Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Relationship between productivity and γ-carboxylation efficiency of recombinant protein C

Summary The relationship between productivity and -carboxylation of recombinant protein C was studied with Chinese hamster ovary (CHO) cell line producing human protein C (huPC) by the use of two types of enzyme-linked immunosorbent assay (ELISA). Productivity could be varied by the addition of different levels of serum. An inverse correlation was found between the degree of -carboxylation and productivity of recombinant protein C.     

Localization of Various Forms of the γ Subunit of G Protein in Neural and Nonneural Tissues

Abstract: For a study of the localization of various forms of the γ subunit of G proteins, antibodies were raised in rabbits against peptides that corresponded to partial amino acid sequences of bovine γ₂, γ₃, γ₅, and γ₇. Affinity-purified antibodies against γ₂, γ₃, and γ₅ reacted specifically with γ₂, γ₃, and γ₅, respectively, but the antibody against γ₇ reacted with γ₂, γ₃, and a novel γ subunit, designated γ_s1, as well as with γ₇. Because these antibodies reacted with the respective forms of the γ subunit from rat brain, we investigated the localization of γ subunits in the rat. γ₂ and γ₃ were abundant in all regions in the brain, whereas the concentration of γ₅ and γ₇ was relatively low with the single exception being a high concentration of γ₇ in the striatum. The concentration of γ₂ was consistently high during ontogenic development in the rat brain, whereas γ₃ appeared about a week after birth and their concentrations then increased until a month after birth. In tissues other than the brain, γ₃ was observed only in the pituitary gland, whereas γ₂, γ₅, and γ₇ were found in a variety of tissues. In addition, most tissues contained relatively high concentrations of some other γ subunit, which was detected with an antibody against a γ₇-derived peptide and appeared to be γ_s1. Among cloned cells tested, γ₃ was detected only in PC12 pheochromocytoma cells. Taken together, the results indicated that γ₃ was expressed specifically in neuronal cells, and γ_s1 was the major γ subunit in most nonneural cells. γ₂, γ₅, and γ₇ were distributed in a variety of tissues, but γ₂ was dominant in the brain.     

Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

Genotypic analysis of families with lactate dehydrogenase A(M) deficiency by selective DNA amplification

Summary Genomic DNA prepared from LDH-A-deficient whole blood was amplified by the polymerase chain reaction technique using two primers specific for the active human LDH-A gene. The amplified fragment was examined by direct agarose gel electrophoresis, and a deletion of 20 base pairs (bp) in exon 6 of the LDH-A gene was found. The results permitted a clear distinction between the homozygous mutant, the heterozygous mutant, and wild-type genotypes. Moreover, HinfI digestion and direct sequencing of the amplified product confirmed the results from direct agarose gel electrophoresis. Four families, including 18 individuals, were shown to contain the same mutation, that is a 20-bp deletion in exon 6. All genotypes were consistent with their biochemical phenotypes as evaluated by the ratio of LDH-B to LDH-A subunits in erythrocytes. Thus, all four known affected families in Japan have been shown to carry the same mutant gene, which may have been derived from a single mutational event.     

Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k_cat / K_m = 0.19 and 0.0023 M^− 1 S^− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.     

