Biomedical subjects

T Kambara

Publications and source records attributed to T Kambara.

At least 37 records · Page 2Linked to original sources

PubMed1999

Registration of the rod is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

A recent report has suggested that the interaction between the head and the rod region of smooth muscle myosin at S2 is important for the phosphorylation-mediated regulation of myosin motor activity [Trybus, K. M., Freyzon, Y., Faust, L. Z., and Sweeney, H. L. (1997) Proc. Natl. Acad. Sci. U.S.A. 74, 48-52]. To investigate whether specific amino acid residues at S2 or whether the registration of the 7-residue/28-residue repeat appearing in the alpha-helical coiled-coil structure of the rod are critical for such an interaction, two smooth muscle myosin mutants were constructed in which the N-terminal sequences of S2 were deleted to various extents. One mutant contained a deletion of 71 residues at the position immediately C-terminal to the invariant proline (Pro849) linking the S1 domain directly to the downstream sequence of the rod, while in another mutant, 53 residues were deleted at a position 56 residues downstream of Pro849. Despite these alterations which change the registration of both the 28-residue repeat and the 7-residue repeat found in myosin rod sequence, both myosin mutants showed a stable double-headed structure by electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the two mutant myosins was the same as the wild-type recombinant myosin. Furthermore, the head configuration critical for myosin filament formation (extended or folded) was unchanged in either mutant. These results indicate that neither the specific amino acid residues nor the registration of the amino acid repeat in S2 is critical for the head configuration. These results indicate that neither a specific amino acid sequence at the head-rod junction nor the rod sequence registration is critical for the regulation of smooth muscle myosin.

Actins↗

PubMed1999

Functional significance of the conserved residues in the flexible hinge region of the myosin motor domain.

Analysis of the three-dimensional crystal structure of the Dictyostelium myosin motor domain revealed that the myosin head is required to bend at residues Ile-455 and Gly-457 to produce the conformation changes observed in the ternary complexes that resemble the pre- and post-hydrolysis states (Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., and Rayment, I. (1995) Biochemistry 34, 8960-8972). Asp-454, Ile-455, and Gly-457 of smooth muscle myosin were substituted by Ala, Met, and Ala, respectively, and the mechano-enzymatic activities were determined to study the role of these residues in myosin motor function. Whereas the basal steady-state Mg2+-ATPase activity of D454A was higher than that of the wild type, the rate of the hydrolytic step is reduced approximately 2,000-fold and becomes rate-limiting. M-ATP rather than M-ADP-P is the predominant steady-state intermediate, and the initial Pi burst and the ATP-induced enhancement of intrinsic tryptophan fluorescence are absent in D454A. D454A binds actin in the absence of ATP but is not dissociated from actin by ATP. Moreover, actin inhibits rather than activates the ATPase activity; consequently, D454A does not support actin translocating activity. I455M has normal actin-activated ATPase activity, Pi burst, and ATP-induced enhancement of intrinsic tryptophan fluorescence, suggesting that the enzymatic properties are normal. However, the actin translocating activity was completely inhibited. This suggests that the side chain at Ile-455 is critical for myosin motor activity but not for relatively normal enzymatic function, which indicates an apparent uncoupling between enzymatic activity and motile function. Although G457A has normal ATP-dependent actin dissociation, ATP hydrolytic step is reduced by approximately 10(5)-fold in the presence or absence of actin; consequently, G457A does not have actin translocating activity. These results indicate the importance of these conserved residues at the hinge region for normal myosin motor function.

Actins↗

PubMed1999

Electroreceptor model of weakly electric fish Gnathonemus petersii: II. Cellular origin of inverse waveform tuning.

