Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Budgets of major ionic species and nutrients on a dam reservoir in forested watershed.

To evaluate the role of a dam reservoir in the runoff of pollutant loadings from a forested watershed, the input-output budgets in the Ikuno dam reservoir had been investigated for eight years since 1996. The T-N, T-P, TOC and major ionic species in the bulk precipitation, stream water, and outflow were measured. The residence time calculated by using the data of the inflow and outflow was 0.3 year. The average precipitation was 1,772 mm during the investigation period (1996-2004). The direct deposition to water surface was less than one percentage to total loadings of nutrients and major ionic species. The ratios of output to input of TOC, TN, and TP were 1.04 to 1.42, and those of major ionic species were from 0.83 to 0.99 except for NO3(-), which was 1.12. However, the ratios of output to input of major ionic species except for NO3(-) at the Ikuno dam reservoir will be larger, and those of NO3(-), TOC, TN, and TP will be smaller, if we also include rain events. These results suggested that the dam reservoir played a role as a sink for pollutants in forested watershed, and that the pollutant loadings to downstream may decrease.

Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides.

We report a strategy for efficient post-translational modification of a library of ribosomally-translated peptides by activation and elimination of cysteine to dehydroalanine then conjugate addition of a range of exogenous thiols, with an emphasis on carbohydrates. These reactions are selective for cysteine, and do not interfere with amplification of the nucleic acid component of an mRNA-displayed peptide. Furthermore, these reactions are shown to be compatible with two different macrocyclisation chemistries, and when applied to a peptide containing an N-terminal cysteine give a ketone that can be functionalised in an orthogonal manner. This new strategy can overcome a limitation of ribosomal translation, providing a means to incorporate untranslatable groups such as carbohydrates in amino acid side chains, and will allow for the ribosomal generation of glycopeptides, requiring only the introduction of a free thiol in the molecule to be incorporated. In combination with in vitro selection techniques, this strategy is envisaged to allow the discovery of biologically-active glycopeptides with a near-natural, but hydrolytically stable, thioglycosidic bond.

Angiotensin II type 1 receptor antagonist downregulates nonmuscle myosin heavy chains in spontaneously hypertensive rat aorta.

The aim of this study was to clarify the differences between the angiotensin II type 1 (AT1) receptor antagonist and the angiotensin-converting enzyme (ACE) inhibitor on smooth muscle and nonmuscle myosin heavy chain isoforms in aortic smooth muscle cells of Wistar-Kyoto rats and spontaneously hypertensive rats. All 4 myosin heavy chain isoforms are heterogeneously expressed in the smooth muscle cells of the aortic tunica media in 20-week-old rats, and the contractile-type myosin heavy chains are highly expressed in smooth muscle cells of the aortic tunica media compared with the synthetic-type myosin heavy chains. Both the AT1 receptor antagonist and the ACE inhibitor had the same effects on hemodynamics, smooth muscle cell hypertrophy and proliferation, fibrosis, and vascular remodeling in spontaneously hypertensive rats. However, the AT1 receptor antagonist had a more potent effect on the downregulation of the synthetic-type myosin heavy chains than the ACE inhibitor in spontaneously hypertensive rat aortic tunica media. In contrast, these effects of the AT1 receptor antagonist and the ACE inhibitor on hemodynamics, morphology, fibrosis, and expression of myosin heavy chain isoforms in smooth muscle cells of the aortic tunica media were not observed in Wistar-Kyoto rats. Thus, within 6 weeks, the AT1 receptor antagonist might modulate the cellular composition of myosin heavy chain isoforms in smooth muscle cells more efficiently than the ACE inhibitor, without morphological changes in the spontaneously hypertensive rat aorta.

Dilazep, a nucleoside transporter inhibitor, modulates cell cycle progression and DNA synthesis in rat mesangial cells in vitro.

The direct effects of the nucleoside transporter inhibitor dilazep on the cell cycle of mesangial cells have not before been investigated. The purpose of this study was to elucidate whether dilazep can inhibit the proliferation of mesangial cells and how it interferes with the cell cycle of these cells. DNA histograms were used and BrdUrd uptake rate was measured by flow cytometry. There was no significant difference in the cell numbers among the untreated group and the 10(-5) M, 10(-6) M or 10(-7) M dilazep-treated groups at 24 h of incubation. However, at 48 and 72 h, the cell numbers in the dilazep-treated groups were significantly lower compared with that of the untreated group (P < 0.005). The DNA histograms of cultured rat mesangial cells at 12, 24, and 48 h of incubation with 10(-5) M dilazep showed that the ratio of the S phase population in the dilazep-treated group decreased by 2.2% at 12 h, by 9.6% at 24 h, and by 18.9% at 48 h compared with the untreated group. The ratio of the G0/G1 phase population in the dilazep-treated group significantly increased: 6.8% at 12h (P < 0.05), 13.9% at 24 h (P < 0.001), and 76.5% at 48 h (P < 0.001) compared with the untreated group. A flow cytometric measurement of bivariate DNA/BrdUrd distribution demonstrated that the DNA synthesis rate in the S phase decreased after 6 h (P < 0.005) and 12 h (P < 0.05) of incubation compared with the untreated group. These results suggest that dilazep inhibits the proliferation of cultured rat mesangial cells by suppressing the G1/S transition by prolonging G2/M and through decreasing the DNA synthesis rate.

