Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

Generation of specific helper cells and suppressor cells in vitro for the IgE and IgG antibody responses.

Normal splenic lymphocytes from BDF1 mice were cultured on ovalbumin (OA)-bearing syngeneic peritoneal adherent cells for 5 days and their subsequent helper function was tested by an adoptive transfer technique. Lymphocytes harvested from the culture were mixed with DNP-KLH-primed spleen cells and transferred into irradiated syngeneic mice followed by challenge with DNP-OA. The results showed that the cultured lymphocytes has helper function for both IgE and IgG anti-DNP antibody responses. Depletion of mast cells and T cells in the peritoneal adherent cell preparations did not affect the generation of helper cells in the culture. The helper function of the cultured lymphocytes was abolished by the treatment with anti-theta antiserum and complement and was specific for ovalbumin. The OA-specific helper T cells were generated in vitro by the culture of a T cell-rich fraction of normal spleen on T cell-depleted OA-bearing peritoneal cells. If the normal splenic lymphocytes or T cell-rich fraction were cultured with 10 mug/ml of OA in the absence of macrophages, cultured lymphocytes lacked helper function. The transfer of splenic lymphocytes or splenic T cells cultured with soluble OA to normal non-irradiated mice, however, suppressed both IgG and IgE antibody responses of the recipients to subsequent immunization with DNP-OA. The suppressor cells were sensitive to anti-theta antiserum and complement and their activity was specific for OA. The cultured cells transferred into normal mice did not suppress anti-hapten antibody response to DNP-KLH. Normal lymphocytes cultured on OA-bearing macrophages and had helper function in adoptive transfer experiments failed to suppress antibody response of non-irradiated recipients to DNP-OA. The results indicate that OA-bearing macrophages primed T cells and generated helper T cells, whereas the culture of normal lymphocytes with soluble OA in the absence of macrophages generated suppressor T cells.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Regulation of arylsulfatase synthesis by sulfur compounds in Klebsiella aerogenes.

In Klebsiella aerogenes, arylsulfatase synthesis was repressed by inorganic sulfate, sulfite, sulfide, thiosulfate, and cysteine, but not by methionine under normal growth conditions. We isolated cysteine-requiring mutants (Cys minus), and mutants (AtsS minus, AtsR minus) in which the regulation of arylsulfatase synthesis was altered. In the cysteine auxotroph, enzyme synthesis was also repressed by inorganic sulfate or cysteine. Kinetic studies on mutants of the cysteine auxotroph showed that inorganic sulfate repressed arylsulfatase synthesis and that this was not due to cysteine formed by reduction of sulfate. Arylsulfatase synthesis in the AtsS minus mutant was not repressed by inorganic sulfate but was repressed by cysteine. This mutant strain had a normal level of inorganic sulfate transport. Another mutant strain, defective in the inorganic sulfate transport system, synthesized arylsulfatase in the presence of inorganic sulfate but not in the presence of cysteine. The AtsS minus mutant could synthesize the enzyme in the presence of inorganic sulfate but not cysteine. The AtsR minus mutant could synthesize the enzyme in the presence of either inorganic sulfate or cysteine. These results suggest that there are two independent functional corepressors of arylsulfatase synthesis in K. aerogenes.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Genome‐wide distribution and functions of the AAE complex in epigenetic regulation in Arabidopsis

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1‐AIPP1‐EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin‐containing genes. However, the genome‐wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome‐wide level and preferentially interacts with genetic heterochromatin. Polyadenylation (poly(A) sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin‐containing genes, including not only intronic heterochromatin‐containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin‐overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin‐containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation.     

Cell shape controls rheotaxis in small parasitic bacteria

Mycoplasmas, a group of small parasitic bacteria, adhere to and move across host cell surfaces. The role of motility across host cell surfaces in pathogenesis remains unclear. Here, we used optical microscopy to visualize rheotactic behavior in three phylogenetically distant species of Mycoplasma using a microfluidic chamber that enabled the application of precisely controlled fluid flow. We show that directional movements against fluid flow occur synchronously with the polarized cell orienting itself to be parallel against the direction of flow. Analysis of depolarized cells revealed that morphology itself functions as a sensor to recognize rheological properties that mimic those found on host-cell surfaces. These results demonstrate the vital role of cell morphology and motility in responding to mechanical forces encountered in the native environment.     

Identification of asymptomatic Entamoeba histolytica infection by a serological screening test: A cross-sectional study of an HIV-negative men who have sex with men cohort in Japan

BackgroundAmebiasis, caused by Entamoeba histolytica, is spreading in developing countries and in many developed countries as a sexually transmitted infection. Here, we evaluated the efficacy of serological screening to identify asymptomatic E. histolytica infection as a potential epidemiological control measure to limit its spread.Methodology/Principal findingsThis cross-sectional study was carried out between January and March 2021 in an HIV-negative men who have sex with men (MSM) cohort at the National Center for Global Health and Medicine. Serological screening was performed using a commercially available ELISA kit. For seropositive individuals, we performed stool polymerase chain reaction (PCR) to determine current E. histolytica infection. We performed E. histolytica serological screening of 312 participants. None had a history of E. histolytica infection prior to the study. The overall E. histolytica seropositivity was 6.7% (21/312), which was similar to that found by the rapid plasma reagin test (17/312). We identified current infection in 8 of 20 seropositive participants (40.0%) by stool PCR.Conclusions/SignificanceOur serological screening approach constitutes a potentially practical epidemiological strategy. Active epidemiological surveys, in combination with an effective screening strategy for asymptomatically infected individuals, should be applied to help reduce sexually transmitted E. histolytica infections.