg is most abundant in adult mouse brain, especially in synapse-rich regions. arg(-/-) mice develop normally but exhibit multiple behavioral abnormalities, suggesting that arg(-/-) brains suffer from defects in neuronal function. Embryos deficient in both Abl and Arg suffer from defects in neurulation and die before 11 days postcoitum (dpc). Although they divide normally, abl(-/-)arg(-/-) neuroepithelial cells display gross alterations in their actin cytoskeleton. We find that Abl and Arg colocalize with each other and with actin microfilaments at the apical surface of the developing neuroepithelium. Thus, Abl and Arg play essential roles in neurulation and can regulate the structure of the actin cytoskeleton.

Effect of the presence or absence of J chain on expression of recombinant anti-Kell immunoglobulin M.

The aim of this study was to determine the effect of expressing a recombinant anti-Kell immunoglobulin (Ig) M from two cell lines, CH0 and NS0, on its ability to function as a diagnostic antibody. As a polymeric immunoglobulin, IgM is able to directly agglutinate red blood cells (RBCs), making it a useful blood grouping reagent. To simplify expression, recombinant human IgM (rIgM) from NS0 (a mouse myeloma line) and CHO (Chinese hamster ovary line) cells was expressed in the absence of human J chain. Whereas NS0 expresses mouse J chain, rIgM expressed from CH0 cells lack J chain. Although the ability to polymerize resides within the tailpiece of IgM heavy chain, J chain can influence the polymeric state. This in turn could affect the ability of rIgM to bind its antigen. The variable region of the heavy chain of an anti-Kell IgG was grafted onto the constant region of human IgM and co-expressed with light chain derived from the same antibody. rIgM was purified from each cell line and the strength of direct agglutination assessed. Both cell lines produced polymeric rIgM that was able to specifically bind the target antigen and to directly agglutinate RBCs to the same degree. The presence or absence of J chain did not affect the ability of the rIgM to bind the Kell antigen or the strength of agglutination. The presence of J chain is not required for the production of a functional rIgM for use as a diagnostic reagent. CHO and NS0 lines are both suitable for production of such a reagent.

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.

The bcl-3 proto-oncogene encodes a nuclear I kappa B-like molecule that preferentially interacts with NF-kappa B p50 and p52 in a phosphorylation-dependent manner.

The product of the putative proto-oncogene bcl-3 is an I kappa B-like molecule with novel binding properties specific for a subset of the rel family of transcriptional regulators. In vitro, Bcl-3 protein specifically inhibited the DNA binding of both the homodimeric NF-kappa B p50 subunit and a closely related homolog, p52 (previously p49), to immunoglobulin kappa NF-kappa B DNA motifs. Bcl-3 could catalyze the removal of these proteins from DNA. At concentrations that significantly inhibited DNA binding by homodimeric p50, Bcl-3 did not inhibit binding of reconstituted heterodimeric NF-kappa B (p50:p65), a DNA-binding homodimeric form of p65, or homodimers of c-Rel. Phosphatase treatment of Bcl-3 partially inactivated its inhibitory properties, implicating a role for phosphorylation in the regulation of Bcl-3 activity. Bcl-3, like p50, localizes to the cell nucleus. In cells cotransduced with Bcl-3 and p50, both molecules could be found in the nucleus of the same cells. Interestingly, coexpression of Bcl-3 with a p50 mutant deleted for its nuclear-localizing signal resulted in the relocalization of Bcl-3 to the cytoplasm, showing that the proteins interact in the cell. These properties contrast Bcl-3 to classically defined I kappa B, which maintains heterodimeric NF-kappa B p50:p65 in the cytoplasm through specific interactions with the p65 subunit. Bcl-3 appears to be a nuclear, I kappa B-related molecule that regulates the activity of homodimeric nuclear p50 and its homolog p52.

Regulation of phospholipid synthesis by intracellular phospholipases in fetal rabbit type II pneumocytes.

