Effects of 8 weeks of vibration training at different frequencies (1 or 15 Hz) in senior sportsmen on torque and force development and of 1 year of training on muscle fibers.

OBJECTIVE: To examine the effects of 8 weeks of vibration training at different frequencies (1 and 15 Hz) on maximal isometric torque and force development in senior sportsmen, and of 1 year of heavy-resistance and vibration trainings on muscle fibers. METHODS: Seven healthy senior sportsmen (mean age: 69.0 +/- 5.4 years) performed an 8 weeks of strength training of knee extensors. Vibrations were applied vertically to the axis of movement during training. One leg of each subject was trained at a frequency of 1 Hz, while the other leg was trained at 15 Hz. Measures of isometric peak torque (at knee-angles of 60, 90 and 120 degrees ) and force development were recorded before and after training. Four sportsmen continued a year-long heavy-resistance training adding every second week a session of vibration training. After training, muscle biopsies were harvested from their quadriceps muscles and used for structural analyses. Morphometry of muscle fibers was performed by light microscopy. Immunohistochemistry using anti-MHCemb and anti-N-CAM antibodies was performed to measure potential muscle damage. Data from muscle morphometry were compared to that of a series of vastus lateralis biopsies harvested from 12 young sportsmen and four healthy elderly. RESULTS: Our results showed a significant increase in isometric peak torque at both 1 and 15 Hz vibration frequency in all three measured angles of the knee. There was no significant difference between the two frequencies, but we could find a higher increase in percentage of maximum power after the 1 Hz training. The results of force development showed a slight increase at the 1 Hz training in measured time frames from 0 to 50 and 200 ms, without statistical significance. A trend to significance was found at the 1 Hz training at the time window up to 200 ms. The 15 Hz training showed no significant changes of force development. Muscle biopsies show that the muscles of these well trained senior sportsmen contain muscle fibers which are 35% larger than those of sedentary elderly and, unexpectedly, 10% larger than those of young sportsmen. Despite 1 year of heavy resistance and vibration training, no evidence of muscle damage or denervation/reinnervation could be observed by light microscopy analyses, ATPase histochemistry and immunohistochemistry using anti-N-CAM or anti-MHC-emb antibodies. DISCUSSION: Integration of vibration to conventional strength training in elderly sportsmen induces similar improvement of isometric peak torque and force development independently from the vibration frequency after 8 weeks of training, and long-term results in the surprising evidence of hypertrophic muscle fibers larger than those of young active sportsmen. The observation that the vibration training with low frequency is safe opens the possibility to test these rehabilitation procedures in sedentary elderly.

Subclinical myopathy in patients affected with newly diagnosed colorectal cancer at clinical onset of disease: evidence from skeletal muscle biopsies.

OBJECTIVE: To evaluate skeletal muscle biopsy from asymptomatic patients affected with newly diagnosed colorectal cancer and to identify pathological features which may be indicative of tumor-associated muscle disorders, potentially leading to cachexia. METHODS: Patients affected with newly diagnosed colorectal cancer at clinical onset of disease underwent biopsy of the rectus abdominis muscle during elective laparoscopic tumor resection, before chemotherapeutic treatment. Morphometric analyses, ATPase histochemistry and immunohistochemical studies using antibodies directed to N-CAM and to MHC-emb, two sound makers of muscle denervation and injury-induced muscle regeneration, were performed on intraoperative muscle biopsies from ten patients. Muscle biopsies from rectus abdominis of seven subjects affected with non-neoplastic condition, which underwent laparoscopic surgery, were used as controls. RESULTS: In patients' biopsies, we observed a surprisingly high percentage of myofibers with internalized or central nuclei compared to controls (9.15 +/- 8.9 versus 0.6 +/- 0.9, p<0.0003). In addition, in the 30% of patients, small myofibers expressing the MHC-emb have been identified (0.4 +/- 0.5 positive fibers/mm(2)), while in 50% of patients, larger fibers positive for N-CAM have also been detected (0.7 +/- 1.1 positive fibers/mm(2)), suggesting that investigated muscle biopsies exhibit other evidence of muscle fiber injury/regeneration and/or denervation. Among the 10,000 analysed myofibers in control biopsies, no MHC-emb and N-CAM-positive muscle fibers have been detected. Thus, patients affected with newly diagnosed colorectal cancer at clinical onset of disease display early signs of a subclinical myopathy. DISCUSSION: Factors and mechanisms of this cancer-associated myopathy are yet unknown. The facts that the great majority of the abnormally nucleated myofibers are of the fast type and that regenerating myofibers are present, suggest a myogenic response to the colorectal cancer and not to the laparoscopic modalities of the biopsy harvesting. Follow-up of the patients will elucidate the clinical relevance of our observation, and further studies investigating the molecular mechanism underlying this early cancer-associated myopathy will hopefully provide some pathogenetic clues leading to the identification of potential specific targets for therapeutic intervention to prevent tumor cachexia.

