Phosphocreatine resynthesis during recovery in different muscles of the exercising leg by 31P-MRS.

To investigate the high-energy phosphate metabolism by (31) P-nuclear magnetic resonance spectroscopy during off-transition of exercise in different muscle groups, such as calf muscles and biceps femoris muscles, seven male long-distance runners (LDR) and nine untrained males (UT) performed both submaximal constant and incremental exercises. The relative exercise intensity was set at 60% of the maximal work rate (60%W max) during both knee flexion and plantar flexion submaximal constant load exercises. The relative areas under the inorganic phosphate (Pi ) and phosphocreatine (PCr) peaks were determined. During the 5-min recovery following the 60%W max, the time constant for the PCr off-kinetics was significantly faster in the plantar flexion (LDR: 17.3 ± 3.6 s, UT: 26.7 ± 6.7 s) than in the knee flexion (LDR: 29.7 ± 4.7 s, UT: 42.7 ± 2.8 s, P < 0.05). In addition, a significantly faster PCr off-kinetics was observed in LDR than in UT for both exercises. The ratio of Pi to PCr (Pi /PCr) during exercise was significantly lower during the plantar flexion than during the knee flexion (P < 0.01). These findings indicated that the calf muscles had relatively higher potential for oxidative capacity than that of biceps femoris muscles with an association of training status.

Lower-lobe shrinkage relative to total lung volume in collagen vascular diseases.

PURPOSE: It has been reported that the prognosis differs between patients who have collagen vascular diseaseassociated interstitial pneumonia (CVD-IP) and those with idiopathic IP (IIP). In this study, chest computed tomography (CT) findings were compared between patients with CVD-IP and IIP. MATERIALS AND METHODS: A retrospective analysis was performed of 47 consecutive patients (23 with CVD-IP and 24 with IIP). The lower-lobe volume (LLV), total lung volume (TLV), and their ratio (LLV/TLV) were determined by volumetry using three-dimensional computed tomography (CT). RESULTS: There was no significant difference of the LLV/TLV ratio between the CVD-IP and IIP groups. However, the LLV/TLV ratio was <0.33 in 9/23 patients with CVD-IP versus 2/24 patients with IIP, and there was a significant difference in the percentage of patients with a ratio<0.33 between the CVD-IP and IIP groups (p = 0.01). The LLV/TLV ratio was not influenced by the severity of lung disease. CONCLUSIONS: Measuring the LLV/TLV ratio by threedimensional CT can help distinguish between CVD-IP and IIP at initial diagnosis, especially in patients with CVD-IP who have pulmonary involvement before other organ diseases and symptoms caused by CVD.

Clock gene dysfunction in patients with obstructive sleep apnoea syndrome.

Clock genes regulate mammalian circadian rhythms, and dysfunction of clock genes can contribute to various disorders. To investigate whether obstructive sleep apnoea syndrome (OSAS) influences clock gene function, the present authors examined Period1 (Per1) mRNA expression in vitro and in vivo. In eight healthy subjects and eight OSAS patients, plasma noradrenaline, serum interleukin (IL)-6, high-sensitivity C-reactive protein (hsCRP) and Per1 mRNA expression in peripheral whole blood were measured. Expression of Per1 mRNA in cultured cells was examined under IL-6 or noradrenaline stimulation in vitro. After noradrenaline was administered to mice in vivo, Per1 mRNA expression in the brain was examined. The concentrations of serum IL-6, hsCRP and plasma noradrenaline were elevated in OSAS patients, but improved by continuous positive airway pressure (CPAP) therapy. Per1 mRNA expression in the peripheral blood significantly decreased at 02:00 h by CPAP in OSAS patients. Stimulation with IL-6 did not directly induce Per1 mRNA in vitro. Administration of noradrenaline induced Per1 mRNA in the cerebral cortex of mice in vivo. The current study revealed that obstructive sleep apnoea syndrome caused clock gene dysfunction, and continuous positive airway pressure helped to improve it. Sympathetic activation and elevation of the plasma noradrenaline concentration in obstructive sleep apnoea syndrome may be one of the factors involved in disorders of Period1 mRNA expression.

