Human papillomavirus infection status of single cells isolated from cervical cytology specimens by simple manual microdissection.

Human papillomavirus (HPV) testing with cytology triage for cervical cancer screening has proven to be useful. It is considered that a significant percentage of HPV-positive women followed by reflex cytology have had multiple-type HPV infections rather than single-type infections. However, the effects of multiple-type infections on changes in the cytomorphology of exfoliated cervical cells have not been investigated. The aim of this study was to validate simple manual microdissection (MMD) maneuver and investigate the HPV infection status of single cells isolated from Papanicolaou (Pap) smears prepared from women with multiple-type infections. Using cytology samples from 90 patients with abnormal Pap smear results, we evaluated the efficiency of the MMD procedure and determined the HPV infection status of single squamous intraepithelial lesion (SIL) cells microdissected from patients with multiple-type infection. When validating the MMD procedure, the HPV-positive rate was 81.5% using 119 MMD samples from the Pap smear in 61 cases with single-type infection. This MMD procedure was able to efficiently collect single cells. Of 119 MMD samples from 29 cases with multiple-type infection, the HPV-positive rate was 42.9%, and most (96.1%) MMD samples exhibited only one genotype. Our MMD maneuver successfully identified HPV genotypes using single cells isolated from cytology specimens. A majority of single SIL cells prepared from multiple-type infection cases turned out to contain only one genotype. In the future, the MMD method could be applied while studying the relationship between the morphological changes exhibited by SIL cells on Pap smear and the infected HPV genotype.

Koilocytic changes are not elicited by human papillomavirus genotypes with higher oncogenic potential.

Koilocytes are considered a common cytopathological effect in patients with human papillomavirus (HPV) infection. Thus, we aimed to elucidate whether koilocytes are common to all HPV infections. Liquid-based cytology samples from 651 patients with abnormal Papanicolaou (Pap) test results were used to analyze the presence of koilocytes and HPV genotype. HPV genotype was determined in complete liquid cytology samples and microdissected cell samples from Pap smear slides using the uniplex E6/E7 polymerase chain reaction method, which can detect 39 mucosal HPV genotypes. Koilocytes were found in 29.3% (191) of all patients. Logistical regression analysis of diverse HPV genotypes revealed that infections with low-risk HPV types (HPV-6b, HPV-40, HPV-42, HPV-61, HPV-74, HPV-89, and HPV-90), probably high-risk HPV types (HPV-53 and HPV-66), and high-risk types (HPV-39 and HPV-56) were significantly associated with the presence of koilocytes. However, HPV-16, HPV-18, and HPV-52, which have higher oncogenic potential, were not found to be associated with koilocytes. These results were confirmed by HPV genotyping using microdissected koilocytes in 27 patients.Most common high-risk types belonging to α-9 and α-7 genotypes appear to rarely induce koilocytic changes. Therefore, koilocytes may provide additional useful information for predicting the risk of progression to high-grade lesions.

Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-ionization tandem mass spectrometry.

Pyrroloquinoline quinone (PQQ) is believed to be an important factor for mammalian growth and development and has, therefore, been declared a vitamin by some researchers. However, this issue remains controversial, and from a nutritional viewpoint, accurate determination of PQQ levels in a variety of foods is very important. Here, we describe a simple, highly sensitive, and highly selective method for quantitative analysis of PQQ. Liquid foods or aqueous extracts of solid foods were analyzed using high-performance liquid chromatography (HPLC) combined with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS). (15)N-labeled PQQ was added to the samples as an internal standard. Quantitative analyses of PQQ were performed by multiple reaction monitoring (MRM) with LC/MS/MS. Free PQQ was detected in almost all food samples in the range 0.19-7.02 ng per g fresh weight (for solid foods) or per mL (liquid foods). This method will enable the rapid and simple determination of PQQ levels in many samples.

A 9-cis-epoxycarotenoid dioxygenase inhibitor for use in the elucidation of abscisic acid action mechanisms.

The plant hormone abscisic acid (ABA) accumulates in response to drought stress and confers stress tolerance to plants. 9-cis-Epoxycarotenoid dioxygenase (NCED), the key regulatory enzyme in the ABA biosynthesis pathway, plays an important role in ABA accumulation. Treatment of plants with abamine, the first NCED inhibitor identified, inhibits ABA accumulation. On the basis of structure-activity relationship studies of abamine, we identified an inhibitor of ABA accumulation more potent than abamine and named it abamineSG. An important structural feature of abamineSG is a three-carbon linker between the methyl ester and the nitrogen atom. Treatment of osmotically stressed plants with 100 microM abamineSG inhibited ABA accumulation by 77% as compared to the control, whereas abamine inhibited the accumulation by 35%. The expression of AB A-responsive genes and ABA catabolic genes was strongly inhibited in abamineSG-treated plants under osmotic stress. AbamineSG is a competitive inhibitor of the enzyme NCED, with a K(i) of 18.5 microM. Although the growth of Arabidopsis seedlings was inhibited by abamine at high concentrations (>50 microM), an effect that was unrelated to the inhibition of ABA biosynthesis, seedling growth was not affected by 100 microM abamineSG. These results suggest that abamineSG is a more potent and specific inhibitor of ABA biosynthesis than abamine.