Polymyositis, dermatomyositis and malignancy: a further intriguing link.

The association between malignancy and autoimmune myositis has been largely described and confirmed by numerous epidemiological studies. The temporal relationship between the two pathologic conditions can vary: malignancy may occur before, at the same time or following the diagnosis of myositis. Beside these observations, the molecular mechanisms underlying this association are still unknown, even though it has been demonstrated a possible antigenic similarity between regenerating myoblasts and some cancer cell populations. To better identify peculiar histopathologic features common to cancer and myositis, we screened muscle biopsies from patients affected with polymyositis, dermatomyositis, myositis in association to cancer, and from patients affected with newly diagnosed cancer, but without myositis. Similarly to the histopatologic features that were observed in the muscle from myositis patients, especially in those with cancer associated myositis, in patients affected with malignancy at the clinical onset of disease we observed early sign of myopathy, characterized by internally nucleated and regenerating myofibers, most of them expressing the neural cell adhesion molecule. The hypothesis that in a particular subset of individuals genetically predisposed to autoimmunity, an initial subclinical tumor-induced myopathy may result in an autoimmune myositis, represents a further intriguing link behind the association of these two conditions.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2-24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (beta-dystroglycan, alpha-sarcoglycan, and gamma-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.

Ecto-ATPase activity of alpha-sarcoglycan (adhalin).

alpha-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of alpha-sarcoglycan. alpha-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin.dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that alpha-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.

Myotonic dystrophy protein kinase expressed in rat cardiac muscle is associated with sarcoplasmic reticulum and gap junctions.

Myotonic dystrophy (DM) is one of the most prevalent muscular diseases in adults. The molecular basis of this autosomal disorder has been identified as the expansion of a CTG repeat in the 3' untranslated region of a gene encoding a protein kinase (DMPK). The pathophysiology of the disease and the role of DMPK are still obscure. It has been previously demonstrated that DMPK is localized at neuromuscular junctions, myotendinous junctions, and terminal cisternae of the sarcoplasmic reticulum (SR), in the skeletal muscle, and at intercalated discs in the cardiac muscle. We report here new findings about specific localization of DMPK in the heart. Polyclonal antibodies raised against a peptide sequence of the human DMPK were used to analyze the subcellular distribution of the protein in rat papillary muscles. Confocal laser microscopy revealed a strong although discontinuous reactivity at intercalated discs, together with transverse banding on the sarcoplasm. At higher resolution with immunogold electron microscopy, we observed that DMPK is localized at the cytoplasmic surface of junctional and extended junctional sarcoplasmic reticulum, suggesting that DMPK is involved in the regulation of excitation-contraction coupling. Along the intercalated disc, DMPK was found associated with gap junctions, whereas it was absent in the two other kinds of junctional complexes (fasciae adherentes and desmosomes). Immunogold labeling of gap junction purified fractions showed that DMPK co-localized with connexin 43, the major component of this type of intercellular junctions, suggesting that DMPK plays a regulatory role in the transmission of signals between myocytes.

Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans.

