Search

Cyclic 3', 5'-guanosine monophosphate (cGMP) was measured at eight stages of forelimb regeneration in adult newts and compared with the cGMP levels of non-regenerating control limbs. There was a significant increase in cGMP content during dedifferentiation followed by a sharp decrease to minimal levels at the cone stage. A second smaller increase in cGMP occurred between the cone stage and mid-differentiation, followed by a decrease to relatively constant levels approaching control values as differentiation progressed. The changes in cGMP during dedifferentiation and during the period of highest cell proliferation indicate that cGMP may play a role in these processes. The smaller increases in cGMP levels during differentiation may reflect a reduced rate of cell division in the differentiating tissues.

The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examing competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5 mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s). The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP. Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.

The regenerating amphibian limb serves as a useful model for studying factors influencing cell proliferation and differentiation. In particular, peripheral nerves are thought to provide a stimulus for growth of the blastema, presumably via the elaboration of an as yet unidentified neurotrophic factor. In the present study, pressure ultrafiltration coupled with chromatofocusing have proven to be effective methods of partially purifying a neurotrophic factor from adult chicken brains. This chick brain growth factor (CBGF) appears to be a heat-stable, basic peptide of low molecular weight (less than 6,000). It is a potent mitogen in vitro, at nanomolar concentrations, for both blastema cells and Swiss mouse 3T3 fibroblasts. CBGF is apparently distinct from other peptide mitogens and/or neuromodulators that have been reported to stimulate blastema growth in vivo and in vitro. These include substance P, FGF from bovine brain and pituitary, EGF, transferrin (sciatin), and spinal cord growth factor (SCGF). The possible relationship of CBGF to other neural regulatory molecules is discussed.

The activities of the membrane-bound protein kinases of the human erythrocytes membrane that phosphorylate spectrin, band-3 protein, and phospholipids were compared in patients with myotonic muscular dystrophy and normal age- and sex-matched controls. These activities tended to be lower in the patients, but the differences were not statistically significant. In contrast, the temperature responses (the increase in activity in response to an increase in temperature from 30 degrees C to 37 degrees C) of the spectrin and band-3 protein kinase activities were significantly lower in the patients. Although they do not eliminate an alteration of one of the substrates, these results are consistent with the proposal that differences in erythrocytes from myotonic muscular dystrophy (MyD) patients are due to a membrane lipid change. Cholesterol is unlikely to be the altered lipid, as no difference in membrane cholesterol content was found.

The phosphorylation of spectrin, band 3 protein, and the phospholipids of erythrocyte membranes (ghosts) was examined in 10 patients with Duchenne muscular dystrophy (DMD) and in healthy age- and sex-matched controls. The rates of phosphorylation of spectrin and band 3 protein were significantly higher in ghosts prepared from patient blood than from control blood at both 30 degrees C and 37 degrees C. However, the mean increases in the rate of phosphorylation of both spectrin and band 3 protein in response to a temperature change from 30 degrees C to 37 degrees C were identical in ghosts from patient and controls. Phosphorylation of phospholipid and its temperature response did not differ between patients and controls. These results complement previous observations of differences in erythrocytes from patients with DMD. The similarity of the changes in phosphorylation of both spectrin and band 3 protein indicates a common cause, possibly their lipid environment.

We have examined the development of the brachial lateral motor column in White Leghorn chickens that are homozygous for muscular dystrophy. We found an accelerated loss of motor neurons between days 6 and 11 in ovo in the dystrophic embryos such that at the end of this time they had only 80% of the population in age-matched controls. After day 11 in ovo the rate of motor neuron loss was the same in both normal and dystrophic birds. To determine whether the accelerated motor neuron loss was due to expression of the dystrophic gene within the spinal cord itself or whether it was secondary to abnormalities in some other tissue, we exchanged the brachial region of the spinal cord between normal and dystrophic embryos at 2 days in ovo, allowed the resulting chimeras to develop until 11 days in ovo and estimated the motor neuron population in the transplanted segment of cord. We found that spinal cords transplanted into normal hosts had significantly higher populations of motor neurons than spinal cords transplanted into dystrophic hosts. We concluded that the accelerated motor neuron loss seen in dystrophic birds is not intrinsic to the cord but is influenced by other tissues in the embryo.

Corynebacterium fascians causes a fasciation disease in a number of dicotyledons and this disease appears to be caused by compounds with cytokinin activity elaborated by the infecting bacteria. Extractions of C. fascians in late logarithmic phase under conditions where the pH never falls below 7.0 yield about 2 μg/l of N (6)-(Δ(2), a potent cytokinin. If a mild acidification step is included in the extraction procedure the yield increases to about 12 μg/l. This is due to release of N (6)-(Δ(2)-isopentenyl)adenine from C. fascians tRNA during the extraction procedure. In terms of total cytokinin activity present in C. fascians cultures, N (6)-(Δ(2)-isopentenyl)adenine appears to be a minor component.