Potential use of embalmed cadavers to study mast cell presence.

BACKGROUND: Embalmed cadavers in medical classes represent a potential source for collecting human tissues without the inherent problems of obtaining fresh or surgical specimens. Although the manner of fixation and vagaries of embalming techniques eliminate many such tissues for histological assessment, other techniques can be applied successfully to embalmed tissues. Pertinent to the present study, mast cells contain granules that are preserved under good fixation in formaldehyde, a main ingredient in embalming fluids. Visualization of these granules is possible, even though the ultrastructure of these cells is not preserved. METHODS: Two techniques for the visualization of connective tissue mast cells were compared using embalmed and fresh specimens: Alcian blue and avidin conjugated to fluorescein isothiocyanate (FITC-Avidin). Both will bind to mast cell granules, even in the presence of formaldehyde. RESULTS: Although mast cell numbers in the connective tissue did not differ between embalmed and fresh tissues, comparisons between the techniques involved showed the FITC-Avidin technique to be possibly more sensitive, perhaps because of the increased contrast from the fluorescent dye. CONCLUSIONS: Thus for some studies, human cadavers may provide a valuable source of tissue. However, use of embalmed tissue necessitates ensuring good embalming, checking for dehydration, and proper storage until embedment.

Vasoactive intestinal polypeptide nerve fibers in human and monkey (Macaca fascicularis and Macaca mulatta) kidneys.

Nerve fibers immunoreactive for vasoactive intestinal polypeptide (VIP) were demonstrated for the first time by the indirect immunofluorescence technique in human and monkey kidneys. VIP-immunoreactive nerve fibers showing varicosities were observed in the adventitia of arcuate arteries and their branches. The density of VIP-immunoreactive nerve fibers decreased from the juxtamedullary region to the cortex. Occasionally a VIP-immunoreactive varicose nerve fiber was observed near the vascular pole of a glomerulus, but no direct innervation of afferent or efferent arterioles in either monkey or human kidney was found. The distribution of VIP-immunoreactive nerve fibers in the monkey and human kidneys was similar to that reported in other species, with less density. The functional role of VIP in the innervation of the kidney is not known, but various suggestions have been made regarding the possible involvement of VIP on vasodilation of selective intrarenal blood vessels, renin secretion, and/or effects on tubules. While none of these questions were established at this time they would appear to be logical areas for further study.

Neuropeptide Y (NPY)-like immunoreactive nerve fibers in the human and monkey (Macaca fascicularis) kidney.

Nerve fibers immunoreactive for neuropeptide Y (NPY) are demonstrated for the first time by the indirect immunofluorescence technique in the human and monkey kidney. NPY-like immunoactivity (NPY-LI) is shown in a bundle of nerve fibers in the surrounding connective tissue of arteries and to a lesser extent, veins, mainly at the juxtamedullary region. Varicose nerve terminals are shown associated with blood vessels and passing between tubules in the mid and lower cortex. NPY-LI nerve fibers are also seen surrounding afferent and occasionally efferent arterioles at the vascular pole of the glomeruli. The distribution of NPY-LI nerve fibers in the monkey and human kidneys is similar to that of other species, only the quantity of the nerve fibers varies.

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation.

Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the 45Ca-exchange method to determine free cellular Ca++, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca++). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca++] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca++ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca++] results in a release of the fusion-block and an increase in cellular Ca++ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca++] must exist. The latter suggests further that cell surface Ca++ was also critical.

Ascorbic acid facilitates chicken myoblast fusion in vitro.

Addition of low concentrations of ascorbic acid (5 micrograms/ml to 10 micrograms/ml) to myogenic chick embryo cultures resulted in an early fusion. At 30 h cultures receiving small amounts of ascorbic acid presented fusion rates 3 times that of the control. However, control rates of fusion were not different from those of experimentals at 50 h. No such effect was seen with ascorbic acid added at 24 h of culture, or with ascorbic acid addition to a calcium-deprived system. These findings demonstrate that the calcium binding properties of ascorbic acid can induce precocious myogenic fusion.

Chicken lactose lectin: cell-to-cell or cell-to-matrix adhesion molecule?