Influence of work rate on dynamics of O2 uptake under hypoxic conditions in humans.

AIM: It was the purpose of the investigation to determine whether an altered work rate could influence the oxygen uptake (V.O(2)) and heart rate (HR) dynamics at hypoxia and normoxia. METHODS: Ten males performed a cycle exercise with 2 repetitions of 6 min each at a constant work load while breathing one of two inspiratory O(2) fractions (FIO(2)): 0.12 (moderate hypoxia) and 0.21 (normoxia). Each test began with unloaded pedaling. This was followed by three constant loads, which were 40%, 60%, and 80% of the subject's gas exchange threshold (GET) in hypoxia (F(I)O(2) = 0.12), with the 80% GET load repeated under normoxia (room air). V.O(2) was measured on a breath-by-breath basis and beat-by-beat HR via ECG, and the half time (t1/2) of each parameter was established, following interpolation data. RESULTS: There were no remarkable differences in t1/2 V.O(2) dynamics among the 40%, 60% and 80% GET; however, the differences became significant at hypoxia compared with normoxia. The HR dynamics were significantly faster in normoxia compared with hypoxia, independent of work rates. During steady-state exercise, the alterations in HR and cardiac output (Q) using the acetylene rebreathing method depended on increases in the work rate, and a significantly increase in at 80% GET was observed when compared with normoxia. Increases of stroke volume (SV) were unaffected by altered work rates and inspired O(2) concentrations. The arteriovenous oxygen difference (Ca-vO(2)) at a steady-state of exercise increased proportionally with the work rate under hypoxia, and a much greater Ca-vO(2) was observed during normoxic exercise than under hypoxia. CONCLUSION: These results seem to suggest that in humans, O(2) uptake dynamics are affected by lower O(2), not by changing work rates at hypoxia, to which the interaction between lower O(2) utilization in exercising muscles and hypoxic-induced greater blood flow can be attributed.

Preliminary evidence supports circulating microRNAs as prognostic biomarkers for type 2 diabetes.

Background: Circulating microRNAs are emerging as potential prognostic biomarkers for the development of type 2 diabetes. However, microRNAs are also associated with complications from impaired glucose metabolism (e.g. endothelial cell function). Prior studies have not evaluated for associations between trajectories of circulating microRNAs with trajectories of fasting blood glucose over time and the responses to behavioral interventions to reduce risk. This study performed longitudinal assessment of microRNAs and fasting blood glucose and identified relationships between microRNAs and behavioral risk reduction interventions. Methods: MicroRNAs (n = 353) were measured in subsets (n = 10, n = 8) of participants from previously completed clinical trials that studied behavioral risk reduction interventions. Fasting blood glucose trajectories were associated with changes in 45 microRNAs over 12 months. Results: Following a 3-month physical activity and dietary intervention compared with baseline, 13 microRNAs were differentially expressed. Seven microRNAs (i.e. miR-106b, miR-20b, miR-363, miR-486, miR-532, miR-92a and miR-93) were commonly identified between the two analyses. Conclusions: Further studies are needed to determine which microRNAs are prognostic biomarkers of risk for type 2 diabetes versus consequences of impaired glucose metabolism. Additional future directions of this research are to differentiate whether microRNAs are prognostic and/or diagnostic biomarkers for risk for type 2 diabetes and predictive biomarkers of responses to risk reduction interventions.

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor.

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Cellular receptors to the anaphylatoxins C3a and C5a.

