Role of oregano on bacterial enzymes in 1,2-dimethylhydrazine-induced experimental colon carcinogenesis.

Colon cancer incidence is higher in developed countries than in developing countries. We determined the effect of oregano (Origanum vulgare L.) on fecal bacterial enzyme activities in 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis in rats. Male Wistar albino rats were divided into 6 groups and all animals were fed with a high-fat diet (20% fat in the diet). Group 1 served as control and group 2 animals received 60 mg.kg(-1) body weight (b.w.) oregano daily for 15 weeks. To induce colon cancer, DMH (20 mg.kg(-1) b.w.) was injected subcutaneously once a week for the first 4 weeks (groups 3-6). In addition, oregano was administered at 20, 40, or 60 mg.kg(-1) b.w. each day orally for the entire 15 weeks (groups 4-6). We analyzed the fecal bacterial enzyme activities and found it to be significantly higher in the group treated with DMH alone than in the control group. Oregano supplementation at all 3 doses significantly suppressed the bacterial enzyme activities and modulated oxidative stress significantly compared with the unsupplemented DMH-treated group. Results of our present investigation therefore revealed that oregano markedly inhibited DMH-induced colon carcinogenesis and that the optimal dose of 40 mg.kg(-1) b.w. was more effective than either the higher or lower doses.

Purification and characterization of myosins from human and rabbit skeletal muscles by using specific monoclonal antibodies.

By using immunoaffinity column chromatography slow (I) and fast (IIA, IIB) myosins were isolated from human (vastus lateralis) and rabbit (tibialis anterior, psoas and conoidal bundle) skeletal muscles. The peptide pattern revealed that slow (I) and fast (IIA, IIB) myosin heavy chains are quite distinct, as are those from pure slow (conoidal bundle) and fast (psoas) rabbit skeletal muscles. Unlike Billeter et al. (1981) the authors observed that fast human myosins were always associated with a small amount of slow myosin light chains. The fast myosins (IIA, IIB) from rabbit tibialis anterior muscle did not appear very distinct and contained only fast myosin light chains. These myosins were different from the IIB myosin from the psoas muscle. Ten per cent of the fibres revealed histochemically as fast IIA also reacted with an anti-slow myosin antibody. The classical histochemical techniques appear inadequate to demonstrate the existing differences among fibre types, but the monoclonal antibodies hold promise.

Myosin isoenzymes in human hypertrophic hearts. Shift in atrial myosin heavy chains and in ventricular myosin light chains.

The myosin light chain complement and proteolytic peptide patterns of myosin heavy chains were studied by two-dimensional and one-dimensional electrophoretic techniques respectively, in a total of 57 samples from ventricular and atrial tissues of normal and hypertrophied human hearts. Hypertrophies were classified haemodynamically as due to pressure-overload and volume-overload. In addition to the occurrence of ventricular light chains in hypertrophied atria we also observed the atrial light chain-1 (ALC-1) in hypertrophied ventricular tissues. On average over 6% of total light-chain-1 comprised ALC-1 in pressure-overloaded ventricles and around 3% in volume-overloaded ventricles. In single cases of pressure-overload ALC-1 amounted up to over 20% of total light chain-1. With regard to the myosin heavy chains limited digestion by two different proteinases produced over 200 clearly resoluble peptides. The absence of any detectable differences in the peptide patterns between myosin heavy chains from normal and hypertrophic tissues of left or right ventricle is in line with the findings of J. J. Schier and R. S. Adelstein (J Clin Invest 1982; 69: 816-825). In atrial tissues however, reproducible qualitative differences in the peptide patterns indicated that during hypertrophy a different type of myosin heavy chains becomes expressed. No differences were seen between the myosin heavy chains from normal left and right atria.

Changes of myosin and its ATPase in "neuronally" and "mechanically" contralateral muscles after cross-reinnervation in normal and capsaicin-treated rats.

