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Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained. Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation. Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies. Conclusion: In particular, the flexible structure was induced by the A30P mutation.

Introduction Developing prognostic markers can be useful for clinical decision‐making. Peripheral blood (PB) examination is simple and basic that can be performed in any facility. We aimed to investigate whether PB examination can predict prognosis in coronavirus disease (COVID‐19). Methods Complete blood count (CBC) and PB cell morphology were examined in 38 healthy controls (HCs) and 40 patients with COVID‐19. Patients with COVID‐19, including 26 mild and 14 severe cases, were hospitalized in Juntendo University Hospital (Tokyo, Japan) between April 1 and August 6, 2020. PB examinations were performed using Sysmex XN‐3000 automated hematology analyzer and Sysmex DI‐60 employing the convolutional neural network‐based automatic image‐recognition system. Results Compared with mild cases, severe cases showed a significantly higher incidence of anemia, lymphopenia, and leukocytosis (P

Article, International journal of laboratory hematology. 43(2): 281-289(2021)

Mycolic acid-containing bacteria (MACB) are known to activate cryptic natural product biosynthesis in co-cultures with actinobacteria. We cultured Actinosynnema mirum NBRC 14064, a producer of the mono-cyclic polyene macrolactam mirilactam A (6), with the MACB Tsukamurella pulmonis TP-B0596. As a result, three novel compounds (mirilactams C-E, 1-3) were produced in the co-culture conditions. Compounds 1-3 were likely derived from 6 by epoxidation and subsequent spontaneous cyclization. The chemical structures and stereochemistries of 1-3 were determined by spectroscopic analyses (NMR and MS), conformational searches in the optimized potentials for liquid simulations-3 (OPLS3) force field, and calculations of electronic circular dichroism (ECD).

Nocardia is a potent bacterial producer of bioactive compounds. From a culture of Nocardia beijingensis NBRC 16342, we isolated four aromatic compounds, named beijinchromes A-D (1-4). We purified them by silica gel chromatography and reverse phase HPLC, and identified their structures by NMR and high resolution (HR)-MS analyses. 1, 2, and 4 are novel 1,2,3,8-tetrasubstituted naphthalenes, and 3 is a novel 3,8-disubstituted ortho-naphthoquinone. 1 and 2 exert antioxidant activities, and 3 exhibits antibiotic activity. Remarkably, the putative biosynthetic gene clusters for 1-4 are widely distributed in 37 Nocardia species, implying their potential to produce this family of compounds and important biological functions of beijinchromes.

Three new lignoids, premnan A (1), premnan B (2), and tauntangyiol C (3), were isolated from Premna serratifolia wood, a traditional cosmetic plant in Myanmar, together with a new lignoid, premnan C (4) assumed to be an artifact, one natural new lignoid (5), and three known lignoids (6–8). The structures of the new compounds 1–4 were elucidated based on 1D and 2D NMR, IR spectroscopy, and HRESIMS. The absolute configurations of 1–4 were also determined by optical rotation, circular dichroism (CD) data analyses, and comparisons with the reported literature. All isolated compounds were tested for their melanogenesis activities against the B16-F10 mouse melanoma cell line. Compounds 1 and 4 showed melanogenesis enhancing activities of 31% and 50%, respectively, at a 50 μM concentration. Compounds 2, 3, and 6 increased melanin production by 67%, 30%, and 45%, respectively, at a 100 μM concentration, without any cytotoxicity.

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene (1), having a 6-8-6-5-fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope-incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation-initiated second cyclization sequence, 1,2-alkyl migration, and 1,5-hydride shift to generate the novel scaffold of 1.

A medullary-spinal cord preparation without the pons isolated from the neonatal rat was used to investigate the role of inhibitory neurotransmitters in the respiratory depression induced by hypoxia (hypoxic respiratory depression; HRD). The burst frequency (C(4)-f) and peak amplitudes of the integrated activity of the C(4) roots (integral C(4)) and of the hypoglossal nerve (integral XII) were recorded. A marked decrease in C(4)-f (to 36+/-6% of control, P