A single component in the plasma membrane of human polymorphonuclear leukocytes (hPMN) has been identified as the binding site for C5a. Labelled human C5a can be cross-linked to a 48,000-Mr membrane component on the hPMN surface by using the bifunctional reagent ethylene glycol bis(succinimidyl succinate). The membrane component is believed to be the C5a receptor or the binding subunit of a C5a receptor complex. Our ligand-uptake data indicate that the hPMN has high-affinity binding sites for C5a with a Kd of the order of 1-2 nM and an estimated 50,000-113,000 binding sites/cell. Preliminary binding studies of C3a and C5a to rat peritoneal mast cells indicate that non-specific uptake by these cells is so great that it obscures characterization of specific receptor interactions. Data recently reported [Gervasoni, Conrad, Hugli, Schwartz & Ruddy (1986) J. Immunol. 136, 285-292] suggest that non-specific binding of C3a to mast cells is caused by electrostatic interactions between the cationic ligand and anionic heparin-proteoglycan on the cell surface, with an additional complication of the bound ligand undergoing proteolytic degradation. It is therefore proposed that synthetic analogue peptides designed to minimize non-specific interactions with the cell will be useful tools for demonstrating anaphylatoxin receptors on mast cells and may prove essential for receptor isolation.

Neuronal loss in the medullary reticular formation in myotonic dystrophy: a clinicopathological study.

Respiratory insufficiency occurs frequently in patients with myotonic dystrophy (MyD). We have performed a quantitative study of neurons linked to respiratory function in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN) in seven patients with MyD and eight age-matched controls. Alveolar hypoventilation of the central type occurred in three of the MyD patients but not in the remaining MyD patients or controls. The densities of neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in MyD without hypoventilation and controls. These data suggest the neuronal loss of the DCMN, VCMN, and SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Loss of serotonin-containing neurons in the raphe of patients with myotonic dystrophy: a quantitative immunohistochemical study and relation to hypersomnia.

Hypersomnia occurs frequently in patients with myotonic dystrophy (MyD). We performed a quantitative immunohistochemical study of serotonin (5-HT)-containing neurons linked to hypersomnia in the dorsal raphe nucleus (DRN) and the superior central nucleus (SCN) in 8 patients with MyD, 5 of whom showed hypersomnia, and in 12 age-matched controls. The densities of 5-HT neurons in the DRN and the SCN were significantly lower in MyD patients with hypersomnia than in MyD patients without hypersomnia and controls. These data suggest that the loss of 5-HT neurons of the DRN and the SCN is associated with the presence of hypersomnia in MyD.

Loss of catecholaminergic neurons in the medullary reticular formation in myotonic dystrophy.

OBJECTIVE: To clarify the possible relation between the extent of involvement of catecholaminergic neurons and the presence of alveolar hypoventilation in patients with myotonic dystrophy (MyD). BACKGROUND: Respiratory insufficiency has been reported frequently in MyD patients. Recent data support the hypothesis that this respiratory failure results from a primary dysfunction of the CNS. METHODS: The authors performed a quantitative immunoreactive study of tyrosine hydroxylase immunoreactive (TH+) neurons linked to hypoventilation in the dorsal central medullary nucleus (DCMN), the ventral central medullary nucleus (VCMN), and the subtrigeminal medullary nucleus (SMN)--where the autonomic respiratory center is thought to be located--in eight MyD patients and in 10 age-matched control subjects. Alveolar hypoventilation of the central type was present in three of the MyD patients but not in the remaining MyD patients or the control subjects. RESULTS: The densities of TH+ neurons of the DCMN, the VCMN, and the SMN in MyD patients with hypoventilation were significantly lower than in those without hypoventilation (p < 0.02, p < 0.01, and p < 0.01, respectively) and control subjects (p < 0.01, p < 0.01, and p < 0.01, respectively). CONCLUSIONS: These data suggest that the loss of TH+ neurons of the DCMN, the VCMN, and the SMN is associated with the presence of hypoventilation in MyD and may be an important feature of MyD.

Expression of biologically active C3a as fusion proteins.

We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.

Nucleotide sequence analysis of a human monoclonal antibody TONO-1 with cytotoxic potential for T-leukemia/lymphoma cells.