The extensor digitorum longus (EDL) muscle was cross-reinnervated by the soleus (SOL) nerve in normal and neonatally capsaicin-treated rats. After 5 months the muscles were investigated for their myofibrillar ATPase reaction and their myosin light and heavy chain composition. Besides the well known transformation of the cross-reinnervated EDL toward a slow muscle, muscular changes were also found in the contralateral leg. Although these changes were hardly detectable in the EDL muscle, a remarkable (70 to 100%) reduction of the fast type IIA fiber population was found in the SOL. The decrease of the number of IIA fibers (compared with time-matched controls) was paralleled by corresponding changes in the myosin light and heavy chain patterns. After the cross-reinnervation of a muscle, two kinds of contralaterality must be distinguished. In the experiments reported the cross-reinnervated EDL muscle remains "mechanically" contralateral to the EDL muscle of the other leg, while it becomes "neuronally" contralateral to the SOL muscle. Our results are interpreted as a symmetric "slowing down" of these "neuronally" contralateral muscles. Neonatal capsaicin treatment that decreased considerably the number of unmyelinated group IV afferent fibers did not influence the outcome of these experiments.

6-phosphogluconolactonase. Purification, properties and activities in various tissues.

6-Phosphogluconolactonase was purified to apparent homogeneity in a four-step procedure from bovine erythrocytes. The extent of purification and the kinetic properties of the enzyme were evaluated with an optical test that was based on the hydrolysis of synthetic 6-phosphogluconolactone. The active enzyme from bovine erythrocytes is a monomer with an approximate molecular weight of 30000. It exhibits Michaelis-Menten kinetics and cofactors are not required for activity. The enzyme was found in a number of tissues. Its activity, when compared to the activity of the glycolytic enzymes, illustrates the importance of glucose oxidation via the pentose phosphate pathway relative to glycolysis.

Ipsi- and contralateral fibre transformations by cross-reinnervation. A principle of symmetry.

Cross-reinnervation of rabbit soleus muscle by the peroneal nerve induces a 90% transformation of slow into fast fibres. These changes are reflected in corresponding transformations of the enzyme activity pattern of energy metabolism, the isozyme pattern of lactate dehydrogenase and, in confirmation of previous results (Srihari et al. 1981), transitions from a slow to a fast type myosin light chain pattern. The transformation process appears to be complete after 6 months. Similar changes, although less extensive are also found in the soleus muscle of the contralateral leg. Fibre type transitions in the contralateral muscle are not accompanied by fibre type grouping, as seen in the cross-reinnervated muscle and therefore these changes appear to result from a transformation of the motor units themselves. This phenomenon is interpreted as a compensatory process in maintaining symmetry within the neuromotor system.

Changes of myosin and its ATPase in experimentally induced fiber transformation in the rat.

It has been reported that a reversible muscle fiber transformation was effected within a short time in the sternohyoid muscle of the rat by the removal of the contralateral muscle. In the present investigation the muscles were analyzed histochemically and biochemically. The fiber transformation was easily detected histochemically by the appearance of slow type I fibers, as this fiber type is almost lacking in control muscles. In two-dimensional electrophoresis the light chain pattern of the experimental and control muscles was similar. However, the peptide pattern of the heavy chains revealed clear differences between control and experimental muscles. The muscle fiber transformation found was reflected in the myosin heavy chain which is the site of ATPase activity. For transformation of the light chain pattern probably a much stronger stimulus is needed.

Enzyme activities, histochemistry and myosin light chain pattern in extraocular muscles of rabbit.

A study was performed on enzyme activities, myosin light chain pattern and histochemical composition of extraocular muscles (EOM) of rabbit. The results were compared with those from slow-twitch soleus and fast-twitch psoas limb muscles. EOM were found to contain myosin light chains of both slow and fast skeletal muscles, with the latter being predominant. The activities of the enzymes regulating glycolysis and lactic acid fermentation were found to lie between those measured in soleus and psoas muscles, whereas the activities of the enzymes involved in oxidative metabolism were found to be very similar or even higher than in soleus muscle. All five isozymes of lactate dehydrogenase (LDH 1-5) were found in EOM. The predominant species was LDH-3 illustrating that this pattern is uncharacteristic of either slow soleus or fast psoas muscles. The complex structural and functional characteristics of EOM might result from the observed heterogeneity of fibres.