In part I (. Biophys. J. 75:1712-1726), we presented a cellular model of the A- and B-electroreceptors of the weakly electric fish Gnathonemus petersii. The model made clear the cellular origin of the differences in the response functions of A- and B-receptors, which sensitively code the intensity of the fish's own electric organ discharge (EOD) and the variations in the EOD waveform, respectively. The main purpose of the present paper is to clarify the cellular origin of the inverse waveform tuning of the B-receptors by using the receptor model. Inverse waveform tuning means that B-receptors respond more sensitively to the 180 degrees inverted EOD than to undistorted or less distorted EODs. We investigated how the A- and B-receptor models respond to EODs with various waveforms, which are the phase-shifted EODs, whose shift angle is varied from -1 degrees to -180 degrees, and single-period sine wave stimuli of various frequencies. We show that the tuning properties of the B-receptors arise mainly from the combination of two attributes: 1) The waveform of the stimuli (Bstim) effectively sensed by the B-receptor cells. This consists of a first smaller and a second larger positive peak, even though in the original phase-shifted EOD stimuli, the amplitudes of the two positive peaks are reversed. 2) The effective time constant of dynamical response of the receptor cells. It is on the order of the duration of a single EOD pulse. We also calculated the response properties of the A- and B-receptor models when stimulated with natural EODs distorted by various capacitive and resistive objects. Furthermore, we investigated the effect of EOD amplitude on the receptor responses to capacitive and resistive objects. The models presented can systematically reproduce the experimentally observed response properties of natural A- and B-receptor cells. The mechanism producing these properties can be reasonably explained by the variation in the stimulus waveforms effectively sensed by the A- and B-receptor cells and by time constants.

Action Potentials↗

PubMed1998

Effects of mutations in the gamma-phosphate binding site of myosin on its motor function.

The role of the highly conserved residues in the gamma-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.

Actins↗

PubMed1998

A specific amino acid sequence at the head-rod junction is not critical for the phosphorylation-dependent regulation of smooth muscle myosin.

It has been suggested that the structure at the head-rod junction of smooth muscle myosin is important for the phosphorylation-mediated regulation of myosin motor activity. To investigate whether a specific amino acid sequence at the head-rod junction is critical for the regulation, three smooth muscle myosin mutants in which the sequence at the N-terminal end of S2 is deleted to various extents were expressed in Sf9 cells; 28, 56, and 84 amino acid residues, respectively, at the position immediately C-terminal to the invariant proline (Pro849) were deleted, and the S1 domain was directly linked to the downstream sequence of the rod. The mutant myosins were expressed, purified, and biochemically characterized. All three myosin mutants showed a stable double-headed structure based upon electron microscopic observation. Both the actin-activated ATPase activity and the actin translocating activity of the mutants were completely regulated by the phosphorylation of the regulatory light chain. The actin sliding velocity of the three mutant myosins was the same as the wild-type recombinant myosin. These results indicate that a specific amino acid sequence at the head-rod junction is not required for the regulation of smooth muscle myosin. The results also suggest that there is no functionally important interaction between the regulatory light chain and the heavy chain at the head-rod junction.

Actins↗

PubMed1998

Characterization of the interaction of myosin with ATP analogues having the syn conformation with respect to the adenine-ribose bond.

Numerous analytical experiments have shown that, in solution, ATP analogues with bulky substitutions at the eighth position of the adenine ring predominantly assume the syn conformation with respect to the adenine-ribose bond. Two such analogues, 3'-O-(N-methylanthraniloyl)-8-azido-ATP (Mant-8-N3-ATP) and 8-Br-ATP, were synthesized and used to probe the conformation of the ATP-binding site of myosin. In the presence of these analogues, actomyosin was rapidly dissociated; Mg2+-dependent ATP hydrolysis was significantly activated by actin; and Pi bursting was observed. For skeletal myosin, however, these analogues failed to support actin translocation, and they did not significantly enhance the intrinsic tryptophan fluorescence of skeletal muscle myosin subfragment-1 (SKE S-1). These results suggest that although myosin**/ADP/Pi intermediates can be formed with these analogues, the crucial conformational changes required for cross-bridge cycling do not occur in skeletal muscle myosin. The conformations of the ATP-binding sites of skeletal and smooth-muscle myosin were compared using the ternary complexes, myosin-ADP-beryllium fluoride (BeFn) or myosin-ADP-aluminium fluoride (AIF4-). In AlF4- complexes, Mant-8-N3-ADP affinity labeled the N-terminal 29-kDa domain of smooth-muscle myosin subfragment-1 (SM S-1), as did ATP analogues having the anti conformation, whereas it labeled the C-terminal 20-kDa domain of skeletal S-1. In smooth muscle BeFn complexes, Mant-8-N3-ADP was equally likely to cross-link to the 29-kDa N-terminal and the 25-kDa C-terminal domains. These analogues induced smooth muscle actomyosin super-precipitation and increased intrinsic tryptophan fluorescence to the same degree as ATP itself. As was expected from above results, the analogues supported smooth-muscle-myosin-induced actin translocation. These results suggest that smooth-muscle myosin adopts the eight-substituted ATP analogue in the normal conformation, but skeletal muscle myosin does not. This reflects the likely differences in the structures of their respective ATPase sites.