A human monoclonal antibody (HuMab) TONO-1 (IgM, lambda) recognizes cell surface antigens associated primarily with human T-leukemia/lymphoma cells. In this study, we investigated the reactivity against T-leukemia/lymphoma cells in detail, cytotoxic potential and primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMab TONO-1. Expression of the molecules (TONO-1 Ags) detected by a HuMab TONO-1 was significantly heterogeneous even in the same T-leukemia/lymphoma cell lines HPB-MLT and MOLT-4F. The flow cytometric curves showed an unusual broad-based spread of fluorescence intensity. HuMab TONO-1 was shown to have the ability to kill the T-leukernia/lymphoma cells efficiently in the presence of rabbit complements. However, HuMab TONO-1 did not demonstrate significant antibody-dependent cellular cytotoxic activity. Furthermore, HuMab TONO-1 heavy and light chain variable regions were cloned, sequenced and analyzed. HuMab TONO-1 uses a V(H) gene member of the V(H)IV gene family V(H)71-4, and is productively rearranged with the germ line D(H) gene D(XP')1, and the germ line J(H)5 gene with multiple somatic mutations. HuMab TONO-1 Vlambda belongs to the lambda light chain variable subgroup I family and is derived from the Vlambdalc germ line gene Humlv1042, and germ line gene Jlambda1 without somatic mutations. The results reveal that the production of HuMab TONO-1, with cytotoxic potential for human T-leukemia/lymphoma cells, is achieved by rearrangement of the V(H)71-4/Humlv1042 germ line variable region gene combination, that is associated with the autoimmune repertoire.

Nucleotide sequence analysis of a human monoclonal antibody 22-13 reactive with lung tumor-associated antigen.

A human monoclonal antibody (HuMAb) 22-13 (IgG1, kappa) recognizes a cytoplasmic antigen associated primarily with human lung tumors. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMAb 22-13. This HuMAb uses a VH gene member of the V(H)Ia gene family, 51P1 and is productively rearranged with a D-D fusion product of the D(LR)2 and D(XP)2 germ line DH genes and the germ line JH3 gene. HuMAb 22-13 Vkappa belongs to the kappa light chain variable subgroup IIIb family and appears to be derived from the Humkv325 germ line gene and is rearranged with a germ line Jkappa5 gene. The results reveal that production of a HuMAb 22-13 is achieved by rearrangement of the 51P1/Humkv325 germ line variable region gene combination, associated with the autoimmune repertoire and that HuMAb 22-13 has a striking sequence homology to rheumatoid factors (RFs) of the Wa idiotypic family. HuMAb 22-13 and Wa RFs have in common V(H)Ia and VkappaIIIb gene segments, but use different DH, JH and Jkappa gene segments. However, in spite of this structural similarity, HuMAb 22-13 does not display rheumatoid factor activity. Taken together with the reported findings, these data indicate the representation of the shared usage of highly homologous variable region genes in entirely different humoral immune responses in the human system.

Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit.

1 The rabbit receptor for C5a was cloned from a genomic library and found to be 79.5% identical to the human homologue, the highest degree of similarity found so far in nonprimate laboratory animals. 2 The rabbit C5a receptor stably expressed in RBL cells binds human 125I-C5a (2 nM). Unlabelled C5a and the C-terminal analogue N-acetyl-Tyr-Ser-Phe-Lys-Pro-Met-Pro-Leu-D-Ala-Arg (Ac-YSFKPMPLaR) were found to be competitors of that binding, the peptide analogue retaining approximately 0.1% of the affinity of human C5a. 3 The order of potency human C5a>Ac-YSFKPMPLaR was conserved in bioassays based on rabbits (relaxation of the isolated portal vein and pulmonary artery; acute in vivo neutropenia), but with a decreasing potency gap between the two compounds, a likely consequence of the resistance to peptidases of the analogue. 4 The molecular definition of the rabbit C5a receptor evidenced a high preservation degree of sequence and pharmacologic properties relative to the human ortholog receptor, thus defining a set of molecular tools for the investigation of the role of C5a in physiologic and pathologic models based on the rabbit (e.g. atherosclerosis, inflammation).