Exposure of fetal type II pneumocytes to phospholipase A2 inhibitors led to significantly reduced choline uptake and decreased synthesis of total and disaturated phosphatidylcholines from both [methyl-14C]choline and [9,10(n)-3H]palmitate precursors. The percentage of the total synthesized phosphatidylcholine recovered as disaturated phosphatidylcholine was increased when compared to that in control cultures, suggesting that unsaturated phosphatidylcholine synthesis was reduced to a greater extent than that of the disaturated species. Synthesis of sphingomyelin and phosphatidylethanolamine from labeled palmitate was also reduced, whereas that of phosphatidylinositol and phosphatidylglycerol was significantly increased. Addition of phospholipase C resulted in increased synthesis of phosphatidylcholine from both labeled precursors; no significant changes were found in synthesis of most of the other 3H-labeled lipids. Added phospholipase A2 did not lead to any changes in either choline or palmitate incorporation. However, when melittin (a phospholipase A2 activator) was added to the cultures, greater incorporation of both palmitate and choline was observed, along with a significant increase in the percentage of total cellular radioactivity in 14C-labeled lipids, indicating also stimulation of phosphatidylcholine synthesis. A marked increase in CTP: phosphorylcholine cytidylyltransferase activity was found after treatment of the cultures with phospholipase C. Exposure to quinacrine also increased the activity of this enzyme. Addition of phospholipase C and melittin to prelabeled pneumocyte cultures accelerated degradation of cell phospholipids and the release of free fatty acids as the main degradation products. These findings suggest that intracellular phospholipases are regulators of synthesis of surfactant phospholipids in fetal type II pneumocytes, and that activation or inhibition of these phospholipases could represent a mechanism through which hormones and pharmacological agents modify surfactant and other phospholipid synthesis.

Stimulation of phosphatidylcholine synthesis by fatty acids in fetal rabbit type II pneumocytes.

After 24 h exposure to 0.1 mM oleate or 0.1 mM palmitate there was a 2- and 1.7-fold increase, respectively, in the incorporation of choline into the lipids of type II pneumocytes. Palmitate increased the labeling of disaturated phosphatidylcholine (PC) from 23.0% of total labeled PC in control cultures to 56.6% and oleate decreased labeling of disaturated PC to 9.4%. The percentage of total cellular radioactivity found in the lipid fraction was also markedly higher in the fatty acid-treated cells (83.3% for oleate and 78.7% for palmitate) than in control cultures (64.0%). Radioactivity in water-soluble choline metabolites was correspondingly lower, with phosphocholine representing more than 95% of the label in both control and experimental cultures. After a 3 h pulse-chase period, oleate and palmitate significantly increased the percentage of total cellular radioactivity in PC and decreased the percentage in phosphocholine. Similar results were obtained by adding melittin (1-2 micrograms/ml) or phospholipase C (0.05 U/ml) to the culture medium. The stimulation of PC synthesis by fatty acids was demonstrated as early as 1 h after exposure to oleate or palmitate and at all concentrations from 0.025 to 0.25 mM. Cytidylyltransferase activity in total cell homogenates was also enhanced by long-term exposure to fatty acids and short-term addition of fatty acids or phospholipase C and melittin to the culture medium. A similar increase in cytidylyltransferase activity was found in the 100 000 X g particulate fraction of type II cells exposed to fatty acids, whereas no differences were found between the cytosolic fractions of control and treated cells. These results support the concept that an increase in intracellular level of fatty acids either from an exogenous source or following the activation of endogenous phospholipases regulates PC synthesis in fetal type II pneumocytes.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on phospholipid biosynthesis in fetal type II pneumocytes.