Optical antisense imaging of tumor with fluorescent DNA duplexes

Antisense targeting of tumor with fluorescent conjugated DNA oligomers has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. When administered as a linear duplex of two fluorophore-conjugated oligomers arranged in a manner that inhibits fluorescence as the duplex and designed to dissociate only in the presence of the target mRNA, the fluorescence signal should in principle be inhibited everywhere except in the target cell. Optical imaging by fluorescence quenching using linear fluorophore-conjugated oligomers has not been extensively investigated and may not have been previously considered for antisense targeting. We evaluated in cell culture and in KB-G2 tumor bearing nude mice a 25-mer phosphorothioate (PS) anti- mdr1 antisense DNA conjugated with the Cy5.5 emitter on its 3' equivalent end and hybridized as a linear duplex with a shorter 18-mer phosphodiester (PO) complementary DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end. In serum environments, 90% of the DNA25-Cy5.5 fluorescence was inhibited immediately following addition of the cDNA18-BHQ3 and showed only slight loss of inhibition over 24 h at 37 degrees C. As evidence of antisense specific binding, when incubated with the DNA25-Cy5.5/cDNA18-BHQ3 duplex, the fluorescence was lower in KB-31 (Pgp +/-) cells compared to KB-G2 (Pgp++) cells, but when incubated with the control cDNA18-Cy5.5/DNA25-BHQ3 duplex in which the fluorophores were reversed, the fluorescence of both cell types was low. As further evidence of specific binding, the fluorescent intensity of total RNA from KB-G2 cells incubated with the study duplex showed evidence of dissociation and hybridization with the target mRNA. Furthermore, the fluorescence microscopy images of KB-G2 cells incubated with DNA25-Cy5.5 as the singlet or study duplex show that migration in both cases is to the nucleus. The animal studies were performed in mice bearing KB-G2 tumor in one thigh and receiving iv the study or control duplexes. The tumor/normal thigh fluorescence ratio was clearly positive as early as 30 min postinjection in the study mice and reached a maximum at 5 h. By contrast, much lower fluorescence was observed in mice receiving the control duplex at the same dosage. Fluorescence microscope imaging showed that the Cy5.5 fluorescence was much higher in tumor sections from the animal that had received the study rather than control duplex. Thus combining a fluorophore-conjugated antisense DNA with an inhibitor-conjugated shorter complementary cDNA inhibited fluorescence both in cell culture and in tumored animals except in the presence of the target mRNA. This proof of concept investigation of optical antisense targeting therefore suggests that further studies including optimization of this approach are appropriate.     

Population-Based Incidence Rates of Diarrheal Disease Associated with Norovirus,Sapovirus, and Astrovirus in Kenya

BackgroundDiarrheal diseases remain a major cause of mortality in Africa and worldwide. While the burden of rotavirus is well described, population-based rates of disease caused by norovirus, sapovirus, and astrovirus are lacking, particularly in developing countries.MethodsData on diarrhea cases were collected through a population-based surveillance platform including healthcare encounters and household visits in Kenya. We analyzed data from June 2007 to October 2008 in Lwak, a rural site in western Kenya, and from October 2006 to February 2009 in Kibera, an urban slum. Stool specimens from diarrhea cases of all ages who visited study clinics were tested for norovirus, sapovirus, and astrovirus by RT-PCR.ResultsOf 334 stool specimens from Lwak and 524 from Kibera, 85 (25%) and 159 (30%) were positive for norovirus, 13 (4%) and 31 (6%) for sapovirus, and 28 (8%) and 18 (3%) for astrovirus, respectively. Among norovirus-positive specimens, genogroup II predominated in both sites, detected in 74 (87%) in Lwak and 140 (88%) in Kibera. The adjusted community incidence per 100,000 person-years was the highest for norovirus (Lwak: 9,635; Kibera: 4,116), followed by astrovirus (Lwak: 3,051; Kibera: 440) and sapovirus (Lwak: 1,445; Kibera: 879). For all viruses, the adjusted incidence was higher among children aged <5 years (norovirus: 22,225 in Lwak and 17,511 in Kibera; sapovirus: 5,556 in Lwak and 4,378 in Kibera; astrovirus: 11,113 in Lwak and 2,814 in Kibera) compared to cases aged ≥5 years.ConclusionAlthough limited by a lack of controls, this is the first study to estimate the outpatient and community incidence rates of norovirus, sapovirus, and astrovirus across the age spectrum in Kenya, suggesting a substantial disease burden imposed by these viruses. By applying adjusted rates, we estimate approximately 2.8–3.3 million, 0.45–0.54 million, and 0.77–0.95 million people become ill with norovirus, sapovirus, and astrovirus, respectively, every year in Kenya.     

Opposing effects of circadian clock genes bmal1 and period2 in regulation of VEGF-dependent angiogenesis in developing zebrafish

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.     

Enzyme Aided Regioselective Acylation of Nucleosides

Abstract

Selective protection of the three hydroxyl groups of sugar moiety of nucleosides have been studied by enzyme-catalyzed esterification in organic solvents. Selectively protected products were obtained. The reaction provides an efficient method for selective protection of nucleosides.