Four optically active dodecen-4-olides (1a-d) with carbon-carbon double bonds at different positions and their cyclopropanated derivatives (2a-d) were synthesized, and the odour characteristics of the racemic and optically active compounds were evaluated. Compounds 1a-d and 2a-d exhibited typical lactone-like nuances, characterized by strong top notes and oily middle notes. The position of the carbon-carbon double bond had a significant effect on the odour profile. All 1a-d compounds exhibited different odour characteristics: 1a showed fruity notes, 1b and 1c exhibited green and fruity notes, and 1d exhibited green and floral notes. Cyclopropanation substantially altered these characteristics. As a result, the odour characteristics of compounds 2, where the carbon-carbon double bonds were converted to cyclopropane rings, differed significantly from those of compounds 1. Conversion of the double bond in 1a to a cyclopropane ring changed the fruity note to green note, while cyclopropanation of 1b changed the green note to fruity. The antimicrobial activities of 1 and 2 against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were also investigated. Only compound (S)-1b exhibited antimicrobial activity against S. aureus.

The proto-oncogene c-Src is a protein-tyrosine kinase that is associated with increased risk of acute and chronic kidney diseases, cardiovascular diseases, neurological diseases, and cancers. The purpose of this study was to develop a gene delivery carrier responding to activated c-Src and to evaluate its efficacy in vitro and in vivo. The polymeric gene carrier consists of a neutral main chain bearing a peptide substrate of c-Src or negative control peptide. Complexes of the polymer containing c-Src-targeting peptides and DNA were phosphorylated in the presence of c-Src, resulting in release of the DNA. Green fluorescent protein (GFP) expression was observed after microinjection of complexes of polymer containing c-Src-targeting peptides and GFP-encoding DNA into A431 cells at the nitrogen/phosphate (N/P) ratio of 1.0. GFP was not expressed in cells microinjected with negative control polymer/DNA complex. Direct injection of complexes of polymer containing c-Src-targeting peptides and luciferase-encoding DNA at N/P ratios of 1.0 and 2.0 into tumor-bearing mice resulted in luciferase expression in A431 tumors but not in normal skin tissues. No luciferase expression was observed in A431 tumors or skin tissues after injection of control polymer/DNA complex. These results indicate that our gene delivery carrier enables activated c-Src-specific gene expression.

Six optically active (Z)-7-decen-4-olide derivatives (1a-1f) were synthesized in 99% enantiomeric excess using diastereomeric resolution. The odour properties of the racemic and optically active 1a-1f were evaluated in terms of their orthonasal aromas. All of the stereoisomers had different odour characteristics and thresholds. Decen-4-olides (1a-1c) had a strong fruity note, whereas undecen-4-olide (1d and 1e) and dodecen-4-olide (1f) had a strong green note. For 7-alken-4-olides (1a, 1d, and 1f), the (R)-enantiomer had a lower odour threshold than the (S)-enantiomer. In contrast, no difference in the odour threshold was observed for the enantiomers of the 8-alken-4-olides (1b and 1e). Furthermore, the antimicrobial activity against Escherichia coli (E. coli; ATCC 25922) and Staphylococcus aureus (S. aureus; ATCC 29213) were investigated. Although the no differences in the antimicrobial activity of the stereoisomers was observed, 1d and 1e showed slight antimicrobial activity against E. coli, whereas only 1f showed antimicrobial activity against S. aureus. No antimicrobial activity was exhibited by (R)-1f, whereas (S)-1f exhibited strong antimicrobial activity.

Biotinylated oligopeptides from the envelope glycoproteins of dengue fever virus, influenza A and B viruses, and human immunodeficiency virus (HIV) were synthesized and their interaction with curdlan and dextran sulfates was investigated using surface plasmon resonance to evaluate the antiviral mechanisms of sulfated polysaccharides. More than two clusters consisting of basic amino acids in the oligopeptides from dengue fever virus, strongly interacted with the sulfated polysaccharides elucidated by the association- and dissociation-rate constants. Interactions decreased with the decreasing molecular weights of the sulfated polysaccharides. Although oligopeptides from influenza A virus potently interacted with the sulfated polysaccharides, no interaction was detected on a B/Hong Kong virus oligopeptide bearing few basic amino acids. For the C terminus and V3 region short and long oligopeptides from HIV gp120, the interaction was enhanced by the number of clustered basic amino acids and was inhibited by acidic and bulky amino acids.