The discovery of the dystrophin gene, whose mutations lead to Duchenne's and Becker's muscular dystrophy (DMD and BMD), represents the first important landmark by which, in the last ten years, molecular biology and genetic studies have revealed many of the molecular defects of the major muscular dystrophies. Very rapidly, several studies revealed the presence at skeletal and cardiac muscle sarcolemma of a group of proteins associated to dystrophin. This includes a set of five transmembrane glycoproteins, the sarcoglycans, whose physiological role, however, is still poorly understood. Dystrophin and the associated proteins are believed to play an important role in membrane stability and maintenance during muscle contraction and relaxation. However, the absence of sarcoglycans from sarcolemma does not appear to affect membrane integrity suggesting that these components of the dystrophin complex are recipients of other important functions. This review deals with recent advances in the knowledge of sarcoglycan function and organization that may give important insights into the pathogenetic mechanisms of muscular dystrophies.

Gender- and thyroid hormone-related transitions of essential myosin light chain isoform expression in rat soleus muscle during ageing.

In this brief review, the modulatory influence of essential myosin light chain (MLC) isoforms on muscle cell contractility is discussed. Specific interest is focused on the expression of the MLC1Sa and MLC1Sb isoforms in the slow-twitch soleus muscle in male and female rats, during ageing and after thyroid hormone treatment. According to two-dimensional gel electrophoresis analysis, the MLC1Sa/MLC1SB ratio increased during ageing in both males and females in parallel with the age-related decrease in shortening velocity reported in muscle fibres expressing the slow (type 1) myosin heavy chain (MHC) isoform. However, the MLC1Sa and MLC1Sb isoform expression responded to thyroid hormone treatment in a complex manner which did not parallel the age-related changes in shortening velocity reported in hyperthyroid animals. Thus, if MLC1Sa and MLC1Sb isoforms modulate shortening velocity in type 1 fibres, then other modulators of shortening velocity are not regulated by thyroid hormone in co-ordination with these essential MLCs.

Evidence for localization of the myotonic dystrophy protein kinase to the terminal cisternae of the sarcoplasmic reticulum.

Myotonic dystrophy is an autosomal dominant multisystem disease primarily affecting skeletal muscle and is characterized by the presence of an amplified trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. In this study, the subcellular localization of the myotonic dystrophy protein kinase in muscle tissues has been investigated at both morphological and biochemical level, by using antibodies against the myotonic dystrophy protein kinase. Immunofluorescence studies and Western-blot analysis were carried out with antibodies raised against both a synthetic peptide and a recombinant fusion protein fragment specific for the myotonic dystrophy protein kinase. The kinase is localized both to the surface membranes, and within the skeletal fibres in the region of the A-I band boundary. Consistent with the A-I location of the kinase is that Western-blot analysis of purified fractions from sarcoplasmic reticulum show that triads and sarcoplasmic reticulum terminal cisternae are immunoreactive for two myotonic dystrophy protein kinase proteins of different molecular weight (85 and 54 kDa). The relative amount of these two proteins is different in relation to the muscle type, the 85 kDa protein being more evident in skeletal than in cardiac fibres. In addition, immunofluorescence studies of cardiac muscle reveal a heavy concentration of DM-PK localized to the intercalated discs, as well as a weaker reaction in the sarcoplasm. These results taken together suggest that multiple isoforms of the DM-PK may exist and that they may be differentially located in muscle tissues.

A novel muscle protein located inside the terminal cisternae of the sarcoplasmic reticulum.

