Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease.

Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection.     

The Arabidopsis dwarf1 mutant is defective in the conversion of 24-methylenecholesterol to campesterol in brassinosteroid biosynthesis

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2), constitutive photomorphogenesis and dwarfism (cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22alpha-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism.     

Genome analysis of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19

Previously the development of a hyper acetone‐butanol‐ethanol (ABE) producing Clostridium acetobutylicum BKM19 strain capable of producing 30.5% more total solvent by random mutagenesis of its parental strain PJC4BK, which is a buk mutant C. acetobutylicum ATCC 824 strain is reported. Here, BKM19 and PJC4BK strains are re‐sequenced by a high‐throughput sequencing technique to understand the mutations responsible for enhanced solvent production. In comparison with the C. acetobutylicum PJC4BK, 13 single nucleotide variants (SNVs), one deletion and one back mutation SNV are identified in the C. acetobutylicum BKM19 genome. Except for one SNV found in the megaplasmid, all mutations are found in the chromosome of BKM19. Among them, a mutation in the thlA gene encoding thiolase is further studied with respect to enzyme activity and butanol production. The mutant thiolase (thlA^V5A) is showed a 32% higher activity than that of the wild‐type thiolase (thlA^WT). In batch fermentation, butanol production is increased by 26% and 23% when the thlA^V5A gene is overexpressed in the wild‐type C. acetobutylicum ATCC 824 and in its derivative, the thlA‐knockdown TKW‐A strain, respectively. Based on structural analysis, the mutation in thiolase does not have a direct effect on the regulatory determinant region (RDR). However, the mutation at the 5^th residue seems to influence the stability of the RDR, and thus, increases the enzymatic activity and enhances solvent production in the BKM19 strain.     

Brassinosteroid biosynthesis and inactivation

The term brassinosteroids (BRs) refers to the growth-promoting plant steroidal hormones. Various developmental programs including but not limited to cell elongation, stress tolerance, and skoto-/photo-morphogenesis are controlled by subnanomolar concentrations of BRs. Accordingly, BR mutants that are defective in BR biosynthetic or signaling pathways usually display dwarfism. Characterization of numerous BR dwarf mutants isolated from Arabidopsis , pea, tomato, and rice greatly contributed to our understanding of BR biology. Recently, an enzyme that mediates the final step in the BR biosynthetic pathways has been characterized by two different groups. The brassinolide synthases (Cytochrome P450s 85A2 and 85A3) are multifunctional enzymes that catalyze the last three consecutive steps in BR biosynthetic pathways, namely, C-6 hydroxylation, dehydrogenation, and Baeyer-Villiger type oxidation. In addition, many of the previously unknown steps have been genetically characterized. This review aims to summarize the knowledge that has been developed during the last 2–3 years in this field of BR biosynthesis and inactivation research.     

Adaptation of human immunodeficiency virus type 1 to cells expressing a binding-deficient CD4 mutant (lysine 46 to aspartic acid).

Human immunodeficiency virus (HIV-1) was adapted to replicate efficiently in cells expressing an altered form of the CD4 viral receptor. The mutant CD4 (46 K/D) contained a single amino acid change (lysine 46 to aspartic acid) in the CDR2 loop of domain 1, which results in a 15-fold reduction in affinity for the viral gp120 glycoprotein. The ability of the adapted virus to replicate in CD4 46 K/D-expressing cells was independently enhanced by single amino acid changes in the V2 variable loop, the V3 variable loop, and the fourth conserved (C4) region of the gp120 glycoprotein. Combinations of these amino acids in the same envelope glycoprotein resulted in additive enhancement of virus replication in cells expressing the CD4 46 K/D molecule. In cells expressing the wild-type CD4 glycoproteins, the same V2 and V3 residue changes also increased the efficiency of replication of a virus exhibiting decreased receptor-binding ability due to an amino acid change (aspartic acid 368 to glutamic acid) in the gp120 glycoprotein. In neither instance did the adaptive changes restore the binding ability of the monomeric gp120 glycoprotein or the oligomeric envelope glycoprotein complex for the mutant or wild-type CD4 glycoproteins, respectively. Thus, particular conformations of the gp120 V2 and V3 variable loops and of the C4 region allow postreceptor binding events in the membrane fusion process to occur in the context of less than optimal receptor binding. These results suggest that the fusion-related functions of the V2, V3, and C4 regions of gp120 are modulated by CD4 binding.     

Photoprotective effect of kinetin on pigment content and photochemical activities of wheat chloroplasts agingin vitro

The effects of kinetin (Kn) on pigment content and electron transport activities (ETA) in wheat leavesin vivo and chloroplastsin vitro aging in light was investigated. Excised wheat leaves were infiltrated with Kn for 3 h under irradiation. The treatment increased zeaxanthin (Zx) content by 40% and also increased chlorophyll (Chia, Chib) and major carotenoid (Car) contents in the leaves (per fresh mass unit). Chloroplasts isolated from Kn treated leaves, when incubated in light for 4 h showed relatively lower pigment loss and slower loss of ETA compared to the chloroplasts of untreated leaves. These observations suggest photoprotective action of Kn. The photoprotection was more prominent when Kn was applied directly to the irradiated chloroplastsin vitro. Moreover, chloroplasts agingin vitro under irradiation without Kn treatment lost pigments and ETA. Within 3 h of irradiation, both whole chain (H₂O to methylviologen) electron transport as well as photosystem (PS) 2 activity were completely lost. However, in the chloroplasts treated with Kn, the loss of pigments was slow and even after 4 h of irradiation the chloroplasts retained 15 % of PS 2 and 9 % of whole chain ETA. In the untreated chloroplasts, the loss of Zx after 4 h of irradiation was 49 % whereas in Kn treated samples its level was 1.3 times higher than that of control. Since a higher level of Zx was maintained in Kn treated chloroplasts, photoprotective action of Kn is possibly mediated through Zx. One of us (NKC) thanks Sambalpur University for study leave and Department of Biological Sciences, Mankato State University, Mankato for labortory facilities.     

Disruption of phospholipid asymmetry in erythrocyte vesicles deficient in spectrin

The transbilayer distribution of phospholipids in right-side-out and inside-out vesicles derived from human erythrocytes was studied by phospholipase A2 digestion assays and by staining with the fluorescent dye merocyanine 540. In both types of vesicles, the normal asymmetric distribution of phospholipids characteristic of intact cells was disrupted. Because both types of vesicles are deficient in spectrin, the major protein of the cytoskeletal network which normally underlies the membrane, these results support the contention that spectrin is involved in the maintenance of phospholipid asymmetry.     

Signaling pathways implicated in alpha-melanocyte stimulating hormone-induced lipolysis in 3T3-L1 adipocytes

Melanocortins, besides their central roles, have also recently been reported to regulate adipocyte metabolism. In this study, we attempted to characterize the mechanism underlying alpha-melanocyte-stimulating hormone (MSH)-induced lipolysis, and compared it with that of the adrenocorticotrophin hormone (ACTH) in 3T3-L1 adipocytes. Similar to ACTH, MSH treatment resulted in the release of glycerol into the cell supernatant. The activity of hormone-sensitive lipase, a rate-limiting enzyme, which is involved in lipolysis, was significantly increased by MSH treatment. In addition, a variety of kinases, including protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) were also phosphorylated as the result of MSH treatment, and their specific inhibitors caused a reduction in MSH-induced glycerol release and HSL activity, indicating that MSH-induced lipolysis was mediated by these kinases. These results suggest that PKA and ERK constitute the principal signaling pathways implicated in the MSH-induced lipolytic process via the regulation of HSL in 3T3-L1 adipocytes.