[Gastric SWI/SNF complex deletion-associated undifferentiated carcinoma with rhabdoid phenotype: a clinicopathological and molecular analysis].

Objective: To investigate the clinicopathological features and molecular genetic characteristics of gastric SWI/SNF complex deletion-associated undifferentiated carcinoma with rhabdoid phenotype. Methods: Six cases of gastric SWI/SNF complex deletion-associated undifferentiated carcinoma with rhabdoid phenotype diagnosed at the Henan Cancer Hospital, Zhengzhou, China from January 2019 to December 2021 were collected. Histological observation, immunohistochemical staining, next-generation sequencing, and detection of mismatch repair (MMR), EBER, and HER2 were performed. The clinicopathological and molecular characteristics were summarized and relevant literatures were reviewed. Results: The 6 patients were all male, aged 48-75 years. Their initial symptoms mainly included abdominal pain, melena, and dysphagia. Endoscopic examinations showed gastric ulcer type masses, and the morphology of H&E were similar: the tumor cells showed diffuse infiltrating growth, no specific structural characteristics, obvious cell atypia, obvious mitoses, and rhabdomyoid cells with unequal proportions of eosinophilic cytoplasm. The immunohistochemistry for CKpan was negative in 3 of the 6 cases, while focal expression of other epithelial markers was found, including EMA (6/6), CK8/18 (4/6), and CK7 (1/6). P53 was diffusely strong positive in 4 cases (4/6), and negative in 1 case (1/6). Ki-67 was highly expressed (positive rate range, 60%-90%). Other related markers such as mesenchymal tumors, lymphoma, melanoma and germ cell tumors were all negative. Detection of the SWI/SNF complex subunit, namely INI1 (SMARCB1), BRG1 (SMARCA4), ARID1A protein detection, was detected in 5 cases with no SMARCA4 expression (5/6), 1 case with no ARID1A expression (1/6), and all cases with SMARCB1 expression (6/6). MMR proteins were examined, and dMMR was found in 1 of the 6 cases. HER2 expression was 0 in 3 cases, 1+ in 1 case, and 2+ in 2 cases, while no amplifications of HER2 gene were detected using FISH. EBER was negative in all 6 cases. Among the 4 cases of surgical radical treatment that were subject to next-generation sequencing, 3 cases showed TP53 mutations; 1 case showed ARID1A gene frame shift mutation, and there were also mutations of ATM, PTEN and other genes. There was 1 case with detected SMARCA4 gene copy number variant, and other gene mutations such as ALK, BRAF, CDKN1B, BRCA2, etc. Conclusions: Gastric SWI/SNF complex deletion-associated undifferentiated carcinoma with rhabdoid phenotype is a poorly differentiated and rare tumor. Detection of SWI/SNF complex related proteins is helpful for its diagnosis. Moreover, gene mutations associated with SWI/SNF complex will become a new indicator for its diagnosis and prognostication, and a potential new target for molecular therapy, which deserves more attention and warrants more research.

Lipid profiles as potential mediators linking body mass index to osteoporosis among Chinese adults: the Henan Rural Cohort Study.

This study is to examine the relationship between body mass index (BMI) and osteoporosis in rural areas of China, and then explore whether this association was mediated by lipid profiles. Results showed that with the increasing of BMI, the risk of osteoporosis was reduced, and lipid profiles mediate this association. INTRODUCTION: To examine the relationship between BMI and osteoporosis, and explore whether this association was mediated by lipid profiles. METHODS: A total of 8272 participants (18-79 years) were enrolled from the Henan Rural Cohort Study. The bone mineral density of the calcaneus was measured using an ultrasonic bone density apparatus. Logistic regression and restricted cubic splines were used to evaluate the odds ratio (OR) and 95% confidence intervals (95% CI). Mediation analysis using bootstrap was performed to examine the contribution of lipid profiles to BMI-related osteoporosis. RESULTS: The crude and age-standardized prevalence of osteoporosis were 15.93% and 11.77%, respectively. The mean BMIs were 24.12 kg/m2 for participants with osteoporosis and 25.06 kg/m2 for non-osteoporosis participants (P < 0.001). After adjusting for potential confounders, subjects with obesity had a lower OR of osteoporosis (0.493 [95% CI: 0.405-0.600], Ptrend < 0.001) compared with normal-weight individuals. Mediation analysis showed that lipid profile partly mediated the relationship between BMI and osteoporosis with indirect effect OR (95% CI) of 0.985 (0.978-0.992), and the proportion explained of BMI was 15.48% for lipid profile. CONCLUSION: With the increasing of BMI, the risk of osteoporosis was reduced in the Chinese adult population, and lipid profiles may be a potential mediator linking reduced risk of osteoporosis. Elucidating the underlying mechanisms will facilitate developing feasible preventive and therapeutic measures for osteoporosis. Chinese clinical trial register: ChiCTR-OOC-15006699.

