Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Isolation and characterization of heparan sulfate from various murine tissues

Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes, has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin was purified from 6–8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and ¹H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental understanding of certain aspects of protein-carbohydrate interactions.     

Fast Pseudo-Periodic Oscillation in the Rat Brain Voltage-gated Sodium Channel α Subunit

In the work reported here, we have investigated the changes in the activation and fast inactivation properties of the rat brain voltage-gated sodium channel (rNa_v 1.2a) α subunit, expressed heterologously in the Chinese Hamster Ovary (CHO) cells, by short depolarizing prepulses (10 – 1000 ms). The time constant of recovery from fast inactivation (τ_fast) and steady-state parameters for activation and inactivation varied in a pseudo-oscillatory fashion with the duration and amplitude of a sustained prepulse. A consistent oscillation was observed in most of the steady-state and non-inactivating current parameters with a time period close to 225 ms, although a faster oscillation of time period 125 ms was observed in the τ_fast. The studies on the non-inactivating current and steady-state activation indicate that the phase of oscillation varies from cell to cell. Co-expression of the β1 subunit with the α subunit channel suppressed the oscillation in the charge movement per single channel and free energy of steady-state inactivation, although the oscillation in the half steady-state inactivation potential remained unaltered. Incidentally, the frequencies of oscillation in the sodium channel parameters (4–8 Hz) correspond to the theta component of network oscillation. This fast pseudo-oscillatory mechanism, together with the slow pseudo-oscillatory mechanism found in these channels earlier, may contribute to the oscillations in the firing properties observed in various neuronal subtypes and many pathological conditions.     

Two types of modified cardiac Na+ channels after cytosolic interventions at the α-subunit capable of removing Na+ inactivation

Failure of inactivation is the typical response of voltage-gated Na⁺ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the α-subunit. The present patch clamp experiments with cardiac Na⁺ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na⁺ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481–1496 of the cardiac Na⁺ channel α-subunit) eliminated fast Na⁺ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at –40 mV). NBA-modified and iodate-modified Na⁺ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate P_o of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na⁺ channels: after cytosolic proteolysis with α-chymotrypsin, trypsin or pronase, mean P_o during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100–200 ms at –40 mV, as a minimum) or was virtually non-operating. In-vitro cleavage of the synthetic linker sequence 1481–1496 confirmed that this part of the α-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na⁺ channels are based on a structural alteration of still another region which is also involved in Na⁺ inactivation, besides the linker between domains III and IV of the α-subunit. Endogenous peptidases such as calpain did not affect Na⁺ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na⁺ channels, mode-switching to a non-inactivating mode. Received: 25 May 1996 / Accepted: 12 September 1996     

Identification of two type IIa IgG-binding proteins expressed by a single group A streptococcus.

Functional heterogeneity associated with Ig-binding proteins expressed by group A streptococci is well documented. In this study we have demonstrated that treatment of group A streptococcal isolate 64/14 with CNBr resulted in the solubilization of two different sized proteins that displayed identical functional reactivity with human IgG1, IgG2, and IgG4 (characteristics of a type IIa binding protein). Monospecific polyclonal antibodies to each form of type IIa molecule were prepared and no antigenic cross-reactivity between the two m.w. forms of type IIa binding protein could be detected. The smaller m.w. protein was shown to be identical or closely related to the recombinant type IIa protein cloned from strain CS110. These studies provide further evidence for the heterogeneity of type II Ig-binding proteins expressed by pathogenic group A streptococci.     

α-Helix peptides designed from EBV-gH protein display higher antigenicity and induction of monocyte apoptosis than the native peptide

We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.     

