Mutagenesis studies with four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: G->T mutations dominate in each case

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts. Four stereoisomers of B[a]P-N2-dG (designated: [+ta]-;, [+ca]-, [-ta] and [-ca]) were studied by NMR in duplex 11mers in a 5'-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G-->T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [-ta] and [-ca]-B[a]P-N(2)-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P-N2-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5'-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P-N2-dG stereoisomers in E.coli reveals that B[a]P-N2-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G-->T versus G-->A mutations) than does DNA sequence context, which is discussed.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Supradiaphragmatic ectopic adrenocorticotropic hormone-secreting adenoma

A 22-year-old woman with Cushing's syndrome, caused by an extremely rare suprasellar ectopic pituitary adenoma, is presented. Magnetic resonance imaging and computed tomography revealed a well-circumscribed mass in the right suprasellar region. Endocrinological tests showed elevated s-adrenocorticotropic hormone level and hypercortisolemia. The tumor was totally removed by right subfrontal approach. At the time of the operation, the tumor was in continuity with the distal pituitary stalk but not with the pituitary gland. The diaphragma sellae was intact. Histologic diagnosis of the tumor specimen was confirmed as a pituitary adenoma. After surgical removal of the tumor, continued improvement in the patient's laboratory results and disappearance of her endocrine symptoms strongly indicated the absence of adenoma cells in the pituitary gland or stalk. Six years post-surgery, there was no evidence of recurrence in the patient's clinical and laboratory examination. This tumor probably originated from aberrant anterior pituitary cells of the pituitary stalk.     

PD-1-Expressing SARS-CoV-2-Specific CD8+ T Cells Are Not Exhausted,but Functional in Patients with COVID-19

1. Download : Download high-res image (218KB)
2. Download : Download full-size image

     

Clear cell sarcoma of the kidney--a case report

Clear cell sarcoma of the kidney (CCSK) is a highly malignant childhood tumor, distinguished from classic Wilms' tumor by its propensity to metastasize to the skeletal system. Authors described a case of CCSK from a 3-year-old boy in the right kidney, showing various histologic features, such as classic, epithelioid, trabecular, neurilemmoma-like, cystic and entrapped collecting tubular pattern. Ultrastructurally epithelial differentiation was absent. Immunohistochemically, none of the intrinsic tumor cells showed positive staining with the antibodies against the keratin, S-100 protein, carcinoembryonic antigen, vimentin, desmin and myoglobin, suggesting primitive mesenchymal cell in origin.     

Microcystic adenomas of the pancreas. Report of three cases with two of multicentric origin

Three cases of microcystic adenomas of the pancreas with special reference to multicentric origin are described. The gross features and light microscopic findings were consistent with those described as being microcystic adenomas, but in two cases the gross examination and gelatin-embedded giant slices revealed multiple, isolated development of tumors ranging from submacroscopic nodules to tumors 4.5 cm in diameter. The larger tumors often showed ragged margins with small satellite nodules around the masses. A central fibrolamellar stellate core with centrifugally radiating septation was found in most of the tumor masses, even in the smaller ones. Ultrastructural and immunohistochemical findings revealed a single row of glycogen-rich epithelial cells, but participation of myoepithelial cells was not confirmed. Instead, vimentin-positive cells (pericytes) within the interstitial space incorporated closely with the basal lamina of the cyst wall. This study suggests that a small percentage of microcystic adenomas of the pancreas develop in multiple tumors, and some appear as a single tumor by their confluence.     

Estrogen affects vascular tone differently according to vasoactive substances in ovariectomized Sprague-Dawley rat

The favorable effects of estrogen on cardiovascular diseases can be explained by several mechanisms such as changes in serum lipid profiles and thrombogenecity. Estrogen also affects the vascular tone, but there has been no report in which the effect of estrogen was tested comprehensively for several vasoactive substances, especially after long-term administration. Two weeks after bilateral ovariectomy in 8-week old female Sprague-Dawley rats, placebo or 17 beta-estradiol (E2) pellets (0.5 mg; released over 3 weeks) were implanted subcutaneously. Two weeks after pellet implantation, organ chamber experiments were performed using aortae. Compared with control, E2-treated vessels showed impaired endothelium-dependent relaxation to acetylcholine. E2 enhanced the contraction to norepinephrine and U46619 and had no effect on endothelin-1-induced contraction. In contrast, the contraction to angiotensin (AT)-II was inhibited by E2. Northern blot analysis for AT1 receptor expression using cultured aortic smooth muscle cells showed no difference between control and E2-treated cells, suggesting that AT1 receptor downregulation is not the likely mechanism. These results suggest that E2 affects the vascular tone variably according to vasoactive substances.     