Adenosine↗

PubMed1998

A hinge at the central helix of the regulatory light chain of myosin is critical for phosphorylation-dependent regulation of smooth muscle myosin motor activity.

The motor function of smooth muscle myosin is activated by phosphorylation of the regulatory light chain (RLC) at Ser19. However, the molecular mechanism by which the phosphorylation activates the motor function is not yet understood. In the present study, we focused our attention on the role of the central helix of RLC for regulation. The flexible region at the middle of the central helix (Gly95-Pro98) was substituted or deleted to various extents, and the effects of the deletion or substitution on the regulation of the motor activity of myosin were examined. Deletion of Gly95-Asp97, Gly95-Thr96, or Thr96-Asp97 decreased the actin-translocating activity of myosin a little, but the phosphorylation-dependent regulation of the motor activity was not disrupted. In contrast, the deletion of Gly95-Pro98 of RLC completely abolished the actin translocating activity of phosphorylated myosin. However, the unregulated myosin long subfragment 1 containing this RLC mutant showed motor activity the same as that containing the wild type RLC. Since long subfragment 1 motor activity is unregulated by phosphorylation, i.e. constitutively active, these results suggest that the deletion of these residues at the central helix of RLC disrupts the phosphorylation-mediated activation mechanism but not the motor function of myosin itself. On the other hand, the elimination of Pro98 or substitution of Gly95-Pro98 by Ala resulted in the activation of actin translocating activity of dephosphorylated myosin, whereas it did not affect the motor activity of phosphorylated myosin. Together, these results clearly indicate the importance of the hinge at the central helix of RLC on the phosphorylation-mediated regulation of smooth muscle myosin.

Actins↗

PubMed1998

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy.

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.

Affinity Labels↗

PubMed1998

An olfactory recognition model based on spatio-temporal encoding of odor quality in the olfactory bulb.

In order to study the problem how the olfactory neural system processes the odorant molecular information for constructing the olfactory image of each object, we present a dynamic model of the olfactory bulb constructed on the basis of well-established experimental and theoretical results. The information relevant to a single odor, i.e. its constituent odorant molecules and their mixing ratios, are encoded into a spatio-temporal pattern of neural activity in the olfactory bulb, where the activity pattern corresponds to a limit cycle attractor in the mitral cell network. The spatio-temporal pattern consists of a temporal sequence of spatial firing patterns: each constituent molecule is encoded into a single spatial pattern, and the order of magnitude of the mixing ratio is encoded into the temporal sequence. The formation of a limit cycle attractor under the application of a novel odor is carried out based on the intensity-to-time-delay encoding scheme. The dynamic state of the olfactory bulb, which has learned many odors, becomes a randomly itinerant state in which the current firing state of the bulb itinerates randomly among limit cycle attractors corresponding to the learned odors. The recognition of an odor is generated by the dynamic transition in the network from the randomly itinerant state to a limit cycle attractor state relevant to the odor, where the transition is induced by the short-term synaptic changes made according to the Hebbian rule under the application of the odor stimulus.

Animals↗

PubMed1998

Effect of syncytium structure of receptor systems on stochastic resonance induced by chaotic potential fluctuation.

To study a role of syncytium structure of sensory receptor systems in the detection of weak signals through stochastic resonance, we present a model of a receptor system with syncytium structure in which receptor cells are interconnected by gap junctions. The apical membrane of each cell includes two kinds of ion channels whose gating processes are described by the deterministic model. The membrane potential of each cell fluctuates chaotically or periodically, depending on the dynamical state of collective channel gating. The chaotic fluctuation of membrane potential acts as internal noise for the stochastic resonance. The detection ability of the system increases as the electric conductance between adjacent cells generated by the gap junction increases. This effect of gap junctions arises mainly from the fact that the synchronization of chaotic fluctuation of membrane potential between the receptor cells is strengthened as the density of gap junctions is increased.

