Favourable associations between the myosin heavy-chain and light-chain isoforms in human skeletal muscle

Histochemical and biochemical analyses were performed in order to examine the relationship between myosin light-chain (LC) isoforms and fibre-type distributions in whole human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis muscle in six healthy men, and analysed for the relative area occupied by each fibre type (percentage of fibre type area) and the molar ratio of each LC isoform. The percentage of type I fibre area was positively correlated with the molar ratio of slow LC (LC1s and LC2s) to total LC. The regression line was located below the line of unity. Also, the ratio of percentage of type II fibre area to that of type II fibre area was positively correlated with the molar ratio of the fast alkali LC LC1f to fast alkali LCs LC1f and LC3f. These results support previous study, having shown that in human skeletal muscle some type I fibres express various amounts of fast LC in addition to slow LC and suggest that fast myosin heavy-chain HCII a is favourably associated with LC1f, whereas HCIIb is favourably associated with LC3f.     

Primary structure of a variable region of the V kappa I subgroup (ISE) in light chain deposition disease.

Although structural abnormalities of monoclonal immunoglobulin light chains (LC) are suspected to play a determinant role in non-amyloid light chain deposition disease (LCDD), this condition is as yet poorly documented at the molecular level, since only three sequences have been reported to date. In a case of myeloma-associated LCDD, the patient's urine contained an unglycosylated kappa Bence Jones protein made up of dimers and monomers with an apparent molecular mass of 25,000 which was assigned to the V kappa I subgroup by N-terminal amino acid sequencing. The complete variable region sequence of the monoclonal kappa chain produced by the malignant plasma cells was amplified by polymerase chain reaction (PCR) using small amounts of material obtained by bone marrow aspiration. The sequence of three independently amplified cDNA clones derived from a normal-sized kappa messenger RNA was identical to that of the urinary kappa chain. The kappa mRNA had an overall normal structure made up of a V kappa I sequence rearranged to J kappa I. Several unusual features of the variable region (the first complete V kappa I sequence reported in LCDD) included three substitutions that introduced hydrophobic residues at spatially close positions. The strategy associating N-terminal sequence determination and cDNA cloning by PCR could help in accumulating new sequence data and improving our understanding of LCDD pathogenesis.     

Contractile units in stress fibers of fetal human astroglia in tissue culture

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca2⁺-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed ‘contractile units’, suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.     

Functional characterization of the human atrial essential myosin light chain (hALC-1) in a transgenic rat model

Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using skinned fibers from the hypertrophied ventricles of human hearts.To study the functional importance of hALC-1 in the intact perfused heart, we generated a transgenic rat model (TGR) overexpressing hALC-1 in the heart. Twelve-week-old TGR rats expressed 17±4 g hALC-1 per mg of whole SDS-soluble protein. Their perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (P<0.001) in the contractile parameters of the hearts of the TGR compared to the age matched control (WKY) animals, represented by increases from 20.8±2.3 to 45.1±3.6 mmHg/g heart weight in the developed left ventricular pressure, 1,035.7±89.8 to 2,181±135.4 mmHg/s in the contraction rate, and 713±60.2 to 1,364±137.4 mmHg/s in the relaxation rate in the WKY and the TGR groups respectively. Characterizing the functional effects of hALC-1 at the whole organ level represents a step towards gene therapy of heart failure.     

Sequencing-based typing reveals six novel MHC class I chain-related gene B (MICB) alleles

Abstract: The MHC class I chain-related gene A (MICA) is highly polymorphic. In this paper we demonstrate polymorphism in the other expressing member of the MIC family of genes: MICB. Using a sequencing-based typing approach on cDNA, analysis of exons 2 through 6 revealed eight polymorphic sites resulting in six unique MICB sequences. Although MICB has high nucleotide homology with MICA, its polymorphism is restricted and at different sites compared to those in MICA.     

