A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

Summary We prepared various TNF- derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro⁸Ser⁹Asp¹⁰ was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-. The additional substitution of Ala¹⁵⁶ or Leu¹⁵⁷ by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF- are effective for the improvement of antitumor activity.     

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO₃-pyridine complex in a heterogeneous phase. It was shown from ¹³C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.     

Bulb canker of garlic caused byEmbellisia allii,newly found in Japan

Bulb canker of garlic caused byEmbellisia allii was newly detected in Japan. Symptoms of the disease are described and the morphological characteristics of the causal fungus are illustrated and described.     

Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector

A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosysnthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.     

Na+/H+ exchanger and reperfusion-induced ventricular arrhythmias in isolated perfused heart: possible role of amiloride

The roles of the Na⁺/H⁺ exchange system in the development and cessation of reperfusion induced ventricular arrhythmias were studied in the isolated perfused rat heart. The hearts were perfused in the working heart mode with modified Krebs Henseleit bicarbonate (KHB) buffer and whole heart ischemia was induced by a one-way ball valve with 330 beat/min pacing. Ischemia was continued for 15 min followed by 20 min of aerobic reperfusion (control). Amiloride (1.0mM), an inhibitor of the Na⁺/H⁺ exchange system, was added to the KHB buffer only during reperfusion (group B) or only during ischemic periods (group C). Electrocardiographic and hemodynamic parameters were monitored throughout the perfusion. Coronary effluent was collected through pulmonary artery cannulation and PO₂, PCO₂, HCO ₃ ^– and pH were measured by blood-gas analyzer.The incidence of reperfusion induced ventricular arrhythmias was 100%, 100% and 0% in control, group B and group C, respectively. The mean onset time of termination of reperfusion arrhythmias was significantly shorter in group B than in control. PCO₂ increased from 39.0±0.9 to 89.3±6.0 mmHg at the end of ischemia in control and from 40.6±0.4 to 60.5±5.8 in group C, the difference between groups was statistically significant. HCO ₃ ^– level decreased from 21.8±0.1 to 18.3±0.5 mmol/l in control, however, this decrease was significantly inhibited in group C (from 22.0±0.5 to 20.3±0.2). The increase in PCO₂ and the decrease in HCO ₃ ^– in group B were similar over time to those observed in control. The decrease in pH produced by ischemia was marked in control (from 7.35±0.01 to 6.92±0.04) and group B (from 7.34±0.01 to 6.94±0.02), whereas a decrease in pH was significantly prevented in group C (from 7.34±0.01 to 7.15±0.04). There were no significant differences in PCO₂, HCO ₃ ^– or pH among the three groups during reperfusion.These experiments provide evidence that amiloride significantly prevented the incidence of reperfusion arrhythmias when added only during ischemia and significantly terminated reperfusion arrhythmias when added only during reperfusion. Amiloride may prevent a decrease in pH, due to alterations in PCO₂ and/or HCO ₃ ^– . These changes in PCO₂ and HCO ₃ ^– might be indirectly influenced by inhibition of the Na⁺/H⁺ exchange system via Cl^–/HCO ₃ ^– exchange. The mechanism by which amiloride terminates reperfusion arrhythmias seems to involve electrophysiological effects which were not directly addressed in this experiment.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

The second phantom aquatic leaf beetle in Japan: Macroplea mutica rediscovery in the wetlands (Coleoptera: Chrysomelidae)

Wetland biodiversity is currently declining on a global scale. Wetland biodiversity understanding is critical for determining the wetlands' conservation value. In this study, Macroplea Samouelle, 1819 (Coleoptera: Chrysomelidae) was discovered in Aomori Prefecture, Honshu Island, Japan. Only two Macroplea species have been recorded in Japan, M. japana (Jacoby, 1885) and M. mutica (Fabricius, 1792). Macroplea japana had been unrecorded for 60 years before being rediscovered in Honshu Island in 2022, and a single adult M. mutica female was discovered in Hokkaido Prefecture in 2003. The discovered individuals were concluded to be M. mutica based on morphological and molecular analyses. Although morphological differences were observed with the Eurasian M. mutica individuals, the male genitalia was nearly identical to M. mutica. For the molecular phylogenetic analysis based on COI and 28S sequences, Macroplea individuals in Japan were clustered with M. mutica on the Eurasian Continent. This is the first record of this species on Honshu Island (and the second in Japan), as well as the first record of adult males. This species would require conservation policies and additional distributional surveys.