Fetuin: the bovine homologue of human alpha 2HS glycoprotein

The fetal protein fetuin has previously been considered to be confined to species of the order Artiodactyla (cattle, sheep, etc.) in spite of demonstrable biological in vitro effects in tissues of other species [(1983) Comp. Biochem. Physiol. 76A, 241-245]. We have determined the partial amino acid sequence of bovine fetuin and compared it with the published sequence of human alpha 2HS glycoprotein. The N-terminal 105 residues and a segment aligned with residues 170-225 of alpha 2HS glycoprotein revealed 109 of 161 residues to be identical between the two proteins (68% homology). Mouse polyclonal antibodies to fetuin, and trypsin digest fragments of this protein have been prepared and used for a comparison of native and digested proteins. Polyclonal antibodies to native protein showed little if any cross reactivity. However, antibodies to trypsin digest fragments of fetuin showed obvious cross reactivity with alpha 2HS.     

Scorpion toxins from Buthus martensii Karsch all possess a predicted α-tight-turn

We have purified a new toxin (BmK 17[4]) from Asian scorption (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8–12) that is similarly found in two other previously described α-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K⁺ and Ca²⁺ channels were blocked, BMK 17[4] acts to selectively alter Na⁺ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC—. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N-terminal motif (KPENC), which introduces a reverse turn in residues 8–12, does not appear in previously characterized BmK α-toxins and may be characteristic of α-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of α-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (α-turn) that is conserved. Sequence similarities with putative polypetides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon 38, 1653–1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their α-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.     

Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4

Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin.     

Structural analysis of glycosaminoglycans derived from axonally transported proteoglycans in regenerating goldfish optic nerve

Structural characteristics of glycosaminoglycans (GAGs) derived from axonally transported proteoglycans (PGs) were compared in 21 day regenerating and intact goldfish optic tracts. Twenty one days following unilateral optic nerve crushes, fish received intraocular injections of³⁵SO₄. Eight hours post injection, tracts were removed and the³⁵SO₄-labeled GAGs, chondroitin sulfate (CS) and heparan sulfate (HS), isolated. The HS from regenerating optic tracts had a DEAE elution profile indicative of decreased charge density, while heparitinase treatment of HS followed by Sephadex G50 analysis of the resulting fragments showed a change in the elution pattern, suggesting reduced overall sulfation. HPLC analysis of HS disaccharides revealed a difference in the sulfation pattern of regenerating tract HS, characterized by the reduced presence of tri-sulfated disaccharides. Other structural features, such as the sizes of CS and HS, and the sulfation of CS, showed no changes during regeneration. These results indicate that changes in the structure of axonally transported HS accompany regeneration of goldfish optic axons.     

Delayed recovery of β-glucuronidase activity driven by an Arabidopsis heat shock promoter in heat-stressed transgenic Nicotiana plumbaginifolia

 The expression of the Arabidopsis heat shock protein (HSP) 18.2 promoter-β-d-glucuronidase (GUS) chimera gene was investigated in transgenic Nicotiana plumbaginifolia plants during the recovery phase at normal temperatures (20–22  °C) after a heat shock (HS) treatment. GUS activity increased during the recovery phase after HS at 42  °C for 2 h, and maximal GUS activity was observed after 12 h at normal temperatures, at levels 50–100 times higher than the activity immediately after HS. After HS at 44  °C, little GUS activity was observed during the first 20–24 h at normal temperatures, but the activity increased gradually thereafter, to reach a maximum at 40–50 h. After HS at 45  °C, no GUS activity was observed throughout the experimental period. RT-PCR analysis showed that GUS mRNA remained for 10 h after a 2-h HS at 42  °C and for 40 h after a 2-h HS at 44  °C. These findings demonstrate that brief HS treatment, especially at a sublethal temperature, induces a long-term accumulation of HSP-GUS mRNA during the recovery phase. Received: 31 July 1998 / Revision received: 4 November 1998 / Accepted: 19 February 1999     

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.     

Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate

During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope than properdin. Factor H was present on the urinary side of renal tubular cells in proteinuric, but not in normal renal tissues and colocalized with properdin in proteinuric kidneys. Factor H dose-dependently bound to proximal tubular epithelial cells (PTEC) in vitro. Preincubation of factor H with exogenous heparin and pretreatment of PTECs with heparitinase abolished the binding to PTECs. Surface plasmon resonance experiments showed high affinity of factor H for heparin and HS (K(D) values of 32 and 93 nm, respectively). Using a library of HS-like polysaccharides, we showed that chain length and high sulfation density are the most important determinants for glycosaminoglycan-factor H interaction and clearly differ from properdin-heparinoid interaction. Coincubation of properdin and factor H did not hamper HS/heparin binding of one another, indicating recognition of different nonoverlapping epitopes on HS/heparin by factor H and properdin. Finally we showed that certain low anticoagulant heparinoids can inhibit properdin binding to tubular HS, with a minor effect on factor H binding to tubular HS. As a result, these heparinoids can control the alternative complement pathway. In conclusion, factor H and properdin interact with different HS epitopes of PTECs. These interactions can be manipulated with some low anticoagulant heparinoids, which can be important for preventing complement-derived tubular injury in proteinuric renal diseases.     

Heparan sulphate epitope–expression is associated with the inflammatory response in metastatic malignant melanoma

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope–specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3⁺ cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3⁺ cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3⁺ cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3⁺ cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3⁺ cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.     

Defining Structural Domains of an Intrinsically Disordered Protein: Sic1, the Cyclin-Dependent Kinase Inhibitor of <Emphasis Type="Italic">Saccharomyces</Emphasis><Emphasis Type="Italic">cerevisiae</Emphasis>

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell–cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1^C-ter) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1^1–186) was complementary to the most stable C-terminal fragments (Sic1^Δ186). Both Sic1^1–186 and Sic1^C-ter fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.     

Complex nonlinear dynamics of the Hodgkin-Huxley equations induced by time scale changes

 The Hodgkin–Huxley equations with a slight modification are investigated, in which the inactivation process (h) of sodium channels or the activation process of potassium channels (n) is slowed down. We show that the equations produce a variety of action potential waveforms ranging from a plateau potential, such as in heart muscle cells, to chaotic bursting firings. When h is slowed down – differently from the case of n variable being slow – chaotic bursting oscillations are observed for a wide range of parameter values although both variables cause a decrease in the membrane potential. The underlying nonlinear dynamics of various action potentials are analyzed using bifurcation theory and a so-called slow–fast decomposition analysis. It is shown that a simple topological property of the equilibrium curves of slow and fast subsystems is essential to the production of chaotic oscillations, and this is the cause of the large difference in global firing characteristics between the h-slow and n-slow cases. Received: 9 August 2000 / Accepted in revised form: 10 January 2001     

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.     

Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Antibodies against a putative heparin receptor slow cell proliferation and decrease MAPK activation in vascular smooth muscle cells

Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti-heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti-heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti-heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.     

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression

We examined the novel interaction ofhyperthyroidism and hindlimb suspension on the pattern of myosin heavychain (MHC) expression (mRNA and protein) in skeletal muscles. FemaleSprague-Dawley rats were assigned to four groups:1) normal control (Con);2) thyroid hormone treated[150 µg 3,5,3'-triiodothyronine(T₃) · kg¹ · day¹](T₃);3) hindlimb suspension (HS); or4)T₃-treated and HS(T₃ + HS). Results show for thefirst time the novel observation that the combinationT₃ + HS induces a rapid andsustained, marked (80-90%) downregulation of type I MHC geneexpression that is mirrored temporally by concomitant markedupregulation of type IIb MHC gene expression, as evidenced by the denovo synthesis of type IIb MHC protein in the soleus. The fast type IIxMHC isoform showed a differential response among the experimentalgroups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing inthe plantaris muscles. The fast type IIa MHC was the least responsiveto suspension of the MHCs and reflected its greatest responsiveness toT₃ treatment while also undergoingdifferential adaptations in slow vs. fast muscle (increases vs.decreases, respectively). These results confirm previous findings thatall four adult MHC genes are sensitive toT₃ and suspension in amuscle-specific manner. In addition, we show thatT₃ + HS can interactsynergistically to create novel adaptations in MHC expression thatcould not be observed when each factor was imposed separately.