PDF file - 128K, The effect of the number of ELP tyrosine residues, ELP MW and concentration on retention radioactivity in FaDu tumor as a function of time post-injection of labeled ELP

Bisphenol A (BPA; 4,4′-isopropylidenediphenol) is a well-known endocrine disruptor. Most human exposure to BPA occurs through the consumption of BPA-contaminated foods. Cardiovascular or cardiometabolic diseases such as diabetes, obesity, hypertension, acute kidney disease, chronic kidney disease, and heart failure are the leading causes of death worldwide. Positive associations have been reported between blood or urinary BPA levels and cardiovascular or cardiometabolic diseases. BPA also induces disorders or dysfunctions in the tissues associated with these diseases through various cell signaling pathways. This review highlights the literature elucidating the relationship between BPA and various cardiovascular or cardiometabolic diseases and the potential mechanisms underlying BPA-mediated disorders or dysfunctions in tissues such as blood vessels, skeletal muscle, adipose tissue, liver, pancreas, kidney, and heart that are associated with these diseases.

Three sulfated 1-alkyl-triazole-maltoheptaosides bearing long-chain alkyl groups (C6, C12, and C18) at the reducing ends were synthesized and subjected to surface plasmon resonance with liposomes to elucidate the cytotoxic mechanism of sulfated alkyl oligosaccharides having potent anti-HIV activity. With increasing the length of the alkyl groups, the cytotoxicity of nonsulfated and sulfated 1-alkyl-triazole-maltoheptaosides against MT-4 cells increased to CC50 values of 102 and 72 mu g/mL from a low cytotoxicity of CC50>1000 mu g/mL. Nonsulfated and sulfated 1-alkyl-triazole-maltoheptaosides bearing a C18 alkyl chain showed the highest apparent association-rate constant (k(a)=1.20x10(5) and 1.00x10(7) 1/M, respectively) and lowest dissociation-rate constant (k(d)=7.30x10(-7) and 1.39x10(-4) 1/s, respectively) constants with the liposomes, indicating that the alkyl groups bearing longer-chains interact tightly with the liposomes. The results suggest that the longer-chain alkyl groups penetrate into the lipid bilayer of MT-4 cells to induce cytotoxicity. Considering the proposed cytotoxic mechanism, the antibacterial activity of the 1-alkyl-triazole-maltoheptaosides against Gram-negative Escherichia coli and Gram-positive Bacillus subtilis was preliminarily investigated, finding certain antibacterial activity.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.

With the appearance of biotin-avidin, digoxigenin (DIG), fluorescent reagents with high molar absorption coefficients, and chemiluminescence systems, the use of P-32-labeled nucleoside reagents has decreased dramatically, and radioisotopes have long since disappeared from the field of nucleic acid research. However, radiometric assays using [gamma-P-32]ATP in protein/peptide phosphorylation studies are still widely used for measuring protein kinase activity, and supports the basis of intracellular signal transduction studies. Protein kinases catalyze the transfer of the gamma-phosphoryl group of ATP to an acceptor protein substrate. Using [gamma-P-32]ATP, we have successfully developed a peptide substrate for protein kinase C alpha, which is constantly and extraordinarily activated in cancer cells. The [gamma-P-32]ATP is an excellent tracer, and the method of analyzing phosphorylation by radioisotopic procedure will be a useful method now and in the future.

Background: Pyoktanin blue (PB) is used for staining tissues and cells, and it is applied in photodynamic therapy due to its potent bactericidal activity. However, clinical application of PB as an antiviral and antitumor agent has been limited due to its potent toxicity. For clinical application, the antitumor and antiviral activity as well as the neurotoxicity of PB were re-evaluated with a chemotherapeutic index. Methods: Tumor-specificity (TS) was determined by the ratio of CC50 against normal oral cells/oral squamous cell carcinoma (OSCC); neurotoxicity by that of normal oral/neuronal cells; antiviral activity by that of mock-infected/virus-infected cells; and potency-selectivity expression (PSE) by dividing TS by CC50 (OSCC). Results: Antitumor activity of PB (assessed by TS and PSE) was comparable with that of DXR and much higher than that of 5-FU and melphalan. PB induced caspase-3 activation and subG1 cell accumulation in an OSCC cell line (Ca9-22). PB and anticancer drugs showed comparable cytotoxicity against both neuronal cells and OSCC cell lines. PB showed no detectable anti-HIV/HSV activity, in contrast to reverse transferase inhibitors, sulfated glucans, and alkaline extract of leaves of S.P. Conclusions: PB showed first-class anticancer activity and neurotoxicity, suggesting the importance of establishing the safe treatment schedule.