Isoforms of heavy and light chains of cardiac myosins from rat and rabbit.

The light chains of myosin from atrial and ventricular tissues from rat and rabbit were examined by one- and two-dimensional polyacrylamide gel electrophoresis. The myosin heavy chains were electrophoretically isolated, digested after denaturation in sodium dodecyl sulfate with papain and proteinase from S. aureus V8, and the resulting peptides resolved in one-dimensional gel electrophoresis. The peptide patterns of myosin heavy chains from atrial and ventricular tissues of adult rabbits were different, indicating differences in their primary structures. No such differences could be detected in a total of around 180 peptides produced by the two proteinases from the myosin heavy chains of adult rat atrial and ventricular tissues. With regard to light chains, the same migration pattern was observed for atrial and ventricular tissues from both rat and rabbit. The atrial light chains ALC1 and ALC2 migrated with molecular weights lying between those of the ventricular light chains VLC1 and VLC2. In two-dimensional electrophoresis, the corresponding light chains from rat and rabbit co-migrated. An additional light chain was observed in foetal ventricles, which exhibited identical electrophoretic properties to ALC1 from adult atrial tissues. In rat myofibrillar preparations from atrium and ventricle, an unidentified protein (x) occurred in the region of light chain-1 but with a more acidic isoelectric point, which seems to be related to the developmental stage of these tissues and which could not be detected in rabbit heart tissues or in any skeletal muscles.

The effect of different patterns of long-term stimulation on contractile properties and myosin light chains in rabbit fast muscles.

Fast rabbit skeletal muscles (tibialis anterior and extensor digitorum longus) were stimulated for 2-28 days by electrodes implanted in the vicinity of the peroneal nerve to produce maximal contractions at two different frequency patterns: that occurring naturally in nerves to slow muscles (10 Hz continuously) or three bursts of tetani (40 Hz) per minute, each 5s in duration. Both types of frequency produced muscles more resistant to fatigue during isometric twitch contractions, and led to a prolongation of contraction time greater and more consistent with 10 Hz than with 40 Hz. The twitch/tetanus ration was significantly higher in muscles stimulated at 10 Hz for 3-4 weeks but was not different from controls in muscles stimulated at 40 Hz. Both types of stimulation led to the appearance of myosin light chains characteristic of slow muscles. Muscles stimulated for 4 weeks at 40 Hz developed greater twitch tension per gram, and had significantly smaller cross-sectional area of myofibrils than control muscles. It is concluded that long-term electrical stimulation of fast muscles can affect some muscle contractile properties to resemble those of slow muscles irrespective of frequency of stimulation, provided the total number of stimuli is comparable, the duration of stimulation is long enough (minimum 2 weeks) and all motor units are activated.

Electrophoretic analyses of atrial and ventricular cardiac myosins from foetal and adult rabbits.

1. Rabbit cardiac myosins from atrium and ventricle were found to differ in their native form in pyrophosphate gel electrophoresis as well as in their light chain pattern in one- and two-dimensional electrophoretic systems. 2. The myosin light chain pattern in sodium dodecylsulfate gel electrophoresis differs between atrial and ventricular tissues in the mammalians (rabbit, dog, human, pig, sheep and rat) but not in the chicken. 3. In foetal rabbit ventricle an additional light chain is observed which, in two-dimensional electrophoresis with adult cardiac light chains, was found to be the same as atrial light chain-1.

Electrophoretic analyses of myofibrillar proteins from the body wall muscles of Ascaris suum.