Studies on 1-substituted 4-(1,2-diphenylethyl)piperazine derivatives and their analgesic activities. 1.

The preparation and analgesic activities of a series of the entitled compounds (5-22) and the optical isomers of the 1-cyclohexyl derivative 5 are described. Reactions of N,N-bis(2-chloroethyl)-1,2-diphenylethylamine (3) with ammonia and primary amines gave N-(1,2-diphenylethyl)piperazine (4) and N1-substituted derivatives (5-20, 22), respectively. The alkylation of 4 afforded 12-21. Compounds 5-18 and 22 were also obtained by the reactions of 1,2-diphenylethylamine (23) and N-substituted 2,2'-dichlorodiethylamine. Racemate 5 was resolved with (+)- or (-)-2'-nitrotartranilic acid into its optical isomers [(+)-5 and (-)-5], and the absolute configuration of (+)-5 was determined to be S configuration by the synthesis and optical rotatory dispersion measurements. The most active members in this series of compounds were 5-7, which were approximately as potent as (-)-morphine. In the case of 5, the more potent enantiomer (S)-(+)-5 has the opposite configuration to that of (-)-N,N-dimethyl-1,2-diphenylethylamine (Spa) or (-)-morphine with respect to the (C-9) asymmetric center and belongs to a new series of compounds having potent analgesic activity.

Chronic glucocorticoid administration as well as repeated stress affects the subsequent acute immobilization stress-induced expression of immediate early genes but not that of NGFI-A.

We reported that repeated immobilization for six days attenuates the subsequent acute immobilization stress-induced expression of the immediate early genes c-fos, fos B, jun B and nerve growth factor-induced gene-B (NGFI-B), but not of NGFI-A, in the rat paraventricular hypothalamic nucleus. In this study, we confirmed these findings by means of a time-course study, and further investigated whether the elevated plasma basal glucocorticoid level induced by repeated stress underlies the attenuated response of immediate early genes and the preserved reactivity of NGFI-A. Rats implanted with 100, 200 or 400 mg corticosterone or placebo pellets (control), were immobilized for 1 h and decapitated seven days later. In control rats acute immobilization induced c-fos, fos B, jun B, NGFI-A and NGFI-B messenger RNA in the paraventricular hypothalamic nucleus, whereas all of them except NGFI-A, were significantly reduced in rats given 200 and 400 mg corticosterone implants. The similarity of the results from the two procedures suggests that glucocorticoid is involved in regulating immediate early genes in the paraventricular hypothalamic nucleus under repeated stress and that the NGFI-A gene is not regulated by this mechanism. However, the plasma basal corticosterone level in repeatedly stressed rats was lower than that of rats implanted with 100 mg corticosterone, suggesting that a repetitive stress-induced corticosterone surge also contributes to this mechanism.

Eosinophil counts and plasma fibrinogen in patients with vasospastic angina pectoris.

Epidemiologic studies have suggested a relation between white blood cell (WBC) counts and the incidence of coronary heart disease. However, the relation between vasospastic angina pectoris (VAP) and WBC counts remains to be elucidated. To clarify the relation between differential and WBC counts in VAP, we compared the hematologic values, blood chemical values, plasma fibrinogen levels, C-reactive protein levels, and coronary risk factors in patients with spontaneous attacks of VAP (n = 39) with those in patients with stable effort angina pectoris (EAP, n = 35) and in control subjects (n = 19). Patients with VAP were further divided into mild VAP (n = 22) and severe VAP groups (n = 17). There were no differences in the coronary risk factors, body temperature, total WBC counts, and C-reactive protein levels among the control, EAP, mild VAP, and severe VAP groups, except that the high-density lipoprotein cholesterol in the EAP group was significantly lower than that in the control group (p <0.01). In contrast, the eosinophil counts were significantly higher in the severe VAP group than in the other 3 groups (p <0.01). Plasma fibrinogen levels were also significantly higher in the severe VAP group than in the other 3 groups (p <0.05). The follow-up study for differential and WBC counts in patients with VAP (n = 23) demonstrated that, after medical therapy, the eosinophil counts were significantly decreased to the some level as those in the control group (p <0.0001). Thus, the eosinophil counts and plasma fibrinogen levels could predict the severity of VAP. Furthermore, a follow-up study in patients with VAP suggests that coronary vasospasm could result in an increase in eosinophil counts.