BACKGROUND:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS:Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS:The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS:The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

Background: Very few studies of the antiviral potential of lignosulfonates have been published. With the aim of oral application, among various groups of natural products, the relative antiviral potency of lignosulfonate and its ability to rapidly inactivate viruses were investigated. Methods: As target cells, MT-4 cells in suspension and attached Vero cells were used for infections with human immunodeficiency virus (HIV) and human herpes simplex type-1 virus (HSV). Mock- or virus-infected cells were incubated for 3–5 days with various concentrations of test samples, and the viable cell number was determined with the MTT method. For the shorter exposure experiments, higher titers of HIV or HSV were exposed to test samples for 10 or 3 min, diluted to a normal multiplicity of infection (MOI), and applied to the cells. Antiviral activity was quantified by using the chemotherapy index. Results: In the long-exposure system, lignosulfonates showed comparable anti-HIV activity with those of AZT, ddC, and sulfated polysaccharides, and it exceeded those of hundreds of tannins and flavonoids. When the exposure time was shortened, the chemotherapeutic index of the lignosulfonates for HIV was increased 27-fold. At a physiological pH, lignosulfonate showed higher anti-HIV activity than commercial alkali-lignin, dealkali-lignin, and humic acid, possibly due to the higher solubility and purity. Conclusions: With their rapid virus-inactivation capabilities, lignosulfonates may be useful for the prevention or treatment of virally induced oral diseases.

Heptapeptide SFLLRNP is a receptor-tethered ligand of protease-activated receptor 1 (PAR-1), and its Phe at position 2 is essential for the aggregation of human platelets. To validate the structural elements of the Phephenyl group in receptor activation, we have synthesized a complete set of S/Phe/LLRNP peptides comprising different series of fluorophenylalanine isomers (F-n)Phe, where n = 1, 2, 3, and 5. Phe-2-phenyl was strongly suggested to be involved in the edge-to-face CH/pi interaction with the receptor aromatic group. In the present study, to prove this receptor interaction definitively, we synthesized another series of peptide analogs containing (F-4)Phe-isomers, with the phenyl group of each isomer possessing only one hydrogen atom at the ortho, meta, or para position. When the peptides were assayed for their platelet aggregation activity, S/(2,3,4,6-F-4)Phe/LLRNP and S/(2,3,4,5-F-4)Phe/LLRNP exhibited noticeable activity (34% and 6% intensities of the native peptide, respectively), whereas S/(2,3,5,6-F-4)Phe/LLRNP was completely inactive. The results indicated that, at the ortho and meta positions but not at the para position, benzene-hydrogen atoms are required for the CH/pi interaction to activate the receptor. The results provided a decisive evidence of the molecular recognition property of Phe, the phenyl benzene-hydrogen atom of which participates directly in the interaction with the receptor aromatic pi plane.

Although a long-chain alkyl group in sulfated oligosaccharides can enhance the anti-HIV activity, the exact mechanism is unclear. To elucidate the role of the long-chain alkyl group, its interaction with a liposome (100 nm) as a HIV model was investigated by surface plasmon resonance and dynamic light scattering. The newly synthesized sulfated 1-(decadecyl-1, 2, 3-triazole)-1-deoxy-maltoheptaoside bearing the long-chain alkyl group was found to interact with the liposome. The particle size increased and the zeta potential was negative, indicating that the sulfated alkyl maltoheptaoside was attached to the liposome by the long-chain alkyl group and the fixed sulfated maltoheptaoside moiety was covered on the liposome. Thus, the long-chain alkyl group penetrates and is fixed into the lipid bilayer of HIV and the sulfated maltoheptaose moiety with negatively charged sulfate groups was electrostatically interacted with HIV gp120 molecule with positively charged amino acids to achieve the inhibition of HIV infection.

Zwitterionic polycarboxybetaines (PCBs) have gained attention as alternative stealth polymers whose liposomal formulation and protein conjugates were reported not to elicit anti-polymer antibodies. Here, we studied the blood retention and antigenicity of liposomes modified with PCBs focusing on their chemical structures and doses. We compared PCBs with either 1 or 3 (PCB1 or PCB3) spacer carbons between the carboxylate and ammonium groups. PCB3-modified liposomes had a short blood retention, whereas PCB1-modified liposomes demonstrated extended blood retention that was somewhat superior to PEG-ylated liposome. This confirmed the excellent non-fouling nature of PCB1 reported previously. Interestingly, PCB1-liposome as well as PCB3-liposome elicited specific IgMs toward each PCB. The dose-dependent production of specific IgMs to PCB-liposomes was similar to that of PEGylated liposome, i.e., high doses of PCB-liposomes reduced the production of specific IgMs, termed immunological tolerance. These results indicate the importance of investigating the effect of dose to clarify the existence of antigenicity of stealth polymers.

