Distribution of type IV collagen during avian limb bud development.

Normal limb development is dependent on an epithelial-mesenchymal interaction between the overlying apical ectodermal ridge (AER) and the underlying mesenchyme. The basement membrane between the epithelium and the mesenchyme has been proposed to play an important role in regulating epithelial-mesenchymal interactions during development. To explore the role basement membrane type IV collagen may play during limb development we investigated the distribution of type IV collagen by immunolocalization. Developing avian leg buds were examined at 2 developmental stages: stage 23, when the AER is inductively active, and stage 28, when the AER is regressing. The proximal basement membrane in stage 23 limb buds stained much more intensely than the distal basement membrane. This proximal-distal immunostaining difference was less in stage 28 limb buds. We used the monoclonal antibody IIB12, which recognizes an epitope adjacent to the initial collagenase cleavage site on the type IV collagen molecule, to explore whether this proximal-distal difference in basement membrane staining could result from the loss of type IV collagen. The distal basement membrane of stage 23 limb buds demonstrated little immunostaining with the IIB12 antibody, suggesting enhanced collagenase-associated degradation. The immunostaining was increased in stage 28 limb buds. Consistent with a loss of type IV collagen, we also found that unfixed stage 23 leg bud cryostat sections stored at 4 degrees C lost their immunostaining for type IV collagen, in contrast to stored stage 28 limb bud cryostat sections. These results demonstrate that type IV collagen is distributed in a proximal-distal pattern in the basement membrane of the developing chick limb bud and suggest that this pattern may be the result of a selective degradation of type IV collagen in the basement membrane underlying the active AER. These results are consistent with the hypothesis that the basement membrane plays a role in regulating the epithelial-mesenchymal interaction responsible for induction of limb outgrowth.

Effects of enzyme isolation on the basal lamina of developing avian limb bud epithelia.

The developing avian limb bud is a classic example of an epithelial-mesenchymal interaction. Numerous attempts at maintenance of the epithelia in culture have been predominantly unsuccessful. The fate of the isolated epithelial sheet of the limb bud [including the apical ectodermal ridge (AER)] in culture may depend at least in part on the integrity of its basal lamina following isolation. In this study the distal epithelium of the stage 23 limb bud was isolated utilizing trypsin and Dispase II in a variety of procedures. The integrity of the basal lamina of limb epithelium immediately upon isolation and after 2 h in culture was determined by immunofluorescent staining for laminin, and electron microscopy. In epithelial sheets isolated with Dispase II a direct relationship was observed between maintenance of the extracellular matrix at isolation and the preservation of the tissue structure and cytoarchitecture following 2 h in culture. In contrast, there was an accelerated deterioration during incubation of the tissue isolated with trypsin, independent of isolation conditions and integrity of basal lamina after isolation. Short-term maintenance of limb bud epithelial structure and cytoarchitecture after enzymatic isolation seems correlative to the maintenance of extracellular matrix at the epithelial basal surface.

Evaluation of EMIT and RIA high volume test procedures for THC metabolites in urine utilizing GC/MS confirmation.

Results of EMIT, Abuscreen RIA, and GC/MS tests for THC metabolites in a high volume random urinalysis program are compared. Samples were field tested by non-laboratory personnel with an EMIT system using a 100 ng/mL cutoff. Samples were then sent to the Army Forensic Toxicology Drug Testing Laboratory (WRAMC) at Fort Meade, Maryland, where they were tested by RIA (Abuscreen) using a statistical 100 ng/mL cutoff. Confirmations of all RIA positives were accomplished using a GC/MS procedure. EMIT and RIA results agreed for 91% of samples. Data indicated a 4% false positive rate and a 10% false negative rate for EMIT field testing. In a related study, results for samples which tested positive by RIA for THC metabolites using a statistical 100 ng/mL cutoff were compared with results by GC/MS utilizing a 20 ng/mL cutoff for the THCA metabolite. Presence of THCA metabolite was detected in 99.7% of RIA positive samples. No relationship between quantitations determined by the two tests was found.

Contact inhibition and malignancy.

The role of contact inhibition in influencing the behaviour of malignant cells is discussed in a review. Although tissue culture cannot simulate the immense complexity of the conditions in vivo, some of the distinctive features of malignant invasion can be conveniently observed with this technique. The evidence derived from this technique indicates that defective contact inhibition of movement of malignant cells does contribute to their invasiveness.

Contact reactions influencing cell locomotion of a mouse sarcoma in culture.

An investigation of the behaviour of cells of the mouse transplantable sarcoma S180 in culture by time-lapse cinematography has been made to determine how the spreading of a population from an explant is affected by contact reactions between the sarcoma cells; and how the invasion of a fibroblast population is affected by contact reactions with the fibroblasts. Although the sarcoma cells show a form of contact inhibition to each other, this has little influence on the spreading of a population, which is brought about very largely by diffusive movement, in sharp contrast to the spreading of a fibroblast population. The invasion of a fibroblast population by S180 cells takes place as a consequence of defective contact inhibition; though contact inhibition (but not contact paralysis) of the S180 cells has an appreciable incidence. During invasion the S180 cells, though moving on the exposed surfaces of the fibroblasts, may be largely using the collagen substratum between the fibroblasts for their locomotion. The arrangement of the fibroblasts produces some degree of contact guidance of the sarcoma cells.

Invasive behavior between sarcoma and fibroblast populations in cell culture.

The mutual invasion in culture of a population of standard fibroblasts (chick embryo) and a population of cells from a transplantable mouse sarcoma (MC)M, BAS/56, or 311) or of neontal mouse fibroblasts has been estimated quantitatively. We arranged the confrontation of the pairs of populations by placing primary explants near each other. After fixation, the distance the cells had migrated from each explant was sampled in the space between the explants where they met and at the sides of the explants where they migrated freely. Measurements of nuclear overlap and orientation were also made. In the sarcoma as in the fibroblast population, homologous contact inhibition of movement probably produced an oriented migration from the explants before the populations met. Abot 12-24 hours after, mutual invasion was considerably greater in the sarcoma versus fibroblast than in the fibroblast versus fibroblast experiments. It is proposed that this difference was due to a difference of heterologous contact inhibition in the two types of experiment.

The difficulties of changing.

In general, our psychological equipment develops in such a way that we get information of predictive value through the senses; we tend to record constancies and consistencies of events and behave as though these are persistent. We are mostly unaware of the power of the stores of relevant experience (assumptions, expectations, attitudes) that condition our perceptions and therefore cannot question them, nor in many circumstances is there need to do so. In fairly constant conditions they are useful in helping us to see, quickly and effortlessly, what we expect to see. But in rapid changes this is not so; we can no longer rely on our psychological equipment and we become uncertain, confused and, in extreme cases, antagonistic, fearful and impotent. Examples of assumptions that influence medical education and medical treatment are given. Ability to respond effectively to change requires confidence to examine and restructure basic assumptions. The value of interaction in small groups in helping to achieve this is indicated.