Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Species- and muscle type-dependence of perinatal isomyosin transitions.

The progressive transition from developmental to adult myosin isoforms during perinatal development was quantified in four muscles (diaphragm, gastrocnemius medialis, masseter and tongue) of four mammals (guinea-pig, hamster, rabbit and rat). It was observed that the timing of transition varied for each muscle, and differed according to the mammal as well. This suggests that the synthesis of adult myosin isoforms may be partly related to the specialized contractile function of a given muscle in a given species.

Electrophoretic separation of developmental and adult rabbit skeletal muscle myosin heavy chain isoforms: example of application to muscle denervation study.

We present the separation by SDS gel-electrophoresis of the six main myosin heavy chains (MHC) present in rabbit skeletal muscle. The separation of the four adult MHC (1, 2A, 2X/2D, 2B) was compared to that of the corresponding rat MHC as described by Talmadge and Roy [J. Appl. Physiol. 99 (1993) 2337-2340]. We found that many rabbit muscles contained mainly one of the four MHC, in some cases the 2B MHC. In addition, we resolved the embryonic E and perinatal P developmental MHC, which should facilitate muscle differentiation and regeneration studies in the rabbit. An example of application to the study of muscle denervation is given.

The same myosin isoforms are found in the female and male sexually dimorphic levator ani muscle of the rat, but their postnatal transitions are not synchronous.

The levator ani of the female adult rat is greatly atrophied in comparison to the same muscle in males. In the present study, the female levator ani was, nevertheless, found to contain type IIb myosin isoforms similar to those contained in the male muscle. These adult type isoforms were, however, synthesized later in the female than in the male levator ani: the half-transition times of the myosin transition curve were 20 days postnatal in the male and 35 days postnatal in the female. The transition curves for castrated and uncastrated male rats were the same. Thus, the presence of male gonadal hormones apparently did not affect the myosin transition.

Opposite regulations by androgenic and thyroid hormones of V1 myosin expression in the two types of rabbit striated muscle: skeletal and cardiac.

The finding that V1 cardiac myosin is expressed in masticatory skeletal muscles of the rabbit provided a unique opportunity for comparing the hormonal regulation of V1 in skeletal and cardiac muscles. Thyroid hormones had no significant effect on the postnatal expression of V1 in masticatory muscles, but increased this expression in cardiac ventricles. In contrast, androgenic hormones reduced V1 expression in masticatory muscles, but did not affect it significantly in cardiac ventricles. Modulation of V1 gene transcription in striated muscle is thus shown here to depend both on the target muscle and on the hormone.

Relationship between muscle myosin isoforms and contractile features in rabbit fast-twitch denervated muscle.

The effects of 8-day-old rabbit fast-twitch gastrocnemius denervation on the type of myosin isoforms and on contractile features (maximum velocity Vmax and contraction time (CT) of the muscle were followed between 15 and 60 days postnatal. The myosin isoforms and the Vmax and CT values of the denervated gastrocnemius displayed large changes during this period. These changes, which led at 2 months postnatal to a muscle displaying the properties of a slow-twitch muscle did not occur in synchrony: complete conversion to slow-type myosin isoforms occurred only at 60 days postnatal, whereas complete conversion to slow-twitch Vmax and CT values occurred as soon as 35 days postnatal. The results address a new question concerning the relationship between muscle myosin and contractile features.

Effect of trimetazidine and verapamil on the cardiomyopathic hamster myosin phenotype.

1. In this study we investigated whether long-term trimetazidine (anti-ischaemic drug) therapy alters the ventricular myosin heavy chain (MHC) isoform composition in a model of cardiomyopathy. 2. MHC isoforms were analysed in the native state by electrophoresis in a pyrophosphate buffer. Myosin isoform patterns were studied in cardiac muscle from cardiomyopathic hamsters (CMH) of the BIO 14:6 strain during the time course of the disease and compared with those of healthy golden hamsters (F1B). The correlation between myosin profile and Ca2+-activated ATPase activity was determined from 220 days. 3. At the stage of insufficiency (350 days), CMH presented the most abnormal phenotype with 53% V1-24% V3 compared to 79% V1-7% V3 (P<0.001), in F1B. Trimetazidine was administered to cardiomyopathic hamsters from the early stage of active disease (30 days) to the congestive stages (220-350 days). Within 65 days, trimetazidine treatment, in CMH and F1B, reduced V1 to a low level (53% and 62%, respectively), which remained constant throughout the treatment. This level was similar to that in 220 and 350 days-old untreated-CMH. In sharp contrast, a standard calcium blocker, verapamil, administered to CMH in the same conditions resulted in a higher V1 (about 70%) and higher global myosin ATPase activity from 220 days. 4. Previous results in terms of hypertrophy and survival, compared to these results, suggest that verapamil and trimetazidine treatments reveal a dissociation between ventricular hypertrophy and isomyosin distribution. In addition, the shift in favour of V3 may not necessarily be an aggravating factor of the disease but an adaptative compensatory event.