Background: Herpes simplex virus (HSV) is usually dormant and becomes apparent when body conditions decline. We investigated the anti-HSV activity of various natural and synthetic compounds for future clinical application. Methods: Mock- and HSV-infected Vero cells were treated for three days with various concentrations of samples. For short exposure, 100-fold concentrated virus were preincubated for 3 min with samples, diluted to normal multiplicity of infection (MOI), before the addition to the cells. Anti-HSV activity was evaluated by the chemotherapy index. Results: Alkaline extracts of the leaves of Sasa sp. (SE) and pine cone (PCE) showed higher anti-HSV activity than 20 Japanese traditional herb medicines (Kampo formulas), four popular polyphenols, and 119 chromone-related compounds. Exposure of HSV to SE or PCE for 3 min almost completely eliminated the infectivity of HSV, whereas much longer exposure time was required for Kakkonto, the most active Kampo formulae. Anti-HSV activity of PCE and Kakkonto could be detected only when they were dissolved by alkaline solution (pH 8.0), but not by neutral buffer (pH 7.4). Anti-HSV activity of SE and povidone iodine was stable if they were diluted with neutral buffer. Conclusions: The present study suggests the applicability of SE and PCE for treatment of oral HSV and possibly other viruses.

Three new spherical sulfated cellobiose-polylysine dendrimers of increasing generations bearing negatively charged sulfate groups were prepared by sulfating the corresponding cellobiose-polylysine dendrimers. The first, second, and third-generation derivatives exhibited potent anti-HIV activity with EC50 values of 3.7, 0.6, and 1.5 mu g/mL, respectively, in constant to sulfated oligosaccharides with low anti-HIV activity, while the second generation sulfated dendrimer was the most active. Surface plasmon resonance measurements with poly-L lysine bearing positively charged amino acids as a model of the HIV surface glycoprotein gp120, indicated that the second-generation dendrimer had the lowest dissociation constant (K-D = 1.86 x 10(-12) M). Both the particle size and zeta potential increased in the presence of poly-L-lysine. It was proven that the moderate distance between the terminal sulfated cellobiose units in the second-generation dendrimer favored the high anti-HIV activity, owing to the electrostatic interactions developed due to the cluster effect.

Sulfated heptakis-6-[4-(beta-D-maltopyranosyloxymethyl)-1H-1, 2, 3- triazoyl-1-yl)-beta-CD (6-O-maltosyl beta-CD) was prepared by the sulfation of 6-O-maltosyl beta-CD that had been synthesized by click reaction of acetylated 1-O-propagyl-beta-D-maltoside with heptakis-6-azido beta-CD and then deacetylation. Sulfated 6-O-glucosyl beta-CD was also synthesized from 6-O-glucosyl beta-CD. These sulfated beta-CDs were found to have potent anti-HIV activity (EC50) of 1.3 and 27.9 mu g/mL, respectively, however, sulfated beta-CD had low anti-HIV activity,>200 mu g/mL. The surface plasmon resonance against poly-L-lysine as a model of HIV surface glycoprotein gp 120 indicated that the apparent association- (k(a)) and dissociation-rate (k(d)) constants increased and decreased, respectively, k(a)=0.21x10(3), 6.24x10(3), 1.46x10(4) 1/Ms and k(d)=6.70x10(-4), 3.92x10(-4), 3.03x10(-4) 1/s, by depending on the number of branched glucose units. The particle size of the sulfated beta-CDs was 13.7 nm, 6.2 nm, and 3.3 nm,respectively, measured by dynamic light scattering. These results suggest that the particle size of the sulfated beta-CDs also played an important role in the strength of anti-HIV activity. The highest anti-HIV activity of sulfated 6-O-maltosyl beta-CD was expressed by the electrostatic interaction with HIV gp 120 putting on sulfated 6-O-maltosyl beta-CD like a cap, because the particle size (13.7 nm) was somewhat larger than that of HIV gp 120 (8 to 10 nm).

The G protein-coupled receptor kinase (GRK) family consists of seven cytosolic serine/threonine (Ser/Thr) protein kinases, and among them, GRK2 is involved in the regulation of an enormous range of both G protein-coupled receptors (GPCRs) and non-GPCR substrates that participate in or regulate many critical cellular processes. GRK2 dysfunction is associated with multiple diseases, including cancers, brain diseases, cardiovascular and metabolic diseases, and therefore GRK2-specific substrates/inhibitors are needed not only for studies of GRK2-mediated cellular functions but also for GRK2-targeted drug development. Here, we first review the structure, regulation and functions of GRK2, and its synthetic substrates and inhibitors. We then highlight recent work on synthetic peptide substrates/inhibitors as promising tools for fundamental studies of the physiological functions of GRK2, and as candidates for applications in clinical diagnostics.