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The purpose of this study was to reveal the presence of Z-helical conformation in normal crystalline lens DNA. Z-DNA antigen was prepared against poly(dG-dC).poly(dG-dC), which had been converted to the Z-helix conformation in high salt and then stabilized by bromination. Circular dichroism (CD) spectra confirmed the presence of left-handed Z-helix DNA. Antibodies to Z-DNA were raised in three rabbits immunized with brominated (Br-) poly(dG-dC).poly(dG-dC). These antibodies do not cross-react with polynucleotides in the B-helical form, but are specific to the left-handed Z-DNA conformation. DNA was isolated from three different regions of the calf lens. Anti-Z-DNA antisera, affinity purified IgG polyclonal anti-Z-DNA antibodies and monoclonal anti-Z-DNA antibodies were used as immunoprobes to detect the presence of S-DNA sequences. DNA from the cortex region of the lens reacted strongly with the anti-Z-DNA antibodies, but no binding could be observed in the DNA from the nucleus region. Digestion of lens DNA with DNase 1 dramatically decreased Z-DNA antibody binding, while RNase A and T1 treatment had no effect on Z-DNA immunoreactivity. This study has demonstrated that: (a) Z-DNA antibodies developed for our study can bind in high salt solutions (4M NaCl) to purified lens DNA sequences isolated from a variety of different calf lens cell types. By this criterion, lens DNA contains sequence determinants which may assume or are in the Z-helix conformation.

In order to establish the presence of Z-DNA sequences in the normal crystalline lens and to define their structure-function relationship, fixed and unfixed calf lens tissue sections were examined immunohistochemically. Polyclonal and monoclonal anti-Z-DNA antibodies were developed as immunoprobes, using brominated (Br-) poly(dG-dC).poly(dG-dC) as an antigen. The structure of the Z-helix antigen was confirmed by circular dichroism (CD) and U.V. spectroscopy. Whole rabbit and goat anti-Z-DNA sera; rabbit and goat IgG polyclonal anti-Z-DNA antibodies; and anti-Z-DNA monoclonal IgG antibodies were utilized as Z-DNA immunoprobes to localize the Z-DNA in calf lens tissue sections. Immunohistochemical examination using the peroxidase-antiperoxidase (PAP) method indicated that the cortex region of the lens reacted strongly with the anti-Z-DNA antibodies, while no immunoreaction could be observed in the nucleus region. Similar immunoreactive patterns were obtained whether whole sera, affinity purified IgG polyclonal antibodies or monoclonal antibodies were utilized. Immunobinding of anti-Z-DNA antibodies was low, effectively background type binding, in unfixed lens tissue sections. Various fixatives were tested to explore the potential antibody-Z-DNA interaction in calf lens tissue. Nuclear fixatives enhanced Z-DNA antibody immunoreactivity, while formalin, microanatomic and cytoplasmic fixatives produced lesser results. Digestion of the lens tissue with DNase I eliminated Z-DNA immunoreactivity, while RNase A and RNase T1 treatment had no effect. Actinomycin D also prevented Z-DNA immunoreactivity.

This study demonstrates, via immunohistochemistry and bioassay, the presence of NGF in embryonic bone and cartilage of the chick. Embryos were killed on days 6-9 of incubation at 12 h intervals, and on days 10-18 at 24 h intervals. Paraffin-embedded sections of hind limbs or buds were immunostained with a polyclonal antibody against NGF and the biotin-avidin-horseradish peroxidase technique. Immunostaining was positive in both bone and cartilage, with cartilage staining more intensely. For bioassay, bones from the hind limbs of 9- and 12-day embryos were fast-frozen, lyophilized, and homogenized with Medium 199 (M199). Dorsal root ganglia from 8-day embryos were cultured for 24-36 h with rooster plasma, M199, and varying concentrations of bone homogenate. Significant neurite outgrowth was seen, with the greatest response elicited by 12-day bone homogenate. Addition of anti-NGF to the cultures abolished neurite outgrowth. The results indicate that NGF is present in cartilage and bone of the chick embryo; it may determine the density of sympathetic innervation to the developing skeletal tissues.

Visual examination of the intensity of Procion dye in hamster femurs revealed that dye incorporation decreased during hibernation. Little or no dye was observed after hibernation lasting longer than 50 days. It seems that hibernation may be associated with increased bone resorption and a loss of bone matrix.

Newt forelimbs were unilaterally amputated and Beta-amino propionitrile (BAPN) was added to the water in which the newts were maintained. Sixty-three days after amputation, animals were injected with either 3H-proline or 3H-lysine. Forelimbs distal to the elbow of both amputated and non-amputated sides were removed 72 hours later. Uptake of 3H-proline and 3H-lysine in whole limbs was assessed by liquid scintillation and in cartilage cells and matrix by autoradiography. Amputation was a potent stimulus to both collagen formation and maturation during regeneration. BAPN reduced the stimulatory effect of amputation on collagen formation but not on its maturation. Collagen synthesis and maturation are independent events and, in intact non-regenerating limbs, BAPN adversely affected collagen maturation while collagen synthesis was unaltered.