Purified type II pneumocytes grown in monolayer cultures after isolation from fetal rabbit lung organotypic cultures were employed to investigate effects of cAMP analogues and phosphodiesterase inhibitors on [methyl-14C]choline and [9-10(n)3H]palmitate incorporation into cell lipids. After 24 h exposure to 0.5 mM N6,O2-dibutyryl-cAMP or 8-bromo-cAMP, a significant increase was found in the rate of incorporation of choline into phospholipids. Addition of 1 mM 1-methyl-3-isobutylxanthine or aminophylline also increased incorporation of choline into phospholipids but did not significantly change the incorporation of choline into sphingomyelin. These effects were not due to increased uptake of choline or changes in the pool size of the precursor. Cyclic AMP analogues also stimulated the rate of incorporation of palmitate into most lipid fractions but did not alter the relative percentages of incorporation of either precursor into any of the phospholipids. Phosphodiesterase inhibitors did not significantly change the rate of incorporation of palmitate into neutral lipids and most phospholipids, except for a decrease into sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. However, they increased the percentage of incorporation of palmitate into phosphatidylcholine and decreased the percentage of incorporation into most other phospholipids. These data clearly indicate that cAMP can stimulate the synthesis of phospholipids within the type II pneumocytes. This effect is probably a general stimulation effect for the cAMP analogues but methylxanthines may selectively increase the synthesis of surfactant lipids such as phosphatidylcholine while decreasing that of other membrane-associated phospholipids.

Insulin receptors in fetal rabbit lung type II cells.

Insulin binding was studied in type II pneumocytes isolated from fetal rabbit lungs (27 days of gestation) and grown in monolayers in tissue culture. The mean high affinity receptor site number was 11.8 +/- 1.4 X 10(3) (+/-SEM)/cell, with a Kd of 0.45 +/- 0.07 nM (n = 6). Low affinity sites averaged 432 +/- 10.7 X 10(3)/cell, with a Kd of 45.6 +/- 11.8 nM. Incubation of the cells with 5 X 10(-10) M (Bu)2cAMP (DBcAMP) and 10(-3) M methylisobutylxanthine (MIX) for 18 h led to significant increases in the number of high affinity receptor sites and Kd (P = 0.025 and 0.05, respectively). Incubation of the cells with insulin (1 microgram/ml) for 18 h led to a significant diminution in the mean number of high affinity sites to 3.23 +/- 0.68 X 10(3)/cell (P = 0.0025). There was no significant change in the Kd of the high affinity sites. There was also no significant change in the number or affinity of the low affinity sites. When the cells were incubated with insulin in the presence of DBcAMP (5 X 10(-4) M) and MIX (10(-3) M), there was a significant increase in high affinity binding sites to a mean of 8.87 +/- 2.18 X 10(3)/cell (n = 4) compared to the value after incubation in the presence of insulin alone. There was no significant increase in the Kd of the high affinity sites. The following conclusions were drawn from these experiments. 1) Fetal type II pneumocytes possess receptors with high affinity for insulin. 2) The up-regulation of insulin receptor binding induced by high ambient concentrations of insulin in vivo in rabbit fetal lungs and circulating human monocytes does not occur in vitro when isolated pneumocytes are grown in tissue culture. 3) Insulin binding to type II pneumocytes is enhanced by DBcAMP and MIX. 4) Insulin down-regulation of receptor binding is significantly counteracted by DBcAMP and MIX.

Diminished in vitro responsiveness of circulating erythroid progenitor cells to insulin as an indicator of insulin resistance.

While insulin resistance is considered characteristic of extreme obesity, it may be more difficult to demonstrate in less severe forms of obesity. We studied five moderately obese individuals [mean body mass index (MBMI), 34.1 +/- 1.85 (+/- SE) kg/m2], one massively obese patient (BMI, 50.2 kg/m2), and seven age-matched normal subjects (MBMI, 22.4 +/- 0.93 kg/m2). While two of the obese patients had normal glucose tolerance, all had fasting hyperinsulinemia (P less than 0.02 vs. normal subjects) and exaggerated insulin responses after oral glucose challenge, as defined by area under the 3-h insulin response curve (P less than 0.01 vs. normal subjects). That this hyperinsulinemia represented in vivo insulin resistance was supported by the glucose and insulin responses in four individuals to an iv glucose bolus analyzed by the minimal modeling technique. Study of monocyte insulin receptors revealed no reduction in total insulin binding in the four obese patients tested. Since physiological concentrations of insulin stimulate the in vitro growth of normal human erythroid progenitor cells (EPC), we reasoned that this response might be blunted in cells from individuals with endogenous insulin resistance. The mean peak EPC proliferative response (26.7 +/- 9.11% above baseline) in the obese hyperinsulinemic group was significantly less than the corresponding mean value in the control group (92.6 +/- 5.24% above baseline, P less than 0.001). These results suggest that the minimal modeling technique is a sensitive method for the in vivo demonstration of insulin resistance in moderately obese individuals and that EPC responsiveness to physiological concentrations of insulin reflects in vivo insulin sensitivity and may be used as an in vitro indicator of insulin resistance.