An immunofluorescence study of adult rat muscle tissues with a polyclonal antibody against the RGD-directed fibronectin receptor of Friend's erythroleukemia cells (alpha5beta1-integrin) unexpectedly revealed a pattern of intracellular antigen distribution. Western blotting analysis of rat and rabbit membrane fractions indicated that the antibody recognizes a 167-kDa protein expressed both in heart and in skeletal muscle (relative abundance: heart > slow muscle > fast muscle), but not in liver and kidney. The 167-kDa protein did not show altered electrophoretic mobility upon reduction and failed to bind several lectins, including wheat germ agglutinin. A study of its subcellular distribution in rabbit skeletal muscle revealed that the 167-kDa protein is mostly associated with the terminal cisternae of the sarcoplasmic reticulum (SR) and, to a smaller extent, with the sarcolemma, while it is absent in the longitudinal tubules of the SR. The 167-kDa protein is not an integral membrane protein since it can be extracted at pH >/=10. This protein can be proteolytically cleaved only in the presence of detergent, indicating that it resides on the luminal side of the SR. The 167-kDa protein could be resolved from the closely spaced sarcalumenin and histidine-rich protein by column chromatography followed by detergent dialysis and two-dimensional gel electrophoresis. The N terminus and the internal sequences did not match any known sequence in protein and DNA data bases, indicating that the 167-kDa protein is a novel muscle protein selectively localized to the SR. Integrins from rat kidney fibroblasts were not recognized by either (i) a polyclonal antiserum against the purified 167-kDa protein or (ii) the anti-alpha5beta1-integrin antiserum after affinity purification onto the 167-kDa protein. These data indicate that the 167-kDa protein is not immunologically cross-reactive with integrins, despite its reaction with a polyclonal anti-integrin antibody.

Increased expression of dystrophin, beta-dystroglycan and adhalin in denervated rat muscles.

To evaluate a potential regulatory role of the nerve, the distribution and expression of dystrophin, of beta-dystroglycan (43DAG) and adhalin (50DAG), two of the dystrophin-associated proteins and utrophin (dystrophin related protein or DRP) were studied in rat muscles after 2 weeks of denervation. We found that dystrophin, beta-dystroglycan and adhalin were overexpressed in denervated muscle, whereas utrophin did not increase and was found only in the post-synaptic membrane. The study of the distribution of dystrophin in the sarcolemma of single muscle fibres indicates that the molecular organization of dystrophin was maintained after denervation. Dystrophin in addition of forming a scaffold around the fibre was found around the clusters of AChR that reappeared in the extra-synaptic membrane after denervation. Also beta-dystroglycan colocalises at these clusters. These results suggest that the increase in dystrophin, beta-dystroglycan and adhalin is correlated with the reappearance of AChRs in the extra synaptic membrane.

Identification and localization of the myotonic dystrophy gene product in skeletal and cardiac muscles.

We have raised a polyclonal antibody against a synthetic peptide chosen within the deduced sequence of the myotonic dystrophy gene product. This antibody binds to a protein whose molecular weight is in the range of 50-54 kDa in Western blotting of rat, rabbit and human muscles. Biochemical studies seem to indicate that this protein is a peripheral component of sarcoplasmic reticulum as well as of plasma membrane.

Ontogenesis of chick iris intrinsic muscles: evidence for a smooth-to-striated muscle transition.

The iris sphincter muscle consists of striated muscle fibers in the adult chicken. The ontogenetic development of this muscle has been studied by immunocytochemistry, from Embryonic Day 7 to the time of hatching. The time course of expression of specific markers of either smooth or striated muscle, i.e., smooth and skeletal muscle myosin heavy chains along with the inositol 1,4,5-trisphosphate (IP3) receptor, the intracellular Ca2+ release channel gated by the second messenger IP3, was investigated. We observed the parallel and transient expression, from about Embryonic Day 8 until hatching, of smooth muscle myosin heavy chain and IP3 receptor in early differentiating cells at the pupillary margin, as well as in migrating cells at subsequent stages; the sequential expression of sarcomeric myosin heavy chain around the end of the second embryonic week; and its spreading throughout the iris sphincter by the time of hatching. The present findings are consistent with a smooth muscle differentiation stage as an intermediate stage in the ontogenic development of the iris sphincter muscle of the chicken.

An age-related type IIB to IIX myosin heavy chain switching in rat skeletal muscle.