Differential expression of fos family and jun family mRNAs in the rat hypothalamo-pituitary-adrenal axis after immobilization stress.

We aimed to clarify the regulatory mechanism of the hypothalamo-pituitary-adrenal axis that plays key roles in initiating stress responses, as well as the roles of immediate early genes in this process. We investigated the stress-induced activation of fos and jun family proto-oncogenes by means of in situ hybridization histochemistry. Immobilization stress induced c-fos and jun B mRNAs in the parvocellular region of the hypothalamic paraventricular nucleus, the anterior and intermediate lobes of pituitary, and in the adrenal gland after 7 min of immobilization, although no c-fos or jun B mRNAs were detected in these and other organs in control rats. The levels of these mRNAs peaked after 30-60 min of immobilization, then declined. A low level of fos B mRNA appeared at 15-30 min and peaked after 60-90 min. On the contrary, c-jun and jun D mRNAs were constitutively expressed in the paraventricular nucleus and adrenal cortex. These findings indicate that the members of the fos and jun family proto-oncogenes play different roles in the transcriptional regulation of genes involved in the hypothalamo-pituitary-adrenal axis, and that monitoring immediate early genes is a useful method for following stress-induced cellular responses in the neuro-endocrine system.

The expression of neuropeptides and their mRNAs in the trigeminal mesencephalic nucleus following masseteric nerve transection.

By in situ hybridization and immunohistochemistry, we examined the expression of neuropeptides such as neuropeptide Y (NPY), galanin (Gal), substance P (SP), vasoactive intestinal polypeptide (VIP) and their mRNAs in the rat mesencephalic trigeminal nucleus (Mes5) following masseteric nerve transection. On the side contralateral to the nerve transection, none of the peptides examined were labeled in Mes5 cell bodies. However, on the side ipsilateral to the lesion, NPY, Gal and preprotachykinin (PPT) mRNAs appeared in Mes5 cell bodies. Double labeling for mRNAs by in situ hybridization and retrograde tracer fluoro-gold (FG) revealed that almost all (96-97%) the FG-labeled neurons which were cut expressed NPY and Gal mRNAs, whereas less neurons (87%) expressed PPT mRNA. NPY and Gal-like immunoreactivities were detected in Mes5 cell bodies ipsilateral to the axotomy. The results suggested that these neuropeptides play roles in adaptive processes after peripheral nerve injury in Mes5 neurons as they are thought to do so in dorsal root ganglion neurons.

A case of acute eosinophilic pneumonia. Evidence for hypersensitivity-like pulmonary reaction.

We examined an 86-year-old man with acute respiratory failure. A chest roentgenogram showed diffuse reticular shadows. Transbronchial biopsy revealed thickening of the alveolar septa accompanied by moderate eosinophil infiltration. After admission to the hospital, the patient's symptoms immediately improved without any medication. Clinical course and pathologic findings suggested acute eosinophilic pneumonia caused by a hypersensitivity reaction.

Differential regulation of IEGs in the rat PVH in single and repeated stress models.

Various kinds of stressors induce immediate early genes (IEGs) in discrete brain regions. We recently reported the reduced response of Fos expression in the hypothalamic paraventricular nucleus (PVH) when rats are repeatedly exposed to immobilization (IMO) stress. In this study, using in situ hybridization histochemistry, we further extended the research to other IEGs, and the results showed that prior exposure to IMO for 6 days suppressed the induction of fosB, junB and NGFI-B, but NGFI-A, mRNAs in response to a challenge IMO on day 7, suggesting that repeated stress has different effects on the transcription of NGFI-A and the other IEGs, in the PVH.

Repeated stress reduces the subsequent stress-induced expression of Fos in rat brain.

Repeated stress is known to potentiate the CNS response to subsequent stress. Various stressful stimuli can induce Fos expression in discrete regions of the brain, such as the lateral septum, the hypothalamic paraventricular nucleus and the locus coeruleus. We investigated by immunohistochemistry the effect of the stress of repeated immobilization on Fos expression in those regions of the brain in adult male rats. Six daily immobilizations suppressed the expression of Fos in all regions when immobilization was subsequently applied, suggesting that Fos does not play a major role in potentiating the stress response under repeated stressed conditions.