Neuromuscular organization of the buccal system in Aplysia californica.

The intrinsic muscles and peripheral nerves in the buccal system of the sea hare Aplysia californica were studied to build a foundation on which to base future investigations of feeding in intact animals. A detailed description of the bilaterally paired intrinsic muscles is given identifying previously unreported muscles. Each of the six buccal nerves (n1-n6) and the cerebrobuccal connective (CBC) have been characterized in several respects. Cell bodies in the buccal ganglion with projections into each of the buccal nerves have been identified via the cobalt backfilling technique. All nerves contain axons of cell bodies in the ipsilateral as well as the contralateral ganglia. For each nerve, there is a consistent pattern in the distribution of cell bodies in the paired ganglia with the number of cell bodies in the contralateral ganglion being less than or equal to the number in the ipsilateral ganglion. Although the total number of backfilled cell bodies varies among the nerves, their size ranges are similar with the majority being small. Nerves 1, 2, 4, 5, and 6 provide motor innervation to the intrinsic buccal muscles in varying degrees with nerve 4 supplying all the intrinsic muscles; nerve 2 supplies only one. The axon composition of each nerve was scrutinized and revealed large numbers of axon profiles, the majority of which were less than 2 microns in diameter. The present study provides a framework for analysis of feeding behavior in Aplysia californica.

Morphological correlates of neural regeneration in the feeding system of Aplysia californica after central nervous system lesions.

Morphological techniques were used to study regeneration of central neural pathways involved in feeding behavior following bilateral crushes of the cerebral-buccal connectives (CBCs). Electron microscopic analysis revealed that CBC crushes completely transect axons within the nerve core while leaving a remnant of the nerve sheath intact. Changes in the ultrastructure of the CBCs at the crush site were determined for 1, 7, 14, 21, and 50 days postlesion. At 1 day postlesion, the crush site was no longer compressed, and the nerve core had assumed a circular shape. In addition, several small axon profiles were evident, and large areas of tissue debris and prominent microglial cells were observed. Membranous debris and hemocytes were also present in sinuses that appeared in the sheath adjacent to the crush site. From 7 to 50 days postlesion, the core of the nerve at the crush site increased in size due to the addition of small diameter axons. Initially, the sheath surrounding the crush site exhibited hyperplasia and contained a few small bundles of processes, apparently due to newly sprouted axons that had strayed from the nerve core. By 50 days postlesion, the crush site appeared nearly normal; the nerve core was reacquiring the normal radial pattern of axon profiles with some medium-sized axon profiles covered with glial sheath and exhibiting invaginations typical of the intact CBC. However, there was still a distinct lack of large diameter axons. Cobalt backfills across the crush site revealed neurons in the cerebral ganglion by postlesion day 9. Positions of stained cell bodies were consistent with those observed in controls, although the numbers of stained neurons did not recover to control levels even by postlesion day 63. The changes in the crush site and return of cell body staining with time postlesion are correlated with the recovery of consummatory feeding.

A fast and efficient method for quantification of monoclonal antibodies in an ELISA using a novel incubation system.

We have developed a sensitive, reliable, optimized ELISA to measure human IgM monoclonal antibodies using a novel shaking incubator system with short incubation periods of 15 min at 37 degrees C for all stages. The shaking incubator is compared with a static incubator over a range of incubation times and temperatures. For each stage using static incubation conditions the system does not reach a saturation level and the results are inconsistent, unlike the shaking incubator. No 'edge effects' are observed in the shaking system due to even heating from beneath and across the plate. The orbital shaking ensures optimal mixing of reagents which eliminates a diffusion limited reaction rate caused by a depletion of reactants at the solid phase as observed in the static system. The optimized shaking system permits economical use of reagents since the coating antibody can be used at high dilutions.