Endurance training affects myosin heavy chain phenotype in regenerating fast-twitch muscle.

The aim of this study was to analyze the effects of treadmill training (2 h/day, 5 days/wk, 30 m/min, 7% grade for 5 wk) on the expression of myosin heavy chain (MHC) isoforms during and after regeneration of a fast-twitch white muscle [extensor digitorum longus (EDL)]. Male Wistar rats were randomly assigned to a sedentary (n = 10) or an endurance-trained (ET; n = 10) group. EDL muscle degeneration and regeneration were induced by two subcutaneous injections of a snake toxin. Five days after induction of muscle injury, animals were trained over a 5-wk period. It was verified that approximately 40 days after venom treatment, central nuclei were present in the treated EDL muscles from sedentary and ET rats. The changes in the expression of MHCs in EDL muscles were detected by using a combination of biochemical and immunocytochemical approaches. Compared with contralateral nondegenerated muscles, relative concentrations of types I, IIa, and IIx MHC isoforms in ET rats were greater in regenerated EDL muscles (146%, P < 0.05; 76%, P < 0.01; 87%, P < 0.01, respectively). Their elevation corresponded to a decrease in the relative concentration of type IIb MHC (-36%, P < 0.01). Although type I accounted for only 3.2% of total myosin in regenerated muscles from the ET group, the cytochemical analysis showed that the proportion of positive staining with the slow MHC antibody was markedly greater in regenerated muscles than in contralateral ones. Collectively, these results demonstrate that the regenerated EDL muscle is sensitive to endurance training and suggest that the training-induced shift in MHC isoforms observed in these muscles resulted from an additive effect of regeneration and repeated exercise.

Molecular characteristics of cocaine-induced cardiomyopathy in rats.

Cocaine abuse induces severe cardiomyopathy. To investigate the molecular effects of acute and prolonged administration of cocaine, mRNAs encoding markers of either mechanical overload, as atrial natriuretic factor (ANF) and alpha- and beta-myosin heavy chains, or fibrosis as type I and III procollagens, were quantitated in the left ventricle of rats 4 h after one injection of cocaine (40 mg/kg, n = 7), or 14 (n = 15) and 28 days (n = 10) after chronic infusion of cocaine (40 mg/kg per day). Plasma cocaine and benzylecgonine concentrations were both significantly augmented during the infusion while plasma levels of triiodothyronine and thyroxine were lowered. Acute injection of cocaine induced ANF gene expression. Cocaine treatment during 28 days resulted in left ventricular hypertrophy (+ 20% after 24 days, P < 0.05) with normal blood pressure, associated with an accumulation of mRNAs encoding ANF and type I and III collagens (+66% and +55%, P < 0.05). Such a chronic treatment also induced a shift from the alpha- to the beta-myosin heavy chain gene expression (-40% and +50%, P < 0.05). In conclusion, cocaine activates markers of both hemodynamic overload and fibrosis. Such an activation may result from direct and/or indirect effects of the drug such as myocardial ischemia, mechanical overload and/or hypothyroidism.

Reinnervation of denervated extensor digitorum longus of the rat by the nerve of the soleus does not induce the type I myosin synthesis directly but through a sequential transition of type II myosin isoforms.

The fast-contracting extensor digitorum longus (EDL) muscle of 1-month-old rats was denervated and reinnervated by the nerve innervating the slow-contracting soleus muscle. After variable periods of time, the myosin isoform content of the EDL was analyzed by sensitive electrophoretic techniques, which allowed to discriminate between the slow-type I and the three, IIA, (IID or IIX) and IIB, fast-type II myosin isoforms. Compared to the control EDL, which contains predominantly the IIB isoform, the operated muscles contained variable proportions of all the isoforms. Analysis of the results leads us to conclude that reinnervation of EDL induces a sequential transition of myosin isoforms: IIB----(IID or IIX)----IIA----I.

Myosin light chains of guinea-pig striated muscles. Similarities and differences with rat myosin light chains.

1. Myosin light chains of guinea-pig striated muscles have been screened by two-dimensional gel electrophoresis and compared to rat myosin light chains. 2. The fast type light chains 1F and 3F, slow type light chains 1S and 2S, and embryonic type light chain 1E are shown to differ in the two rodents; only the fast type light chains 2F co-electrophorese on the gel. 3. In guinea-pig, as in rat, ventricle muscle light chains appear the same as the 1S and 2S light chains and atrial light chain type 1 the same as the 1E light chain. We show that this embryonic light chain of guinea-pig myosin is difficult to identify and may be confused with the adult 1F light chain.

Myosin light chain phosphorylation in intact rat uterine smooth muscle. Role of calcium and cyclic AMP.

Myometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two-dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium 0.8 mM) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a beta-adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproternol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 microM) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation-dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium.

Myosin switches in skeletal muscle development of an urodelan amphibian, Pleurodeles waltlii. Comparison with a mammalian, Mus musculus.