The activation of peripheral chemoreceptors by hypoxia or electrical stimulation of the carotid sinus nerve elicited a hypoxic respiratory response consisting of both stimulatory and subsequent or simultaneous inhibitory components (hypoxic respiratory stimulation and depression). Both components have different time domains of responses (time-dependent response), providing an integrated respiratory response to hypoxia. This review has focused on the neuroanatomical and neurophysiological correlations responsible for these responses and their neuropharmacological mechanisms. Hypoxic respiratory depression is characterized by the initial activation of respiration followed by a progressive and gradual decline in ventilation during prolonged and/or severe hypoxic exposure (biphasic response). The responsible mechanisms for the depression are located within the central nervous system and may be dependent upon activity from peripheral chemoreceptor. Two underlying mechanisms contributing to the depression have been advocated. (1) Change in synaptic transmission: Within the neuronal network controlling the hypoxic respiratory response, hypoxia might induce the enhancement of inhibitory neurotransmission (modulation), disfacilitation of excitatory neruotransmission or both. (2) Change in the membrane property of respiratory neurons: Hypoxia might suppress the membrane excitability of respiratory neurons composing the hypoxic respiratory response via modulating ion channels, leading to hyperpolarization or depolarization blocking of the neurons. However, the quantitative aspects of Pao(2) (degree and duration of hypoxic exposure) to induce these changes and the susceptibility of both mechanisms to the Pao(2) level have not yet been clearly elucidated.

An in vitro brainstem preparation of the neonatal rat with intact right vagal (X) innervation of the right atrium, and intact medullary roots of the left X and glossopharyngeal (IX) nerves for stimulation was developed. The preparation was continuously superfused with artificial CSF at 25 degrees C. The electrical activity of the right atrium was recorded to determine the heart rate. Applications of atropine or propranolol to the superfusate did not alter the heart rate. Electrical stimulation (0.5 ms pulse, 20 Hz) of the left IX and X afferents elicited a reduction in the heart rate from 70.3 +/- 13.2 to 50.6 +/- 13.2 beats/min (mean +/- SD, p < 0.05), which was abolished after division of the right X or application of atropine to the superfusing solution. A similar reflex bradycardia was seen in a preparation with intact left vagal-right atrium innervation during right IX and X afferent stimulation. Cervical spinal cord transection affected neither the baseline heart rate nor the magnitude of the reflex bradycardia. Longitudinal sectioning of the medulla oblongata in the mid-line down to the level of the posterior inferior cerebellar artery abolished the heart rate response. After bilateral cervical vagotomies, electrical stimulation (0.5 ms pulse, 20 Hz, up to 100 microA) of the ventrolateral medulla oblongata, lateral funiculus at C2 or intermediate nucleus of the spinal cord at Th1-4 did not affect the heart rate. These results indicate that the functions in the lower brainstem are preserved in this preparation, at least in regard to the generation of reflex bradycardia. The results also suggest that the laterality of cardiac vagal innervation and sympathetic innervation will develop during the postnatal period. This preparation may be useful for the study of the central neuronal network controlling the heart rate.

Electrophysiological properties of phrenic motoneurons (PMs) were studied by intracellular recordings in anesthetized, paralyzed, and artificially ventilated adult rats. Our results revealed that rat PMs, as compared to cat PMs, had a shorter afterhyperpolarization duration (55 +/- 13 ms; mean +/- SD), a narrower half-width of action potential, a larger membrane input resistance (Rm; 2.0 +/- 0.6 M omega), a smaller rheobase (Irh) and a shorter minimum paired pulse interval to provoke the second spike (1.9 +/- 0.6 ms). These features indicate that the rat PMs possess higher frequency responsiveness than cat PMs. Based on the time of firing onset relative to the onset of whole phrenic nerve activity (relative onset time; ROT) or on the absence of discharge during inspiration, the PMs could be classified into four types: early recruited with ROT10.0%; late recruited with 12.5%ROT37.5%; very late recruited with ROT45%, and quiescent which was not recruited during normal experimental conditions. A number of differences in the membrane properties such as end-expiratory membrane potential, Rm, and Irh among the four cell types are discussed in relation to the recruitment order. Inhibitory postsynaptic potentials (IPSPs) were present during the late expiratory phase (stage II expiration) in all PMs tested.

The stability constants of metal complexes for several pyridone carboxylic acid drugs (ofloxacin, norfloxacin and lomefloxacin) were determined by potentiometry and spectrophotometry. The values of aluminum complexes, magnesium complexes and calcium complexes were Ca Zn). The stability constants of metal complexes for several pyridone carboxylic acids synthesized were also determined and compared with those for pyridone carboxylic adic drugs. The stability constants of these compounds gradually increased with an increasing pKa value of the carboxyl group of pyridone carboxylic acid. In the case of aluminum complexes, the complexes Al(OH)L and Al(OH)2L were formed under weak acidic conditions and the dissociation constants for the hydrolysis of the aluminum complexes were determined. The participation of the carboxyl group and the carbonyl group in the chelating reaction was confirmed by the measurement of carbon-13 nuclear magnetic resonance of the aluminum complex and the magnesium complex. These results suggest that when pyridone carboxylic acids are administered with metallic antacid containing aluminum hydroxide and magnesium oxide, aluminum complexes AIL, Al(OH)L or Al(OH)2L are formed and the adsorption of the drugs in the intestines is reduced.

Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.

Cyanate derivatives such as NaOCN have been known to increase the hypoxia tolerance of animals by increasing the affinity of hemoglobin (Hb) to O(2). To clarify the mechanism of this increase in hypoxia tolerance, we examined changes in metabolic rate and cardiovascular parameters during a hypoxia test in halothane-anesthetized, NaOCN-treated and spontaneously breathing rats (50 mg/kg/d S.C., 10 d). Control animals received saline. The capillary density in the skeletal muscle (sternocleidomastoid muscle), cardiac papillary muscle and medulla oblongata was also examined histologically. The Hb-O(2) affinity index, P(50), decreased from 38 (control rat) to 24 mmHg in NaOCN-treated rats. During hyperoxic gas breathing, the rat treated with NaOCN showed a significantly lower metabolic rate (V(.)O(2), V(.)CO(2)), higher cardiac stroke volume, slower heart rate, lower PvO(2), and lower O(2) extraction ratio than those in control rats. The NaOCN-treated rats exhibited well-maintained arterial blood pressure and a larger cardiac output response to reduction in FIO(2) to 0.10-0.08. The increase in O(2) extraction ratio with reduction in FIO(2) was larger in NaOCN-treated than in control rats. The circulatory and metabolic depressions at FIO(2) 0.05 were effectively attenuated in NaOCN-treated rats. The capillary density of the cardiac muscle and medulla oblongata but not the skeletal muscle was significantly higher in NaOCN-treated rats than in control rats. The greater hypoxia tolerance in NaOCN-treated rats is ascribed to the combined effects of left shift of Hb-O(2) dissociation curve, lower basal V(. )O(2), higher capillary density in the heart, and brain, and other adaptive mechanisms induced probably by prolonged tissue hypoxia.

APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is known to have a role in intrinsic cellular immunity against human immunodeficiency virus type1 (HIV-1). The antiretroviral activity of APOBEC3G (APO3G) is associated with the hypermutation of viral DNA through cytidine deamination. APO3G contains two cytidine deaminase domains that are characterised by highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APO3G (c-APO3G) has the catalytic activity. To shed light on the molecular mechanism of action by which APO3G inactivates HIV-1, we have undertaken the structural and binding studies by NMR. Here, we show the achievement of backbone assignments of c-APO3G and the identification of the secondary structure deduced from chemical shift index (CSI) and NOE data.

To clarify the control mechanism of ventilation during posture change, ventilatory parameters, PETCO2, and ventilatory response to CO2 were examined in 11 healthy male subjects at supine (0 degrees) and 75 degrees head-up tilt positions. Minute expiratory ventilation (V.E), tidal volume (VT), respiratory frequency (f), end-tidal and transcutaneous PCO2 and CO2 output (V.CO2), and ventilatory response to CO2 were measured during a steady state condition. V.E (V.A) and VT increased significantly at 75 degrees tilt with significant decrease in PETCO2 from 40.1 mmHg (0 degrees) to about 36.1 mmHg (75 degrees). Transcutaneous PCO2 also decreased during tilt, by 3.3 mmHg. Physiological dead space (VD/VT) and V.CO2, however, remained unchanged, and ventilatory equivalent (V.E/V.CO2, V.A/V.CO2) increased significantly. The CO2-ventilatory response curve shifted upward (or leftward) without significant change in the response slope. At 75 degrees tilt, EMG activity of gastro-cnemius muscle increased. These findings suggested that PETCO2 decreased because of increased V.E (V.A) with a leftward shift of CO2-ventilatory response curve. Various signals such as afferents from lower extremities might have net stimulatory effects on a CO2-ventilation control system to reset the controlled level of PETCO2 to a lower range, but without significant change in CO2-ventilatory response during upright position.