Proteolytic components of serum IgG preparations

Chemical catalysis, an effector mechanism utilized by fully assembled antibodies, can also be mediated by the isolated antibody subunits. Because trace amounts of free light chains (L chains) are present in IgG preparations, a detailed study was undertaken to identify the constituents responsible for the polyreactive proteolytic activity of IgG purified from human sera, determined as the extent of cleavage of the model peptide substrate Pro-Phe-Arg-methylcoumarinamide. Two proteolytic species with approximate mass of 50 kD and 150 kD were separated by repetitive gel filtration in a denaturing solvent (6 M guanidine hydrochloride). The activity of the renatured 50-kD fraction (in fluorescence units/microg protein) was more than 45-fold greater than of the 150-kD fraction. Both fractions lost the activity following immunoadsorption on immobilized anti-IgG antibody. Fab fragments prepared from the 150-kD IgG fraction retained the activity. Reducing and non-reducing SDS-electrophoresis suggested the 50-kD fraction isolated from the IgG preparations to be a mixture of heavy chain (H chain) monomers and disulphide bonded L chain dimers. Electrophoretically homogeneous monomers of 50-kD H chains and 25-kD L chains were prepared by gel filtration of reduced and alkylated IgG from seven human subjects. Each of the alkylated L chain preparations displayed the proteolytic activity. The activity in alkylated H chains was undetectable or only marginally greater than the background values. L chain dimers appear to be the major species responsible for the polyreactive proteolytic activity of serum IgG preparations, with a smaller contribution furnished by tetrameric IgG.     

Characterization of idiotopes on MOPC 315 IgA using monoclonal antiidiotypic antibodies

The isologous antiidiotypic response in BALB/c mice to immunization with the DNP-binding IgA myeloma protein, MOPC 315, alters the expression of the anti-DNP antibody repertoire and confers immunity against MOPC 315 myeloma tumors. In order to characterize the idiotopes on MOPC 315 IgA which elicit this response we have isolated four monoclonal antiidiotypic antibodies (AIA), D10 (IgG_2a), A2(IgG₁), G3 (IgG_2b) and F1 (IgG_2a), produced by splenocytes of BALB/c mice immunized with MOPC 315 IgA in three independent fusion experiments. These AIA react with MOPC 315 IgA. reassociated H³¹⁵ L³¹⁵ and F³¹⁵_V but not with free H³¹⁵, L³¹⁵, V³¹⁵_H or V³¹⁵₂. In addition the AIA do not react with the closely related DNP-binding IgA myeloma protein, MOPC 460, suggesting that they are directed against private idiotopes on MOPC 315 IgA. These idiotopes can be divided into two groups. Group I, defined by D10, A2 and G3 consists of two overlapping idiotopes, one of which is related to the hapten-binding site. The two idiotopes are formed by an interaction of amino acids in H³¹⁵ and L³¹⁵. Group II defined by F1 consists of one idiotope which is related to the hapten-binding site. This idiotope is comprised of an aminoacid sequence on H³¹⁵ which requires an interaction with either L³¹⁵ or L⁴⁶⁰ for expression. A2 and G3 react identically with the same idiotope but were derived from two independent fusion experiments. This indicates an identity of AIA clonotypes among individual mice and suggests that the isologous AIA response to MOPC 315 IgA is restricted.     

Genetic and light intensity effects on the activity and emigratory behavior of the house fly,Musca domestica (L.)

The emigratory behavior and locomotor activity of yellow-eyed (y/y), wildtype (+/+), and heterozygous (+/y) house flies was examined at 8 fc (86 lx) and 1600 fc (17,223 lx) light intenstities. At 8 fc, emigration rate and activity of the y/y flies was similar to that of the +/+ and +/y flies. However, at 1600 fc, the y/y flies emigrated at twice the rate and showed an activity of about one-third that of the other genotypes. The behavior of the +/+ and +/y flies remained similar regardless of the experimental design or light intensity. The excessive neural stimulation by high-intensity light resulting from reduced shielding pigments led to behavioral modifications in the visual and tactile responses of the y/y flies.This research was supported in part by grants from the CUNY Faculty Research Award Program (No. 1103) and NIH Biomedical Research Support Grant 5-S05-R-07064.     

Components of predation defense behavior in chickens: evidence for endogenous rhythmicity

The manifestation of diurnal periodicity and the extent of its control by the photoperiod was assessed in three predation defense reactions which constitute either components or outcomes of a predator-prey interaction sequence. Two-hundred White Leghorn chicks were reared from hatching for one week in either 24L or 12L and then tested at one of two clock hours previously demonstrated to define peak and trough response for one of the components. Putative evidence was found for an endogenous source of the periodicity manifested in all reactions. Maintenance schedule did not entrain the periodicity, but simple room entry and handling elicited anti-predator reactions, the extent of which varied as a function of clock hour. A general model of predation defense behavior was proposed.