[Effects of resuscitation by different formulation on oxygen metabolism during shock stage of burn injury in swine].

OBJECTIVE: To explore the effects of different fluid resuscitation regimens on oxygen metabolism during shock stage of burn injury in swine. METHODS: Twelve Bama miniature swines were divided into crystal and colloid group (Group 1) and Parkland group (Group 2) according to the random number table. The swine models of burns shock were established. The fluid resuscitation was begun at post injury hour (PIH) 2 according to Chinese formulation or Parkland's formulation, respectively. The blood pressure, urine volume, heart rate, central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP) were recorded. The liquid volume was calculated at the first and second PIH 24. The changes in oxygen delivery (DO2), oxygen consumption (VO2), oxygen extraction (O2Ext) and D-lactate (D-LA) were determined before injury and at PIH 4, 8, 24, and 48. Statistical analyses were performed. RESULTS: There were no statistical differences between the two groups in blood pressure, urine volume, heart rate, CVP, PCWP in every interval (all P>0.05). The resuscitation liquid volume in the two groups during the first and second PIH 24 conformed to the domestic consensus. The VO2 at PIH 8 was significantly higher than that of pre-burn in both groups [(190±29) vs (83±42) L·min(-1)·m(-2,) (149±33) vs (85±15) L·min(-1)·m(-2,) both P<0.05], and the VO2 at PIH 8 was significantly higher in Group 1 than that in Group 2 (P<0.05). The DO2 at PIH 24 in Group 1 was significantly lower than that in Group 2 [(686±72) vs (853±81) L·min(-1)·m(-2,) P<0.05]. There were no significant differences between the two groups in O2Ext at any time points (all P>0.05). The D-LA at PIH 8 was significantly higher in Group 1 than that in Group 2 [(53±4) vs (45±6) mmol/L, P<0.05]. CONCLUSION: There are no significant differences in the resuscitation effects of the crystal and colloid resuscitation regimen and Parkland's formulation on oxygen metabolism during shock stage of burn injury in swine.

Effect of ultraviolet A exposure on transport of compatible organic osmolytes in human lens epithelial cells.

Compatible organic osmolytes, such as betaine, myoinositol, and taurine, are involved in antioxidant defense, protein stabilization, and stress responses. This osmolyte strategy requires the expression of specific osmolyte transporters such as betaine (BGT-1), myoinositol (SMIT), and taurine (TAUT). In contrast to the kidney, keratinocytes, and neural cells, few studies have examined osmolytes in human lens epithelial cells (HLECs). We examined the expression of mRNA specific for BGT-1, SMIT, and TAUT in HLECs. In comparison to normoosmotic (305 mOsM) controls, there was a 3-5-fold time-dependent reaction of BGT-1, SMIT, and TAUT mRNA levels in HLECs exposed to hyperosmotic stress (405 mOsM). Maximal responses were obtained for BGT-1, SMIT, and TAUT mRNA expression after 3, 24 and 9 h of hyperosmotic exposure, respectively. This expression was correlated with increased osmolyte uptake. In contrast, hypoosmotic (205 mOsM) stimulation led to a significant efflux of osmolytes. Exposure to ultraviolet A (340-400 nm) radiation significantly stimulated osmolyte uptake. Increased osmolyte uptake was associated with upregulation of mRNA steady-state levels for osmolyte transporters in irradiated cells. These results demonstrate that ultraviolet A radiation leads to the accumulation of compatible organic osmolytes in HLECs as hyperosmotic pressure, which can maintain cellular environmental homeostasis.

[A consistency comparison between next-generation sequencing and the FISH method for gene rearrangement detection in B-cell lymphomas].

Objective: To compare the consistency of lymphoma multigene detection panels based on next-generation sequencing (NGS) with FISH detection of B-cell lymphoma gene rearrangement. Methods: From January 2019 to May 2023, fusion genes detected by lymphoma-related 413 genes that targeted capture sequencing of 489 B-cell lymphoma tissues embedded in paraffin were collected from Henan Cancer Hospital, and the results were compared with simultaneous FISH detection of four break/fusion genes: BCL2, BCL6, MYC, and CCND1. Consistency was defined as both methods yielding positive or negative results for the same sample. The relationship between fusion mutation abundance in NGS and the positivity rate of cells in FISH was also analyzed. Results: Kappa consistency analysis revealed high consistency between NGS and FISH in detecting the four B-cell lymphoma-related gene rearrangement (P<0.001 for all) ; however, the detection rates of positive individuals differed for the four genes. Compared with FISH, NGS demonstrated a higher detection rate for BCL2 rearrangement, a lower detection rate for BCL6 and MYC rearrangement, and a similar detection rate for CCND1 rearrangement. No correlation was found between fusion mutation abundance in NGS and the positivity rate of cells in FISH. Conclusions: NGS and FISH detection of B-cell lymphoma gene rearrangement demonstrate overall good consistency. NGS is superior to FISH in detecting BCL2 rearrangement, inferior in detecting MYC rearrangement, and comparable in detecting CCND1 rearrangement.