Immobilization stress induces c-fos and c-jun immediate early genes expression in the heart.

Emotional stress is considered to be one of the etiological factors in ischemic heart disease (IHD) and sudden cardiac death (SCD), mechanisms of which are poorly understood. Immediate early genes (IEGs), such as c-fos and c-jun are used as tools for detection of cellular activation. Male Wistar rats were exposed to acute immobilization (IMO). IMO stress for 30 min induced c-fos and c-jun mRNAs expression in the myocardium and the smooth muscle layer of the coronary arteries. IMO stress for 2 h also induced Fos and Jun like-immunoreactivities in the same regions. Distribution of IEG mRNAs and their protein products in the myocardium was not uniform but rather localized. These data provided histological evidence for an early cellular event in the stress response whose consequences could result in activation of tissues in the myocardium and coronary arterial smooth muscle cells which play a role in the pathophysiological changes in IHD and SCD.

Heterogeneous expression of nonmuscle myosin heavy chain-B in mesangial cells of patients with Gitelman's syndrome.

AIMS: It has been suggested that angiotensin II (Ang II) promotes hypertrophy and hyperplasia of mesangial cells. Nonmuscle myosin heavy chain-B (NMHC-B) and alpha-smooth muscle (SM) actin are considered to be molecular markers for phenotypic change ofproliferative mesangial cells. One of the clinical characteristics in Gitelman's syndrome (GS) is the elevation of plasma Ang II. However, little is known about the relation between Ang II and phenotypic change of mesangial cells in patients with GS. In this report, we examined the expression of NMHC-B and alpha-SM actin in mesangial cells of two GS patients. MATERIALS AND METHODS: Plasma renin activity, and the concentrations of Ang II, 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), urinary kallikrein, and 6-keto-PGF1alpha were measured. Immunohistochemical staining of NMHC-B and alpha-SM actin in mesangial cells of GS patients was also performed. RESULTS: Both cases of GS showed normal glomerular function, few histological abnormalities, and higher than normal plasma concentrations of renin and Ang II. Furthermore, one case showed a high urinary concentration of kallikrein and the expression of both NMHC-B and alpha-SM actin in mesangial cells. The other case showed a high urinary concentration of 6-keto-PGF1alpha but not kallikrein and without the expression of NMHC-B and alpha-SM actin. CONCLUSION: Not only plasma kinin-kallikrein and prostaglandins, but the renal expression of NMHC-B and alpha-SM actin may be variable in different patients with GS.

Infection Behavior of Venturia nashicola, the Cause of Scab on Asian Pears.

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.

Application of the fission-track technique to the determination of uranium in natural waters.

A procedure for the determination of the uranium content of natural waters is presented. To 100 ml of natural waters, 200 ml of conc. hydrochloric acid are added and this solution is passed through a 6-ml column of Dowex 1-X8. The uranium is eluted with 60 ml of 0.1M hydrochloric acid, and the eluate is evaporated to dryness. The residue is subjected to the fission-track technique described previously. The uranium content of the river waters in Fukuoka City was determined.

Determination of trace amounts of uranium in silicate minerals by the fission track technique.

The fission track technique has been applied to the determination of uranium on Dowex 1 x 8 after chemical separation from silicates. Standard rock and several stone meteorite samples were treated by this method and satisfactory results were obtained. The procedure is applicable to natural waters.

In vitro studies of determinants of smooth muscle mechanics.

Smooth muscle contraction is dependent upon phosphorylation of the 20,000 Da light chain subunits of myosin. Whereas the kinetics of the hydrolysis of MgATP by smooth muscle myosin suggest a simple phosphorylation-dependent "on-off" mechanism, the contractile response in smooth muscle tissue is complex. Experiments to unravel this complexity have been performed in vitro using a combination of motility assays and kinetic techniques. Some insight into this complexity is obtained, but the mechanism and the regulation of smooth muscle contraction is still not completely known.

Mixed connective tissue disease with multiple organ damage: successful treatment with plasmapheresis.

A 24-year-old-woman with mixed connective tissue disease (MCTD) developed multiple organ failure, disseminated intravascular coagulation (DIC), metabolic acidosis, and respiratory and renal failure resulting from visceral vasospasm, so-called visceral Raynaud's phenomenon. After plasmapheresis, the condition of multiple organ failure was markedly improved. The successful treatment with plasmapheresis was dependent upon the removal of immune complexes in serum and improvement of visceral circulation. Thus plasma exchange is recommended as a possible a treatment for multiple organ damage in MCTD.