A myofibrillar protein extract has been isolated from the muscle of Ascaris suum. Two-dimensional electrophoresis of this extract revealed that the myosin light chain 1 (ALC1) migrates as 3 components with approximate isoelectric points in the range of 5.3-5.6. The most acidic component of ALC1 appeared to be phosphorylated when the myofibrillar extract was incubated for 10 s with catalytic subunit of cAMP dependent protein kinase and [gamma-32P] ATP. The myosin light chain 2 (ALC2) migrated as a single component in isoelectric focusing with an approximate isoelectric point of 5.5 Actin was resolved into 2 components with identical molecular weight but isoelectric points differing by approximatley 0.2 pH units. A protein was tentatively identified in the myofibrillar extract as tropomyosin. It migrated as a single band with an approximate isoelectric point of 5.0 and a molecular weight of 39 000. None of the troponin components could be identified in the myofibrillar extract. It is postulated that muscle contraction in A. suum muscle could be controlled by phosphorylation of myosin.

Ipsi-and contralateral changes in rabbit soleus myosins by cross-reinnervation.

Cross reinnervation of soleus muscle in the adult rabbit induces changes in myosin light chain and isomyosin patterns. The transformation of the light chain pattern consists of a decrease in LC1's and LC2, and an induction of the three fast type myosin light chains. The transition in the isomyosin pattern consists of a decrease of the slow type isomyosin SM and an induction of fast type isomyosins FM1, FM2, and FM3 normally seen in fast-twitch rabbit muscle. An additional isomyosin of intermediate electrophoretic mobility, which resembles isomyosin 4 of rat muscle, is induced. These changes are not restricted to the cross-reinnervated soleus muscle but are also seen, although to a lesser extent, in the contralateral soleus muscle. It is suggested that the altered afferent activity brought about by cross-reinnervation, is neurally transmitted to the opposite side and leads to a change in efferent activity to the contralateral muscle.

The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chick muscles.

The differentiation of neuromuscular junctions of multiply innervated, slow anterior latissimus dorsi (ALD) and focally innervated, fast, posterior latissimus dorsi (PLD) muscles was studied in normal and curarized chick embryos. At 16 days of incubation, fibres of both muscles are contacted by several axon profiles, the number of which falls with age. In 18-day-old embryos individual endplates in ALD are usually contacted by three axon profiles, whereas in PLD, endplates are contacted only by a single large terminal profile. At this time, there is already a significant accumulation of cell organelles in the postsynaptic area. Treatment of embryos with curare during the 7th and 12th day of incubation delays the differentiation of the neuromuscular junction in both muscles. The paralysis dramatically affects the decrease of the number of axon profiles at individual endplates in both muscles. At 16 days the number of axon profiles was greater in embryos treated with curare than in the untreated controls. At 18 days when the number of axon profiles normally decreases, the endplates of both types of curarized muscles have an even greater number of axon profiles than at 16 days. Endplates in curarized PLD had up to 13 and in curarized ALD up to 12 axon profiles. The effects of curare gradually wore off and when the movements of the embryos again became more vigorous, the normal differentiation of neuromuscular junctions continued. At 21 days of incubation many embryos recover from curare and show endplates of normal appearance in both muscles. These results suggest that activity of the muscle is essential for the maturation of the neuromuscular junctions.

Differentiation of slow and fast muscles in chickens.

1. The development of the characteristic histochemical appearance of the slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) was studied in chickens during embryonic development as well as during regeneration of minced muscle. 2. During embryonic development the activity of the oxidative enzyme succinic dehydrogenase (SDH) is higher in the slow ALD muscle already at 16 days of incubation. At this time the fast PLD has a higher activity of the glycolytic enzyme, phosphorylase. Although the histochemical appearance of the two types of muscle is already different at 16 days, their contractile speeds are still similar. No difference in myosin ATP-ase was found in the two muscles in young embryos but in 20-day old embryos the two muscles became distinctly different when stained for this enzyme. 3. When PLD muscles in hatched chickens redeveloped during regeneration in place of ALD the histochemical characteristics of the regenerated muscle resembled ALD, and when ALD regenerated in place of PLD it resembled PLD. 4. It is concluded that the histochemical characteristics of slow and fast muscles become determined during early development, even before any difference in contractile properties can be detected and that they are determined by the nerve.