Endogenous chicken muscle lectin isolated by lactose affinity chromatography inhibits myoblast fusion. Similar lectins isolated from embryonic brain, heart, and liver and from adult intestine exhibit the same ability. Elevated levels of any of these lectins canceled the inhibitory effect. Peanut agglutinin isolated by the same procedure had no effect at any concentration tested. Concanavalin A affected fusion only at high concentrations. Muscle lectin was shown to agglutinate myoblasts in microtiter plates, whereas exogenous addition in culture inhibited alignment as seen by time lapse microcinematography. Cell-to-cell communication between lectin-treated cells was shown by nucleotide exchange, and lectin-coated culture dishes did not affect cell attachment. Our evidence shows a lack of specificity to muscle, but suggests an aggregating capacity between cells, or possibly an interaction between the cell membrane and the extracellular matrix.

The effects of psychosine upon growth of human skin fibroblasts from patients with globoid cell leukodystrophy.

[3H]Thymidine incorporation into cultured skin fibroblasts from patients with globoid cell leukodystrophy (GLD) and from control individuals was utilized to monitor the effects of psychosine (galactosylsphingosine) upon cell replication. The concentration of psychosine necessary to inhibit 50% (ID50) of the growth of cultured skin fibroblasts was approximately 15 microgram/ml for both normal and GLD fibroblasts deficient in the enzyme galactosylceramide beta-galactosidase. Growth inhibition curves for GLD and for control fibroblasts were comparable after 3 days and after 7 days exposure to the glycolipid, so that accumulation of psychosine was not a critical factor affecting toxicity. Galactosylceramide, the major substrate for the enzyme galactosylceramide beta-galactosidase, did not inhibit [3H]thymidine incorporation into either normal or GLD fibroblasts at the concentration tested, in contrast to the highly toxic effects of psychosine at similar concentrations. The comparable inhibitory levels of psychosine in control cells and in GLD fibroblasts which are deficient in ability to hydrolyze this glycolipid suggest that the toxicity of psychosine is nonspecific. Therefore, these results are not consistent with the concept that globoid cell leukodystrophy is primarily a psychosine lipidosis.

Studies of a synthetic substrate in canine globoid cell leukodystrophy.

Gal et al. ((1977) Clin. Chim. Acta 77, 53-59) reported the use of a new synthetic substrate, 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside for the diagnosis of human globoid cell leukodystrophy. Assay of beta-galactosidase in brain homogenates from normal, carrier, and globoid cell leukodystrophy-affected dogs utilizing this new substrate demonstrated overlapping activities. Instead of reflecting specific D-galactosyl-N-acylsphingosine galactohydrolase (EC 3.2.1.46), the 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside beta-galactosidase activity in canine brain is highly correlated with nonspecific 4-methylumbelliferyl beta-galactosidase. Optimization of the 2-hexadecanoyl-amino-4-nitrophenyl-beta-D-galactopyranoside assay system for canine brain and the use of varying concentrations of taurocholate or taurodeoxycholate in the assay mixture did not alter the lack of specificity. These results indicate a significant difference in the nature of the underlying defect in galactosylceramide beta-galactosidase in canine globoid cell leukodystrophy compared to human globoid cell leukodystrophy.

Purified lectin from skeletal muscle inhibits myotube formation in vitro.

A lactose-extractable lectin obtained from 14--16-d embryonic chick pectoral muscle and myotube muscle cultures by affinity chromatography inhibited myotube formation in culture. When applied to muscle cultures at 0.09 micrograms/ml, the purified lectin produced variable effects on the inhibition of myotube formation related to the time and length of application, suggesting that components of the culture medium and/or temperature produced inactivation. Hemagglutination assays showed that the lectin was inactivated by horse serum and by chick embryo extract but not by L-15 salt solution at 4 degrees C. Incubation in L-15 solution at 37 degrees C with or without 2 mM dithiothreitol resulted in inactivation in 2--3 h. To maximize the effect of the lectin on the inhibition of myotube formation, primary muscle cultures were grown in low [Ca+2] medium to inhibit fusion, and then [Ca+2] was increased to elicit fusion in the absence and presence of lectin with solution renewal every 2 h. Without lectin, myotube formation was normal, whereas, with lectin, it was inhibited by 93%. Continued incubation at 37 degrees C. without renewal of lectin resulted in myotube formation, suggesting reversibility by lectin inactivation.