The steady-state ventilatory responses to hypercapnia and hypoxia in 7 assisted breath-hold divers (Funado) were compared with those in 7 normal sedentary controls. Ventilatory response to hypercapnia was measured from the slope of the hyperoxic VN-PETCO2 line, where VN was normalized minute ventilation using the allometric coefficient and PETCO2 end-Tidal PCO2. The slope of this line in the Funado (1.48 +/- 0.54 liters . min-1 . Torr-1) was significantly less than in the control (2.70 +/- 1.08 liters . min-1 . Torr-1) (p less than 0.025). On the other hand, hypoxic sensitivity estimated by hyperbolic and exponential mathematical equations was not found to be significantly different between the two groups, although estimated increments in ventilation using the hyperbolic equation exhibited significantly lower response in the Funado than in the control only when PETO2 decreased lower than 50 Torr (p less than 0.05). These findings in the Funado were different from our previous observations obtained in unassisted breath-hold divers (Kachido), in whom no obvious attenuations in CO2 sensitivity were seen. This difference was assumed to be derived from more hypercapnic and hypoxic conditions produced in the Funado than in the Kachido during diving activities.

Fifty-two active Judo athletes were examined using an isocapnic progressive hypoxia test. The results were analyzed by the hyperbola ventilation equation V = VO + A/(PETO2--C), where V is observed ventilation, VO the horizontal asymptote in ventilation for infinite end-tidal PO2(PETO2), A the slope constant indicating magnitude of hypoxic sensitivity, and C the vertical asymptote in PETO2 for infinite ventilation. Hypoxic sensitivity, A, was positively correlated with body weight (BW). Such correlation in the light and middleweight groups (BW less than 95 kg) and the heavyweight group (BW greater than 95 kg) with Röhrer's index being less than 200 became insignificant when the A value was recalculated after normalization of ventilation for 70 kg body mass. However, the heavyweight group with Röhrer index of higher than 200 still exhibited a significantly higher A value than the light and middleweight groups after normalization of ventilation. These results indicated that body weight plus obesity were determining factors in the increase of hypoxic sensitivity in our subjects.

The ventilatory responses to hypoxia and hypercapnia of 5 Amas (Kachido) were compared with those of 5 controls of similar ages, physical characteristics and lung volumes. The responses to hypoxia and hypercapnia were analyzed by the equations originally proposed [a] by Lloyd et al. and [b] by Kronenberg et al. as follows: (See the formula in the text) The CO2-response slope in hyperoxia, D, of the Ama (1.820 +/- 0.441 liters . min-1 . Torr-1) was slightly higher than that of the control (1.148 +/- 0.586 liters . min-1 . Torr-1), but the difference was not significant. However, the slope of CO2-response in hypoxia at PETO2 = 44 Torr, S44, was almost the same in the two groups (Ama, 1.822 +/- 0.689 liters . min-1 . Torr-1; control, 1.742 +/- 0.902 liters . min-1 . Torr-1). The ratio of S44 to D was significantly lower (p greater than 0.05) in the Ama (1.039 +/- 0.377) than in the control (1.529 +/- 0.249). Comparing the hypoxic response in terms of the ventilation ratio (VR), the elevation of ventilation with augmentation of hypoxia in the Ama was exceeded by that in the control. Thus, it was suggested that the difference in the ventilatory response to hypoxia between the Ama (Kachido) and the control may have been derived from the respiratory adaptation of the Ama (Kachido) acquired by their daily diving activities.

Whether or not stimulation of the peripheral chemoreceptors by hypercapnic-hypoxic exposure results in a long-lasting increase in ventilatory activities was studied using the steady state CO2 response test on 12 human subjects. The degree of hypercapnic hypoxia was end-tidal PCO2 (PETCO2) 42.1 +/- 3.0 and PO2 (PETO2) 39.8 +/- 4.7 mmHg, lasting for 5 min. Minute ventilation values at PETCO2 45 mmHg (V45) and PETCO2 at minute volume 15 liter . min-1 (P15) were calculated from the respective CO2 response curves. The differences in V45 and P15 between the control and the 30 min test group were found to be significant (p less than 0.05). These results suggested the left- and upward-shift of the CO2 response curve of the 30 min test group. On the other hand, in 5 of the 12 subjects, three successive CO2 response tests conducted at 0, 30, and 90 min without hypercapnic-hypoxic exposure showed fairly reproducible results, and no statistically significant differences were found between any of the above trials with the parameters S, B, V45, and P15. These results indicated that the CO2 response curve obtained by using the steady state method can be effected for at least 30 min even if the stimulation of the peripheral chemoreceptors is only for brief periods.