A novel fast-twitch motor unit type, called the IIX-myosin heavy chain (MHC) motor unit, identified by the glycogen depletion technique together with a series of monoclonal antibodies (mAbs) specific for MHCs, has been isolated recently in the rat tibialis anterior muscle. In young animals, this unit has physiological, biochemical and morphometrical properties which separate it from the IIA- and IIB-MHC motor units. In old age, on the other hand, the IIX-MHC units display physiological, biochemical and morphometrical properties resembling the IIB-MHC motor units. Based on these results it was proposed that a transition from IIB to IIX motor units occurs during ageing. In an attempt to clarify this point, the MHC composition was identified by 6% SDS-PAGE and immunoblotting analysis, using specific mAbs antibodies, in the same fast-twitch tibialis anterior muscles in young (3-6 months, n = 9) and old (20-24 months, n = 16) rats from which the single motor units had been identified previously. The IIX-MHC comigrates together with the IIA-MHC band in 6% SDS-PAGE and only two MHC bands are observed in the rat tibialis anterior muscle, i.e. the IIA-IIX- and IIB-MHC bands. A significant increase (P < 0.001) in the average relative amount of the IIA-IIX-MHC was observed in the old (45 +/- 17%) as compared to the young (23 +/- 4%) animals, accompanied by a corresponding decrease in IIB-MHC content. It was demonstrated in immunoblotting analysis that only trace amounts of IIA-MHC were detectable in the IIA-IIX-MHC band in both young and old TA muscles, indicating a substantial increase in the IIX-MHC content in old age. Thus, the present results together with previous observations at the motor unit level strongly support an age-related motor unit transition from type IIB- to IIX-MHC.

Coexistence of two calsequestrin isoforms in rabbit slow-twitch skeletal muscle fibers.

The cardiac and skeletal muscle isoforms of calsequestrin (CS), the low affinity, high capacity Ca2+ binding protein localized in the lumen of sarcoplasmic reticulum, are the products of two different genes (Fliegel, L., Leberer, E., Green, N.M. and MacLennan, D.H. (1982) FEBS Lett. 242, 297-300), and can be both purified from slow-twitch skeletal muscle of the rabbit (Damiani, E., Volpe, P. and Margreth, A. (1990) J. Muscle Res. Cell Motil. 11, 522-530). Here we show that both CS isoforms coexist in slow-twitch muscle fibers as indicated by indirect immunofluorescent staining of cryosections with affinity-purified antibodies specific for each CS isoform.

Immunoelectron microscopic epitope locations of titin in rabbit heart muscle.

The location of cardiac titin epitopes in the sarcomere of rabbit cardiac, atrial and ventricular muscle was studied by using polyclonal antibodies against skeletal muscle titin. The results show that incubation with the antibody leads to the appearance of four electron-dense stripes in the A band of both atrial and ventricular cardiac muscle. The location and intensity of these stripes were identical to those observed in skeletal muscle. In conclusion we demonstrate that titins from skeletal and cardiac muscles share some common antigenic determinants.

Myosin heavy chain composition of muscle fibers in spinal muscular atrophy.

Muscle biopsies from 20 cases of spinal muscular atrophy (SMA), mostly diagnosed as Werdnig-Hoffmann (W-H) disease, were examined for myosin heavy chain (HC) composition. The fetal, fast, and slow heavy chains were characterized in the isolated muscle myosin, and in myosin of single, chemically skinned fibers, by electrophoresis in SDS-6% polyacrylamide gels and by immunoblot techniques, using specific antibodies directed to each main type of myosin HC. The fiber distribution of myosin HC isozymes was further investigated on muscle cryostat sections by an indirect immunofluorescent technique. Fetal myosin HC was found to be expressed in a subpopulation of severely atrophic fibers, alone or together with the slow form of myosin HC. Triangulated fibers of intermediate size contained fetal and fast myosin or fast myosin alone. The hypertrophic fibers were characterized by the predominant expression of slow myosin HC; but in some of these fibers, also low amounts of HC fetal were found to be expressed. These findings are discussed in relation to developmental transitions of myosin heavy chains in human muscle.

Subunit composition of native myosin isoenzymes of some striated mammalian muscles.

Six "native" isomyosins of characteristic electrophoretic mobility are present in various proportions according to the muscle type (fast, slow or mixed). SM2 and SM1 contain MHC1; IM contains MHC2A; FM1, FM2 and FM3 contain MHC2A and MHC2B. SM2, SM1 and IM contain MLC1s; SM1, IM, FM2 and FM3 contain MLC1f; FM2 and FM1 contain MLC3f. The six isomyosins appear to separate on the base of their myosin light chain content.