Animals↗

PubMed1998

Electroreceptor model of the weakly electric fish Gnathonemus petersii. I. The model and the origin of differences between A- and B-receptors.

We present an electroreceptor model of the A- and B-receptors of the weakly electric fish Gnathonemus petersii. The model consists of a sensory cell, whose membrane is separated into an apical and basal portions by support cells, and an afferent fiber. The apical membrane of the cell contains only leak channels, while the basal membrane contains voltage-sensitive Ca2+ channels, voltage-sensitive and Ca2+-activated K+ channels, and leak channels. The afferent fiber is described with the modified Hodgkin-Huxley equation, in which the voltage-sensitive gate of the K+ channels is a dynamic variable. In our model we suggest that the electroreceptors detect and process the information provided by an electric organ discharge (EOD) as follows: the current caused by an EOD stimulus depolarizes the basal membrane to a greatly depolarized state. Then the release of transmitter excites the afferent fiber to oscillate after a certain time interval. Due to the resistance-capacitance structure of the cells, they not only perceive the EOD intensity, but also sense the variation of the EOD waveform, which can be strongly distorted by the capacitive component of an object. Because of the different morphologies of A- and B-cells, as well as the different conductance of leak ion channels in the apical membrane and the different capacitance of A- and B-cells, A-receptors mainly respond to the EOD intensity, while B-receptors are sensitive to the variation of EOD waveform.

Animals↗

PubMed1998

Effect of natural interferon-beta on the growth of melanoma cell lines.

Malignant melanoma is one of the fulminant skin cancers. The 5-year survival of patients with stage III (N0, N1) malignant melanoma treated with multi-agent chemoimmunotherapy, including natural interferon-beta (nIFNbeta), was found in our department to be better than that of patients treated with other forms of therapy. In order to study the effects of nIFNbeta on melanoma, the growth inhibition effect of nIFNbeta was assessed in vitro using the melanoma cell lines, MM8.1, MM28, MM33.1, Bowes and A375-2. The growth of these cell lines was inhibited by nIFNbeta. Incorporation of [3H]thymidine and [3H]uridine was also inhibited by nIFNbeta in a dose-dependent manner. Apoptosis was demonstrated using the TUNEL method in melanoma cell lines cocultured with nIFNbeta. Results showed that nIFNbeta had direct killer activity on melanoma cell lines.

Antineoplastic Agents↗

PubMed1996

Role of proline residue in the channel-forming and catecholamine-releasing activities of the peptaibol, trichosporin-B-VIa.

Trichosporin-B-VIa (TS-B-VIa) has a Pro14-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib14]TS-B-VIa with Pro-->Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib14]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib14]TS-B-VIa had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca(2+)-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terminal domain caused by the Pro-->Aib substitution.

Adrenal Glands↗

PubMed1996

Identification of myosin III as a protein kinase.

Drosophila ninaC gene encodes myosin homologous proteins which are classified as myosin III of the myosin superfamily, yet the physiological and biochemical function of myosin III has not characterized. We report here that myosin III does exhibit protein kinase activity. The kinase homologous domain (MYOIIIPK) of myosin III was expressed in the baculovirus expression system and purified to homogeneity. MYOIIIPK phosphorylated a number of proteins including myosin III p132 and smooth muscle myosin regulatory light chain (LC20), suggesting that myosin III is a multifunctional protein kinase. The phosphoamino acid analysis revealed that myosin III is a serine/threonine kinase but not a tyrosine kinase. The observation that MYOIIIPK phosphorylates myosin III suggests that the autophosphorylation might play a role for the regulation of myosin III function. This is the first direct demonstration of kinase activity for the myosin III class.

Adenosine Triphosphate↗

PubMed1996

Self-organized phase transitions in neural networks as a neural mechanism of information processing.

