Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56-kDa type-specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56-kDa gene sequence homologies, reactivities with type-specific monoclonal antibodies and polymerase chain reaction (PCR) using type-specific primer-pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56-kDa gene sequences, but distant from the known Karp types, such as Karp, JP-1 and JP-2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer-pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer-pair may serve to find this strain type in future studies.     

Effects of light and nutrients on plankton stoichiometry and biomass in a P-limited lake

A field enclosure experiment was performed over 12 weeks in a P-limited lake to test the hypothesis that light:nutrient balance affects pelagic communities by altering the C:P stoichiometry of seston and by influencing exudation of labile DOC by algae. Three levels of light intensity (ambient, 50% of ambient, 25% of ambient) were cross-classified with three levels of nutrients in a factorial design (n=2). Dissolved nutrient concentrations, seston C concentration and C:P ratios in small (<1 m) and larger (1–85 m) size fractions were monitored, along with chlorophyll a concentration, abundance of bacteria and protozoa, and biomass and P-content of macrozooplankton. Algal exudation of recently-fixed C into the dissolved pool was also measured at the end of the experiment in selected enclosures. Treatments had no effect on seston C concentration but reduction of light intensity significantly decreased whole seston and large (1–85 m) seston C:P ratios. However, the magnitude of these effects was modest and not likely to be ecologically significant. There were no effects of nutrient addition or light×nutrient interaction on seston stoichiometry. Algae tended to release a higher percentage of fixed C as DOC in high light enclosures but this difference was not statistically significant. There were no effects of treatments on the abundance of bacteria or protozoa but nutrient enrichment led to a statistically significant but generally modest increase in macrozooplankton biomass. No effects on zooplankton community composition or P-content were observed. Comparison of effect sizes and treatment variances indicated a high probability of type II error and thus our confidence in failing to reject the null hypothesis in most of the above cases was low. Thus, our data provide support for only some aspects of the light:nutrient hypothesis but more appropriate tests of the hypothesis should involve stronger treatments and/or increased replication in order to be better able to evaluate its validity.     

Formation of (R)-2,3-dihydrogeranylgeranoic acid from geranylgeraniol in rat thymocytes

In a metabolic study of [1-(14)C]geranylgeranial involving rat thymocytes, the radioactivity was mainly incorporated into two metabolites, Z1 and Z2, the latter moving slower than the former on a silica-gel thin-layer plate. The time course of Z1 and Z2 formation superficially suggested a precursor-product relationship between Z1 and Z2. The two metabolites were chemically converted to their methyl esters on treatment with trimethylsilyl diazomethane. Z1 was cochromatographed with E,E,E-geranylgeranoic acid (GGA). Z2 was prepared in a large quantity from geranylgeranial using thymocytes, and purified by TLC followed by ESI (negative ion mode) or EI mass-spectrometry. The observation of a negative ion at m/z 305 on ESI and a molecular ion at m/z 306 (C(20)H(34)O(2)) with fragments similar to GGA on EI implied that Z2 was dihydroGGA, which has been detected in the urine and serum of patients with Refsum disease. The EI mass spectrum of (R)-2,3-dihydroGGA was identical to that of Z2. The diastereomeric amide synthesized from metabolite Z2 with (R)-1-(1-naphtyl)ethylamine was cochromatographed with (R acid, R) amide, not with (S acid, R) amide, which were similarly synthesized from (R)- and (S)-2,3-dihydroGGAs, respectively. In another metabolic study on [1-(14)C]geranylgeraniol (GGOH), the radioactivity was similarly incorporated into a metabolite corresponding to (R)-2,3-dihydroGGA. (R)-2,3-DihydroGGA induced DNA ladder formation with a maximum at 15 mciroM in thymocytes. However, 2,3-dihydrofarnesoic acid did not induce it at all.     