Overall gain of the O2-ventilation feedback control system (GO2) was determined in 9 male and one female healthy subjects. GO2 progressively increased with decreasing end-tidal PO2 (PETO2). This value did not exceed the overall gain of the CO2-ventilation feedback system (GCO2) even at a PETO2 level of 40 mmHg, suggesting that hypoxic stimulation did not become predominant in the present experimental condition. With addition of 250 ml of external dead space, PETO2 decrement (delta PETO2 X actual) was experimentally observed. The delta PETO2 X actual thus obtained was found to be in good agreement with the PETO2 decrement deduced from GO2 (delta PETO2 X expected). This result was similar to that found in the PETCO2 change previously seen in normoxia.

SASAKI, K., INOUE, S., YOSHIDA, A., HAYASHI, F., MASUDA, Y. and HONDA, Y. Ventilatory Response and Drive Due to Carbon Dioxide Stimulation in Idiopathic Scoliosis. Tohoku J. exp. Med., 1982, 137 (2), 145-151-Ten patients with idiopathic scoliosis and seven controls were studied for the purpose of investigating the ventilatory response and drive due to CO2 inhalation by the steady state CO2 response test. To evaluate the ventilatory drive for the respiratory output mouth occlusion pressure (P0.1) was used. The ventilatory response to CO2 of the patients was not significantly different from that of the controls. P0.1 for a given PCO2 stimulation or tidal volume in the patients was significantly higher than the control. Ventilatory output against P0.1, however, was not different in the two groups. The small tidal volume of the scoliotic patients seemed to be compensated by the high respiratory frequency with a large ventilatory drive in order to maintain adequate ventilation.

Twenty-four active judo athletes were examined by an isocapnic progressive hypoxia test. The results of ventilatory and heart rate responses to hypoxia were analyzed by the hyperbolic equations, $$\dot V$$ E= $$\dot V$$ o+A VE /( $$P_{ETO_2 }$$ −C VE ) andHR=HR O+A HR /( $$P_{ETO_2 }$$ −C HR ), respectively, where $$\dot V$$ Eand HR are observed ventilation and heart rate, $$\dot V$$ O andHR O, the horizontal asymptote in ventilation and heart rate for infinite endtidal $$P_{O2} \left( {P_{ETO_2 } } \right)$$ ,A VE andA HR the slope constant indicating the magnitude of hypoxic sensitivity, andC VE andC HR the vertical asymptote in $$P_{ETO_2 }$$ for infinite ventilation and heart rate.A VE was further re-calculated after inlineE was normalized for a 70 kg body mass, using an allometric coefficient, and was defined asA VEN . 1)A VE andA VEN significantly increased with increasing body weight (BW) as has been reported previously, but no such correlation was found betweenA HR and BW. 2) $$\dot V_{{\text{O}}_{\text{2}} }$$ at rest was found to be positively correlated withA VE andA VEN but not withA HR . 3) The relationship betweenA VE andA HR was not significant. Thus, the characteristic feature seen in hypoxic ventilatory activity was not accompanied by a similar trend in heart rate response.

Relative contributions of chemical and non-chemical respiratory stimulations to breath-holding time (BHT) were examined in assisted (Funado) and unassisted (Kachido) breath-hold divers (Ama). In the Funado the magnitude of the chemical contribution was reduced, though statistically not significantly. On the other hand, in the Kachido no difference in chemical contribution was seen from the control. This was considered to be due to the fact that ventilatory response to CO2 was reduced in the Funado, but not in the Kachido. Despite the decreased contribution of CO2 drive to BHT, absolute BHT in the Funado was no prolonged. This may be related to sensitization of the respiratory centers to non-chemical stimulation. Such adaptation would be effective for preventing the danger of losing consciousness in the Funado who face extreme hypoxia on returning to the surface from a dive.

Human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) is known to play a role in intrinsic cellular immunity against human immunodeficiency virus type 1 (HIV-1). The antiretroviral activity of APOBEC3G is associated with hypermutation of viral DNA through cytidine deamination. APOBEC3G contains two cytidine deaminase domains that are characterized by a highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APOBEC3G (c-APOBEC3G) is involved in the catalytic activity. Here, we present the solution structure and the interaction with single-stranded DNA of c-APOBEC3G. Furthermore, we have succeeded for the first time in monitoring the deamination reaction of c-APOBEC3G in real-time using NMR signals. The monitoring has demonstrated that the deamination reaction occurs in a strict 3'-->5'