Transitions between dynamically stable activity patterns imposed on an associative neural network are shown to be induced by self-organized infinitesimal changes in synaptic connection strength and to be a kind of phase transition. A key event for the neural process of information processing in a population coding scheme is transition between the activity patterns encoding usual entities. We propose that the infinitesimal and short-term synaptic changes based on the Hebbian learning rule are the driving force for the transition. The phase transition between the following two dynamical stable states is studied in detail, the state where the firing pattern is changed temporally so as to itinerate among several patterns and the state where the firing pattern is fixed to one of several patterns. The phase transition from the pattern itinerant state to a pattern fixed state may be induced by the Hebbian learning process under a weak input relevant to the fixed pattern. The reverse transition may be induced by the Hebbian unlearning process without input. The former transition is considered as recognition of the input stimulus, while the latter is considered as clearing of the used input data to get ready for new input. To ensure that information processing based on the phase transition can be made by the infinitesimal and short-term synaptic changes, it is absolutely necessary that the network always stays near the critical state corresponding to the phase transition point.

Animals↗

PubMed1996

Monocyte chemotactic factor in rheumatoid arthritis synovial tissue. Probably a cross-linked derivative of S19 ribosomal protein.

The extracts of rheumatoid arthritis-synovial lesions from seven patients possessed a strong chemotactic activity for monocytes and a negligible one for polymorphonuclear leukocytes. These results are consistent with a prominent histological feature of the synovial lesion, the mononuclear cell predominant infiltration. The major monocyte chemotactic factor in the synovial tissue extracts was purified to a single protein peak in reverse phase high performance liquid chromatography with a C4 column. NH2-terminal amino acid analysis of the initial 20 residues yielded a single sequence. Surprisingly, this sequence was completely identical to that of S19 ribosomal protein. The purified sample demonstrated two protein bands in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 34 and 68 kDa. These sizes were 2 and 4 times that of S19 ribosomal protein, suggesting that the chemotactic factor would be a dimer or tetramer of S19 ribosomal protein cross-linked by factor XIIIa. A recombinant human S19 ribosomal protein was prepared as a fusion protein with a maltose binding protein in Escherichia coli. After treatment with factor XIIIa, cross-linked recombinant S19 ribosomal protein exhibited the monocyte chemotactic activity, although the untreated recombinant protein did not.

Amino Acid Sequence↗

PubMed1996

IL-8/NAP-1 is the major T-cell chemoattractant in synovial tissues of rheumatoid arthritis.

T-cell infiltration into synovium is a crucial process for rheumatoid arthritis (RA). To investigate the mechanism of T-cell infiltration, we studied T-cell attracting activity in synovial tissue extracts of RA or osteoarthritis (OA) whose synovium lacks T-cell accumulation. RA extracts attracted twofold more T cells than OA extracts. By gel filtration column chromatography the activity of RA extracts was separated into two peaks; one was eluted at the 67-kDa region and the other was at the 12-kDa region, while the latter was absent in OA extracts. The activity eluted at the 12-kDa region was absorbed mostly by an antibody against IL-8/NAP-1, a potent T-cell chemotactic factor. IL-8/NAP-1 concentrations in RA extracts were much higher than those in OA extracts and correlated to T-cell attracting activity eluted at the 12-kDa region. The checkerboard analysis revealed that the 67-kDa activity was chemokinetic but not chemotactic. These results suggest that IL-8/NAP-1 is the major T-cell chemoattractant in RA-synovium.

Aged↗

PubMed1996

Changes in T cell subpopulations of sheep due to age and dietary protein intake; association with protective immunity to Trichostrongylus colubriformis.

Purified lymphocytes from peripheral blood and cannulated intestinal lymph were examined by flow cytometry, to study the effect of age, level of dietary protein, and prior exposure to helminths on their composition. This was then compared with animals resistant to Trichostrongylus colubriformis infection. When young lambs consumed a low protein diet, they developed significantly lower protective immunity to the parasite than age-matched animals offered a high protein diet, or older animals irrespective of diet. In the peripheral blood there was an increased percentage of CD4+, CD5+ and CD8+ cells with increasing age (from 8 to 51 weeks). The percent of CD4+ cells in lambs that repeatedly ingested infective larvae was significantly increased in both peripheral blood and mesenteric lymph, compared with non-immunized animals. There was a significant interaction of dietary protein and immunization, as the percentage of T19+ cells from mesenteric lymph in young lambs that consumed a high protein diet increased when they were sensitized and challenged with larvae, while this was not apparent in those that consumed a low protein diet. The increase of peripheral and local CD4+ cells may contribute to development of protective immunity to the parasite. In addition, local T19+ cell populations in young animals were influenced by the status of protein in the diet, which may in turn influence immunity.

Age Factors↗