Highly enantioselective asymmetric autocatalysis of 2-alkenyl- and 2-vinyl-5-pyrimidyl alkanols with significant amplification of enantiomeric excess

2-Alkenyl- and 2-vinyl-5-pyrimidyl alkanols are highly enantioselective asymmetric autocatalysts with significant amplification of enantiomeric excess in the enantioselective addition of diisopropylzinc to 2-alkenyl- and 2-vinylpyrimidine-5-carbaldehydes. Consecutive asymmetric autocatalysis starting from 7% ee increases the ee of pyrimidyl alkanol up to 99% without the need for any other chiral auxillary.     

Fluctuation of the zooplankton community in Lake Biwa during the 20th century: a paleolimnological analysis

Detailed zooplankton records from a 26-cm sediment core with a time resolution of approximately 3–10 years were obtained from Lake Biwa, Japan, to examine the historical variations in the zooplankton community during the 20th century. In the sediments, selected zooplankton remains have fluctuated over the years. Daphnia – large zooplankton herbivores – did not occur from 1900 to 1920, and formed a very minor component of the zooplankton community in the following 30 years, while Bosmina – small zooplankton herbivores – were common during this period. In the mid-1960s, however, when eutrophication was noticeable in this lake, Daphnia numbers increased dramatically and became the dominant zooplankton thereafter. In contrast, Difflugia brevicolla and D. biwae, two amoeboid protozoans that live in connection with the lake bottom environment, occurred abundantly until the late 1950s, but gradually decreased after the mid-1960s. In particular, D. biwae, a species peculiar to this lake, was not found in sediment dated after 1980, suggesting its extinction. These results indicate that the zooplankton community structure changed greatly in the 1960s, and suggest that the eutrophication occurring at this time altered the relative strength of top-down and bottom-up forces on the zooplankton community in Lake Biwa.     

Histidine-rich glycoprotein plus zinc reverses growth inhibition of vascular smooth muscle cells by heparin

Vascular smooth muscle cell (SMC) hyperplasia is known to be an important component in the pathogenesis of arteriosclerosis and restenosis. Although heparin has been well recognized as the representative molecule suppressing SMC growth in vitro, attempts to use heparin as a therapeutic anti-restenosis drug have not favorably influenced the angiographic or clinical outcome after angioplasty in some clinical trials. In this study, we have examined the effect of histidine-rich glycoprotein (HRG), a relatively abundant serum glycoprotein (~100 micrograms/ml in human serum), on the growth inhibition of cultured vascular SMC by heparin. Vascular SMC growth was significantly inhibited by heparin, giving nearly 85% inhibition with 100 micrograms/ml heparin. HRG reversed heparin-induced SMC growth inhibition in a dose dependent manner; 75% restoration of cell growth was observed when 100 micrograms/ml of HRG was co-added with 100 micrograms/ml heparin. Interestingly, micromolar concentrations of the zinc ion (0-10 microM), compatible with concentrations released from activated platelets, were found to enhance the restorative action of HRG. Western blot experiment demonstrated no significant amounts of the HRG moiety in fetal bovine serum, eliminating the possible contribution of contaminant HRG from culture media. These findings indicate that HRG, in combination with the zinc ion, plays a role in modulating the SMC growth response in pathophysiological states and explain the lack of success of heparin as a therapeutic anti-restenosis drug in clinical trials.     

Self-amplification system for recombinant adeno-associated virus production

A recently reported system for recombinant adeno-associated virus (rAAV) production does not require infection of a helper virus and depends on the transfection with a huge amount of three plasmids: AAV-vector, AAV-helper, and adenovirus-helper plasmids. Toward simplifying rAAV production, as a first step, we tested the use of the rAAV itself instead of the AAV-vector plasmid as a source of rAAV DNA and determined the optimal timing of infection and dose of the input rAAV. When 293 cells were infected just after transfection with 100 particles/cell of rAAV, irrespective of the purity, CsCl-purified or crude, up to 2000 particles/cell of rAAV were produced (9- to 20-fold self-amplification), a yield comparable to that obtained by an adenovirus-free transfection. These results indicate that infection of rAAV can greatly reduce the amount of plasmid DNA for a large-scale transfection. This strategy will also be useful when applied to packaging cell lines inducibly expressing Rep and Cap proteins.