Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Isolation of an antigenically unique methanogen from human feces

A methanogenic bacterium with the morphological and physiological properties of the genus Methanobrevibacter was isolated from the feces of a Japanese man who excreted methane in his breath. Indirect immunofluorescence staining revealed that the isolate had an antigenicity unrelated to that of any known members of the genus Methanobrevibacter.     

Expression in Escherichia coli and properties of the carotene ketolase from Haematococcus pluvialis

Abstract High level expression of the functional β-carotene ketolase gene bkt from Haematococcus pluvialis occurred in Escherichia coli transformants producing β-carotene or zeaxanthin as a result of the presence of additional carotenoid genes from Erwinia uredovora . Requirement of molecular oxygen for the insertion of the keto group was demonstrated. The final product of this two-step ketolase reaction from β-carotene is canthaxanthin (4,4'-diketo-β-carotene) with the 4-monoketo derivative echinenone as an intermediate. A reaction sequence for the formation of astaxanthin from β-carotene was established based on kinetic data on astaxanthin formation in E. coli transformants carrying the hydroxylase gene crtZ from Erwinia along with bkt . We conclude that the carotenoids zeaxanthin and adonixanthin which accumulate in addition to astaxanthin in this transformant are products of side reactions rather than direct precursors of astaxanthin. The possible mechanisms for the formation of the keto derivatives are discussed.     

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.     

Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.

Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential for viral replication. We also confirmed the importance of sites by using an HIV-1-infected animal model, the hu-PBL-SCID mouse system, representing HIV-1 replication and pathogenesis in activated CD4+ T cells in vivo. These results indicate that Nef accelerates viral replication in activated CD4+ T cells.     

Partial tetrasomy 9(9pter to 9q2101) due to an extra iso-dicentric chromosome.

A 3-year-old boy with partial No. 9 tetrasomy is described. The patient showed markedly retarded physical and mental development as well as multiple congenital anomalies. Routine chromosome analysis revealed an extra C-group chromosome. It had a pronounced secondary constriction at the proximal part of its long arm. Based on studies by a variety of banding techniques, the extra chromosome was identified to be an iso-dicentric No. 9 chromosome with inactivation of one of the two centromeres, the karyotype being 47,XY, + DIC (9)(Q2101). The value of BrdUrd treatment was emphasized in the detection of a very small piece of euchromatin within a long stretch of constitutive heterochromatin.     

Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora.

The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.     

Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.