Expression of mucin genes in the human testis and its relationship to spermatogenesis

In this study we investigate the expression pattern of mucin genes in the human testis and evaluate the relationship between the expression of mucin genes and impaired spermatogenesis in the human testis. Thirty human testis tissues were collected from patients undergoing diagnostic testicular biopsy to investigate the cause of infertility. One part of the tissue underwent histological observation, and the other part of the tissue was subjected to semiquantitative RT-PCR of mucin genes, that is, mucin1, 2, 3, 4, and 9. The relative amount of mucin mRNAs was calculated by densitometry using glyceraldehydes-3-phosphate dehydrogenase (GAPDH) as an internal control. The samples were histologically diagnosed as either obstructive azoospermia with normal spermatogenesis (n = 13) or non-obstructive azoospermia with impaired spermatogenesis (n = 17). In the human testis with normal spermatogenesis, mRNA expression of mucin1, 9, 13 and GAPDH were found, but RT-PCR products of mucin 2, 3 and 4 were not detected. In the testis with impaired spermatogenesis, however, RT-PCR product of mucin1 was not found. There was no difference in the other mucin mRNA expression patterns between the testis with either normal or impaired spermatogenesis. To our knowledge, this study is the first that has detected the mRNA of mucin9 and 13 in human testis. This study also shows that mucin1 expression might be closely related to spermatogenesis. Our findings should be substantiated by more direct evidence, such as mucin protein expression and localization.     

Continuous measurements of Na, Li, and Cl in the perfused salivary gland by use of NMR

The present study was undertaken to measure tissue contents of Na, Li, and Cl non-invasively in the isolated perfused organ by nuclear magnetic resonance (NMR) using a broadband tunable probe. The NMR signals of 23Na, 7Li, and 35Cl from the isolated perfused rat mandibular gland were collected continuously and each spectrum was obtained for every 15 or 20 s. The Na concentration in the perfusate was varied by replacement with Li, and the resulting changes were monitored by measuring the signal intensities of the electrolytes. The time constant for Na exchange was slower following complete removal of extracellular Na than following its half replacement, suggesting that the Na extrusion by Na+/K+ ATPase was reduced by lowering the extracellular Na level. The time constant for Li exchange was slower than that for Na exchange. The level of Cl was nearly constant during experiment, except for a very slow increase in Cl, possibly resulting from increasing edema and/or intracellular Li storage.     

Different corticostriatal projections from two parts of the cortical masticatory area in the rabbit

The cortical masticatory area (CMA) elicits rhythmic jaw movements in response to repetitive stimulation and is involved in the control of mastication. Based on jaw movement patterns, the CMA is divided into two parts. One is the part of the CMA in which a T-pattern similar to jaw movements during food transport in natural mastication is evoked by electrical stimulation. The other is more dorsomedially located, and during chewing a C-pattern similar to jaw movements can be induced. However, it is still not known which region of the putamen receives projections from the CMA and whether projections originate from both parts of the CMA. In this study, electrophysiological and histological experiments were undertaken in rabbits to investigate projections from the CMA to the putamen. Both experiments showed that the ventral region of the putamen received projections from the CMA. The density of the projections from the CMA area inducing the T-pattern seemed to be higher than that from the area inducing the C-pattern. Furthermore, the peak latency of the evoked potentials from stimulation of the CMA area inducing the T-pattern was shorter than that from stimulation of the area inducing the C-pattern. The data obtained from the present study indicate the functional role of the ventral region of the putamen in the regulation of mastication, and further suggest that the corticostriatal pathway is involved in the transition between behavioral jaw movement patterns.