Biomedical subjects
Publications and source records attributed to S Jeffery.
Human muscle sections were examined for the presence of carbonic anhydrase using fluorescent second antibody and antisera specific for the three isozymes, carbonic anhydrase I (CAI), carbonic anhydrase II (CAII), and carbonic anhydrase (CAIII). CAIII was present in all fibers, CAII only in the connective tissue, and CAI showed a weak association with the sarcolemma.
Using radioimmunoassay, the concentration of carbonic anhydrase III in the livers of adult male rats was found to be approx. 30-times greater than that observed in mature females. Castration of male rats led to a marked reduction in liver carbonic anhydrase III concentrations which could be partially restored to control levels by testosterone replacement. Administration of testosterone to ovariectomised female rats induced about a 5-fold increase in liver carbonic anhydrase III concentration. Immunoprecipitation analysis of the products of liver mRNA translation in vitro with antiserum specific for carbonic anhydrase III showed that hormonal control of the levels of carbonic anhydrase III in liver is mediated by changes in the amount of translatable carbonic anhydrase III mRNA. Marked changes in liver carbonic anhydrase III concentrations were also observed in developing and ageing male rats.
Plasma carbonic anhydrase III (CAIII) levels determined by radioimmunoassay have been compared, in detail, with creatine kinase (CK) as indices of Duchenne muscular dystrophy (DMD). CAIII levels were markedly elevated in all patients but variability of levels in a number of individual patients was higher than CK.
The concentration of carbonic anhydrase III (CAIII) in male rat liver was found to be 30 times greater than that in the female. Castration of male rats led to marked reduction in liver CAIII concentrations which could be partially restored to control levels by testosterone replacement. Marked developmental and senescence changes in liver CAIII were also observed in male rats.
Carbonic anhydrase III (CA III) was identified in the products of rat soleus muscle RNA translation in vitro by both a radioimmunoassay and a specific immunoprecipitation technique followed by SDS--polyacrylamide gel electrophoresis analysis of the precipitated antigen. The primary translation product has the same Mr-value as the native isoenzyme. CA III mRNA was found to represent approximately 0.55% of the total mRNA present in rat soleus muscle.
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A radioimmunoassay for the human isozyme carbonic anhydrase III (CAIII) has been developed. The assay can detect levels as low as 4 microgram/l of sample. Plasma CAIII levels in patients suffering from Duchenne muscular dystrophy were found to be up to 39 times greater than levels in a control group. Urine CAIII levels in patients suffering from Duchenne muscular dystrophy were not significantly different from the levels found in urine from normal adults. Measurement of plasma CAIII levels may be useful in prenatal diagnosis of Duchenne muscular dystrophy, and in investigation of adult skeletal muscle disease.
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Zinc and carbonic anhydrase III measurement in human and rat muscle extracts indicate that: 1. About one fifth of zinc in human soleus is associated with carbonic anhydrase III isozyme, and even higher levels of zinc and carbonic anhydrase III are found in rat soleus, where about one half of the zinc is in carbonic anhydrase III. Other muscle was also analysed in a similar way, (see text). Heart is notable in containing lower levels of zinc but negligible carbonic anhydrase III. 2. Treatment of muscle with water or phosphate solutions showed that all the carbonic anhydrase III was water extractable, whereas significant zinc remained bound, but was partially extractable by phosphate solutions. 3. Dialysis of muscle extracts showed that whilst some zinc was dialysable, there was no significant contribution from the carbonic anhydrase III in the dialysed extract. EDTA enhanced the release of dialysable zinc from muscle extract. These findings are discussed in relation to muscle disease.
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Carbonic anhydrase III (CAIII), an enzyme recently shown by conventional electrophoresis to be muscle specific, has been qualified by "rocket" immunoelectrophoresis. This more sensitive technique has shown that the enzyme is virtually specific to skeletal muscle, where it occurs at a level of 5 mg per g, with trace levels in smooth muscle, cardiac muscle, and lung. In man there does not appear to be any correlation between CAIII levels and the proportion of red and white muscle fibers. The fetal development of CAIII has also been examined using immunoelectrophoresis, and the enzyme can be detected at 11 weeks' gestation. The CAIII level rises gradually up to 25 weeks, and there is then a more dramatic increase to reach approximately half adult level at birth.
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A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.
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Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.
The presence of electolytes inhibited the transformation of Naegleria gruberi from amoeba to flagellate, the molarity required varying with the salt used, namely 80 mM NaCl, 90 mM KCl, 50 mM CaCl2 or 60 mM MgCl2. Non-electrolytes also prevented this transformation at 250 mM for either sucrose or glucose, and this is known to be an osmotic effect. That the effect of ionic solutions was different was demonstrated by varying the time at which the environemnt was changed from distilled water to salt solution. Experiments with suspensions of either living or heat-killed bacteria in distilled water, together with the supernatants obtained when bacteria were removed by centrifugation, showed that the inhibition of transformation which occurred in bacterial suspensions was not due to any factors produced by the bacteria and present in solution. It appeared that this inhibition was brought about by the physical presence of the bacteria, either living or heat-killed, and some possible interpretations of this 'contact' phenomenon are discussed.