Kinetic studies on the prenyl chain elongation by undecaprenyl diphosphate synthase with artificial substrate homologues

In the undecaprenyl diphosphate synthase reaction, an allylic substrate homologue, (2Z,6E,10E)-4-methyl-geranylgeranyl diphosphate was found to be a potent competitive inhibitor against the allylic primer, (2Z,6E,10E)-geranylgeranyl diphosphate. On the other hand, it acted as a strong noncompetitive inhibitor against isopentenyl diphosphate. On the basis of these facts, the topology of the substrate-binding sites as well as the reason why the synthase reaction with (E)-3-methyl-3-pentenyl diphosphate always stops completely at the first stage of condensation, yielding an allylic diphosphate with a methyl group at the 4-position, are discussed.     

Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.     

Polypeptides coded for by the region pre-S and gene S of hepatitis B virus DNA with the receptor for polymerized human serum albumin: expression on hepatitis B particles produced in the HBeAg or anti-HBe phase of hepatitis B virus infection

There are four polypeptides coded for by the region Pre-S and gene S on DNA of hepatitis B virus that carry the receptor for polymerized human serum albumin (poly-HSA), i.e., P31 and P39, as well as their glycosylated counterparts P35 and P43. With the use of monoclonal antibodies directed to Pre-S(1) sequence and Pre-S(2) sequence (bearing the receptor for poly-HSA), the content of these polypeptides, as well as their expression on the surface, was determined for hepatitis B particles of various categories. P39 and P43, carrying both Pre-S(1) and Pre-S(2) sequences, were contained abundantly in Dane and tubular particles, and to a much lesser extent in small spherical particles, all of which were purified from plasma containing hepatitis B e antigen (HBeAg). P31 and P35, carrying Pre-S(2) but not Pre-S(1) sequence, were contained comparably in these three categories of hepatitis B particles. In remarkable contrast, small spherical particles derived from plasma containing antibody to HBeAg were very low in the content of any Pre-S polypeptides. P31 and P39 showed higher activities for poly-HSA receptor than their glycosylated versions. When Dane particles were digested with trypsin, the poly-HSA receptor was deprived in parallel with the loss of antigenicity for Pre-S(2) sequence. The antigenicity for Pre-S(1) sequence was much less affected, and that for the product of gene S was virtually unchanged by the digestion.     

Biological activity assessment of the vitamin D metabolites 1,25-dihydroxy-24-oxo-vitamin D3 and 1,23,25-trihydroxy-24-oxo-vitamin D3

Two new metabolites of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], namely 1,25(OH)2-24-oxo-vitamin D3 and 1,23,25(OH)3-24-oxo-vitamin D3, have been prepared in vitro using chick intestinal mucosal homogenates. To investigate the binding of 1,25(OH)2-[23-3H]-24-oxo-D3 and 1,23,25(OH)3-[23-3H]-24-oxo-D3 to the chick intestinal receptor we have isolated both metabolites in radioactive form using an incubation system containing 1,25(OH)2-[23,24-3H))-D3 with a specific radioactivity of 5.6 Ci/mmol. Both metabolites were highly purified by using Sephadex LH-20 chromatography followed by high-pressure liquid chromatography (HPLC). Sucrose density gradient sedimentation analysis showed specific binding of both tritium-labeled metabolites to the chick intestinal cytosol receptor. Experiments were carried out to determine the relative effectiveness of binding to the chick intestinal mucosa receptor for 1,25(OH)2D3. The results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1,25(OH)2D3. Whereas the RCI obtained for 1,25(OH)2-24-oxo-D3 was 98 +/- 2 (SE), the RCI for 1,23,25(OH)3-24-oxo-D3 was only 28 +/- 6 (SE). Also, the biological activity of both new metabolites was assessed in vivo in the chick. In our assay for intestinal calcium absorption, 1,25(OH)2-24-oxo-D3 was active at a dose level of 1.63 and 4.88 nmol/bird (at 14 h), whereas 1,23,25(OH)3-24-oxo-D3 showed only weak biological activity in this system. In our assay for bone calcium mobilization, administration of both new metabolites showed modest activity at the 4.88-nmol dose level, which was reduced at the 1.63-nmol dose level. The results indicate that biological activity declines as 1,25(OH)2D3 is metabolized to 1,24R,25(OH)3D3, 1,25(OH)2-24-oxo-D3, and then 1,23,25(OH)3-24-oxo-D3.     

Mechanism of the stimulatory effect of cyclodextrins on lankacidin-producing Streptomyces

Summary Gel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that -CyD had strong, -CyD weak and -CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of l-[methyl-¹⁴C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of -CyD, less with -CyD and the least with -CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by -CyD and their production was not stimulated by -CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by -CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, -CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by -CyD or -CyD. -CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.The second report of the stimulatory effect of cyclodextrins on lankacidin fermentationOffprint requests to: H. Sawada     

Aspergillus taichungensis,a new species from Taiwan

Aspergillus taichungensis isolated from a soil sample collected in Taiwan is described as a new species. The new species is characterized by its restricted growth on Czapek's and malt extract agars and its white to light yellow colonies, radiate conidial heads, smooth and often diminutive conidiophores, hemispherical to elongate vesicles with biseriate aspergilla (conidiogenous cells), globose, micro-verrucose conidia and dark brown sclerotia. The species somewhat resemblesA. versicolor, A. terreus andA. flavipes, but differs in cultural and morphological details, and is considered to represent an interface species in the subgenusNidulantes.     

Materials for the fungus flora of Japan (49)

Two interesting microfungi are described as new to Japan:Talaromyces galapagensis (anam.Penicillium galapagense), isolated from soil in Shizuoka; andPenicillium megasporum, isolated from marine sludge in Nagasaki. Some observations are recorded, particularly on ascomatal initials ofT. galapagensis, which are similar to those described forTalaromyces flavus.(48): Kaneko, S., Mycoscience 36: 359–360, 1995.     

Talaromyces lagunensis,a new species from Philippine soil

A new species ofTalaromyces (Ascomycetes; Trichocomaceae) with aPenicillium anamorph,T. lagunensis, is described and illustrated. This fungus is characterized by its extremely restricted growh on Czapek-yeast extract agar, light yellow to light orange ascomata with a telaperidium, catenate, pyriform or ellipsoidal asci, ellipsoidal or subglobose ascospores with a microtuberculate wall, short conidiophores with an irregular, mostly monoverticillate to biverticillate penicillus, and subglobose to ovoid conidia. The holotype was isolated from forest soil in the Philippines.     

Chain length distribution of the products formed in solanesyl diphosphate synthase reaction.

Factors that affect the termination of isoprenoid chain elongation catalyzed by prenyltransferase were investigated. The chain-length distribution of reaction products of solanesyl diphosphate synthase [EC 2.5.1.11] homogeneously purified from Micrococcus luteus changed dramatically according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ (IPP-Mg) in the reaction mixture. However, the concentration of the complex between farnesyl diphosphate and Mg2+ (FPP-Mg), the priming substrate for this synthase, did not affect the product distribution, provided that the concentration of IPP-Mg was maintained at a certain level. Thus, the level of IPP-Mg is decisive in affecting the chain length distribution of the products of the prenyltransferase reaction, and the Mg(2+)-dependent variability of product specificity so far observed can now be understood in terms of the effect of IPP-Mg concentration.