The effects of cold-stress and hibernation on bone dynamics in the femurs of hamsters were investigated using histometric analyses. Control animals were maintained at 27 degrees C for 90 days; experimental animals were kept at 5 degrees C and hibernated for 7, 15, 21, 50, or 90 days. Histometric analyses of cross sections indicated that bone diameter and cortical thickness at the femoral midshaft increased after 83 days of extreme cold and 7 days of hibernation but decreased significantly after 69 days of cold stress and 21 days of hibernation. Osteoporosis was evident although the number of osteons per unit area of bone increased during hibernation. An initial decrease in the number of non-Haversian longitudinal vessels per unit area of bone was seen in experimental animals which was apparently related to a corresponding reduction in cortical thickness. Lacunar area increased in these animals, suggesting that osteocytic osteolysis may be a significant mechanism for calcium regulation during hibernation.

In Triturus administration of NGF causes hypertrophy of neuronal perikarya of third dorsal root ganglia and post-subclavian sympathetic ganglia. Two classes of neurons were characterized cytologically in the third dorsal root ganglia. Some evidence is presented which indicates that Class I neurons respond more vigorously to NGF. It is suggested that Class I neurons are similar to the medio-dorsal neuroblasts of chick embryos in their response to NGF. Forelimb amputation did not cause neuronal hypertrophy in either third dorsal root ganglia or post-subclavian sympathetic ganglia. The incorporation of (3H)-uridine increases to a highly significant extent in third dorsal root ganglia following NGF treatment or limb amputation. When NGF treatment and limb amputation were combined, no further increase in (3H)-uridine incorporation was noted. This suggests that RNA synthesis is maximally stimulated by either NGF treatment alone or limb amputation alone.

Twenty-four LSH and LVG strain golden hamsters, Mesocricetus auratus, were used. Experimental animals were maintained at 5 C and allowed to hibernate. Control animals were kept at 27 C. Six animals (3 experimental, 3 control) were injected subcutaneously with 1 microCi of 3H-proline/gm body wt. (Spec. act. 3 Ci/mM) after hibernation lasting 12 hours, 1 day, 3 days, or 7 days. Animals were killed 1 hour after injection and autoradiographs were prepared from 5 microns thick decalcified sections of femurs. A greater number of endosteal cells were labeled than periosteal cells and also exhibited a greater magnitude of labeling throughout the study. Differences between endosteal and periosteal cells both in percentage of cells labeled and magnitude of labeling were maximum in control animals and progressively decreased with increasing periods of hibernation. A reduction in synthesis of matrix proteins during the early period of hibernation was seen and was attributed to a significant reduction both in average cell activity and in the number of active cells during hibernation. The latter phenomenon apparently made a large contribution to the reduced matrical synthesis. 3H-proline uptake by osteoblasts probably reflects the reduced requirements of matrical synthesis during hibernation.

This investigation is a light and electron microscopic description of the submandibular duct salivary bladder of the rat, a dilation of the distal end of the main excretory duct. The wall of the bladder consists of a mucosa with pseudostratified epithelium, a submucosal layer of connective tissue, and an underlying layer of striated muscle. The pseudostratified columnar epithelium lining the bladder is composed of three cell types: light cells, dark cells, and basal cells. The lamina propria contains bundles of collagen, attenuated fibrocytes, capillaries with fenestrated endothelia, and nerve fibers which enter the epithelial layer. The capillaries of the submucosa are not fenestrated. The morphology of the wall of this structure provides evidence that the primary fluid of the submandibular gland is modified in the bladder by transepithelial fluid and ion transport.

The persistence of cartilage in the adult newt (Notophthalmus viridescens) forelimb skeletal regenerate has recently been reported by Libbon et al. It is particularly evident in the carpal group, which remains cartilaginous for at least 9 months while all other regrown skeletal parts are either ossified or ossifying. The present investigation was undertaken to determine whether delayed ossification in the adult regenerate recapitulates a pattern evident during the development of the forelimb of the red eft (N. viridenscens' immature terrestrial phase). Right upper limbs of 20 efts were examined at low magnification to establish carpal composition and organization. Among five efts of the smallest size (26.54 plus or minus 2.20 mm snout-to-vent length), and displaying bright orange dorsal skin coloration, all carpal rudiments were cartilaginous. Eleven efts, intermediate in length (33.88 plus or minus 0.81 mm), displaying orange-green coloration, all carpals had ossified. These observations demonstrate that during adult newt forelimb regeneration, the failure of the carpal elements to begin ossification in synchrony with other regrown skeletal parts duplicates a similar schedule of delayed mesopodial ossification which is evident during the development of the red eft's forelimb.