Purification of cat submaxillary kallikrein.

The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.

Activation of guinea pig platelets by a monoclonal antibody.

We herein describe the characterization of a monoclonal anti-guinea pig platelet antibody, termed GT2-12, which causes aggregation, ATP and [3H]serotonin release, and platelet protein phosphorylation. GT2-12 can bind to platelets and megakaryocytes in guinea pig bone marrow cells. A protein of 34,000 daltons was detected by immunoprecipitation of platelet lysates with GT2-12. Incubation of 32P-labeled guinea pig platelets with GT2-12 resulted in rapid phosphorylation of proteins of 40,000 and 20,000 daltons. GT2-12-induced aggregation and protein phosphorylation of platelets were inhibited by diltiazem, TMB-8 and dibutyryl cAMP and partially inhibited by indomethacin. The F(ab')2 fragment of GT2-12 IgG also induced platelet aggregation, indicating that activation is not mediated by Fc receptors. This type of antibody should be useful for studying platelet function in the guinea pig model.

Intracytoplasmic inclusion bodies of the substantia nigra in myotonic dystrophy. Immunohistochemical observations.

We recently reported a significantly higher incidence of intracytoplasmic inclusion bodies (IIBs) of the substantia nigra in patients with myotonic dystrophy (MyD) than in age-matched controls. The changes are, per se, not specific, since a small percentage of disease and normal controls also showed similar inclusions. To elucidate the pathological significance of the inclusion in MyD, we studied immunohistochemical characteristics of IIBs of the substantia nigra in eight patients with MyD. Many IIBs showed moderately intense immunoreactivity for ubiquitin, microtubule-associated protein (MAP) 1 and MAP 2. However, the IIBs did not react with any of the following: anti-neurofilament protein antibodies (Abs) (68, 160 and 200 kDa), anti-neuron-specific enolase antibody (Ab), anti-tau Ab, anti-tubulin Abs (alpha and beta), anti-paired helical filament Ab, anti-actin Ab, anti-phosphorylated epitope of neurofilaments Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-glial fibrillary acidic protein Ab. Our results suggest that IIBs of the substantia nigra in MyD are related to an alteration of neuronal cytoskeleton metabolism affecting microtubular proteins in conjunction with activation of ubiquitin proteolytic systems.

Immunohistochemical study of intracytoplasmic inclusion bodies of the thalamus in myotonic dystrophy.

Intracytoplasmic inclusion bodies of the thalamus in eight patients with myotonic dystrophy (MyD) were studied immunohistochemically. The intracytoplasmic inclusion bodies of the thalamus (thalamic inclusions, TIs) were strongly immunostained with anti-ubiquitin antibody (Ab) and some of them were mildly stained with anti-microtubule associated protein 1 (MAP 1) and anti-MAP 2 antibodies. However, TIs did not react with any of the following: anti-neurofilament protein Ab, anti-tau Ab, anti-paired helical filament Ab, anti-tubulin Abs (alpha and beta), anti-neuron-specific enolase Ab, anti-glial fibrillary acidic protein Ab, anti-synaptophysin Ab, anti-myelin basic protein Ab, anti-actin Ab and anti-phosphorylated epitope of neurofilaments Ab. Thus, our study demonstrates the unique immunohistochemistry of TIs in MyD which differentiates them from other intracytoplasmic inclusions in various neurodegenerative disorders.

Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.

Measurement of low molecular weight urinary monoamines by gas chromatography.

A simple gas chromatographic method for the measurement of low molecular weight urinary monoamines has been developed without use of a prior isolation procedure. The primary and secondary monamines in urine were directly converted into their 2,4-dinitrophenyl derivatives with 2,4-dinitrobenzensulfonate in aqueous alkaline solution, which after extraction with benzene and concentration, were submitted to gas chromatography. The application of this method to the determination of methylamine and dimethylamine in urine from normal subjects and hepatic patients is described.