The isomyosins from dorsal axial muscle, which appear successively through metamorphosis of P.waltlii, are shown to be composed of identical fast-type light chains but of distinct heavy subunits. We observe that this modification goes with a change in ATPase activity as also in the case of mouse. Metamorphosis in amphibian as well as birth in mammalian are thus both accompanied by the synthesis of new myosins of higher catalytic efficiency.

Comparison of myosins from the masseter muscle of adult rat, mouse and guinea-pig. Persistence of neonatal-type isoforms in the murine muscle.

Adult rat, mouse, and guinea-pig masseter muscles display distinct myosin electrophoretic patterns. The rat muscle contains four main forms which by reference to the myosins of the IIB tensor fasciae latae, of the IIA mylohyoid, and of the red and white portions of the sternomastoid muscles, correspond respectively to the intermediate-type and to the three fast-type isoforms. The mouse masseter muscle contains only three main myosins, the intermediate-type and two fast-type isoforms. The guinea-pig muscle also displays only three bands, whose assignment is, however, less straightforward than in the murine species; their electrophoretic mobilities are not strictly the same as those of their homologous forms in rat and mouse. Comparison with the myosins of the tensor fasciae latae and of the sternomastoid muscles of guinea-pig allows their identification as intermediate and fast-type myosins. In addition to these typical adult-type forms, adult murine masseter muscles are observed to contain between zero and 30% of neonatal-type myosins. The comparison of the developmental transitions of myosins in the rat masseter with those in the skeletal muscles of the same animal indicates a delay in the appearance of the adult as well as in the disappearance of the neonatal-type myosins in the masseter muscle. Both the variability in myosin types with the animal species and the atypical presence of neonatal forms in the murine adults suggest that myosin expression in the masseter muscle is subjected to unusual regulations.

Isoforms of myosin and actin in human, monkey and rat myometrium. Comparison of pregnant and non-pregnant uterus proteins.

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non-pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non-dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two-dimensional gel electrophoresis, we found that the 17-kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two-dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the gamma form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non-pregnant uterus. The changes observed in the 17-kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.

Specific programs of myosin expression in the postnatal development of rat muscles.

The expression of myosin during postnatal development was studied in a dozen muscles of the rat. All muscles displayed the usual sequential transitions from embryonic to neonatal and to adult isomyosins. However, we observed that these transitions did not take place uniformly. Thus, half-transition times for the appearance of the adult intermediate and fast myosin extended from seven days for diaphragm, the most precocious muscle of all those examined, to 23 days for male rat masseter. Besides the large differences between their half-transition times, we noticed that the transition curves displayed different slopes, covering different periods. Differences between muscles mainly affected the neonatal-to-adult transition rather than the embryonic-to-neonatal transition, since the embryonic-type myosin disappeared from all muscles examined except for one, at about the same time, by the end of the first week after birth. In addition, the appearance of slow myosin varied for each muscle and did not follow curves parallel to those for intermediate and fast myosins. These results indicate that each muscle of the rat is subjected to a specific program of myosin isoform transitions during postnatal development.

Effect of testosterone and thyroid hormone on the expression of myosin in the sexually dimorphic levator ani muscle of rat.

During postnatal development, the myosin transition from embryonic and neonatal isoforms to adult isoforms has been shown to occur with half-transition times of about 20 and 32 days in the male and female levator ani muscles, respectively. We show that this difference could not be attributed to the testosterone male hormone, since treatment of newborn females by testosterone did not modify the half-transition time. However, treatment of females by thyroid hormone accelerated the myosin transition of the female muscle, which then occurred at almost the same time as the transition of the male muscle. This suggests that the difference between the half-transition times of the male and female levator ani muscles may be largely attributed to different sensitivities of the male and female muscles to thyroid hormone. This is the first example of sexually dimorphic muscle response to thyroid hormone.

Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals. Myosin isoform analysis.

The regeneration of adult rat and mouse slow (soleus) and fast (sternomastoid) muscles was examined after the degeneration of myofibers had been achieved by a snake venom cardiotoxin, under experimental conditions devised to spare as far as possible the satellite cells, the nerves, and the blood vessels of the muscles. Three days after the injury, no myosin was detectable in selected portions of the muscles. New myosins of embryonic, neonatal, and adult types started to be synthesized during the following two days. Adult myosins thus appeared more precociously than in development, which implies that the synthesis of myosin isoforms during regeneration does not entirely 'recapitulate' the sequence of myosin transitions observed during normal development. Two weeks after the injury, the isomyosin electrophoretic pattern displayed by regenerated muscles was already the same as that of control muscles; the normal adult pattern was therefore expressed more rapidly in regenerating than in developing muscles. Except for the synthesis of the slow isoform which was generally inhibited in denervated muscles, the same types of myosins were expressed during the early stages of regeneration in denervated as in innervated muscles; long-term denervation prevented however the qualitative and quantitative recovery of the normal myosin pattern.

Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidine-tagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.