First Report of Burkholderia andropogonis Causing Bacterial Leaf Spot of Betel Palm in Hainan Province, China.

In May 2009, a severe bacterial disease of arecanut (Areca catechu L.) with an incidence of 100% was observed in a plantation of about 8,400 plants in Wenchang City, Hainan Province, China (19°47.171' N, 110°54.335' E). Symptoms consisted of small circular to elongated brown lesions, ranging from 1 to 105 mm in length and 1 to 21 mm in width, surrounded by yellow halos. White colonies, without fluorescent or diffusible pigments, were consistently recovered on King's B Medium plates from lesions surface-sterilized in 70% ethyl alcohol for 1 min. All isolates were gram-negative and each had a single, polar, sheathed flagellum. Isolates were identified as a Burkholderia sp. based on physiological and biochemical tests: oxidase and catalase positive, negative for arginine dihydrolase, gelatin hydrolysis and starch hydrolysis, and negative for acid production from levan (1,3). Sequences (approx. 1,400 bp each) of the 16S rRNA gene amplified from four isolates using primer pair 27F/1492R (2) (GenBank Accession Nos. JX415481, JX415479, JX415482, and JX415483) shared 99% sequence identity with that of Burkholderia andropogonis strain 6369 (DQ786951). Representative isolates Y11 (China General Microbiological Culture Collection Center No. CGMCC 1.12337), Y30 (CGMCC 1.12338), W15, and W20 were compared with B. andropogonis strain NCPPB No. 1012 and all caused a hypersensitive reaction on leaves of Nicotiana benthamiana. Isolate pathogenicity was tested twice with a total of three replications per isolate. Two young leaves each of 2-year-old arecanut plants were infiltrated with a bacterial suspension of 108 CFU/ml, then covered individually with plastic bags for 48 h, and incubated at 100% relative humidity with 16 h of daylight at 25°C by day and 8 h of darkness at 20°C by night. After 7 days, small water-soaked spots with yellow halos were observed and 60 days after inoculation, lesions developed similar to those caused by B. andropogonis in the field. Koch's postulates were fulfilled by reisolating bacteria from typical lesions on inoculated plants. These bacteria were identical to inoculated strains in colony morphology and sequences of the 16S ribosomal RNA gene. To our knowledge, this is the first report of B. andropogonis infection on betel in Hainan Province, mainland China. This disease was first reported in Taiwan, a province of China. Conditions of high humidity and high temperature support disease outbreaks and infection can result in severe economic losses. In 2012, this disease also appeared on a number of plantations located in other counties. As betel is, economically, the second most important crop in Hainan Province, measures should be required to control this disease, especially in typhoon seasons. References: (1) S. H. Hseu et al. Plant Pathol. Bull. 16:131, 2007. (2) D. J. Lane. In: E. Stackebrandt, et al. Nucleic acid techniques in bacterial systematics. John Wiley & Sons, Chichester, United Kingdom, pp. 115-175, 1991. (3) X. Li and S. H. De Boer. Plant Dis. 89:1132. 2005.

[Subcutaneous sparganosis: a case report].

This case report presents the diagnosis and treatment of a case with subcutaneous sparganosis.

Regulated tyrosine phosphorylation at the tips of growth cone filopodia.

Several types of evidence suggest that protein-tyrosine phosphorylation is important during the growth of neuronal processes, but few specific roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry using a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a polylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting material. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdraw rapidly from filopodia, suggesting that the protein associates or interacts with actin filaments. Phosphotyrosine has previously been found concentrated at adherens junctions, where bundles of actin filaments terminate, but video-enhanced contrast-differential interference contrast and confocal interference reflection microscopy demonstrated that the filopodial tips were not adherent to the substrate. Acute application of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed ends of actin filaments in a context other than an adherens junction, indicate an association between changes in phosphorylation and filament dynamics, and provide evidence for tyrosine phosphorylation as a signaling mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.

Antibody response to GD3 ganglioside is independent of NKT cells.

BACKGROUND: NKT cells recognize glycolipids presented by CD1d on antigen-presenting cells (APC) and have been largely characterized by their ability to be activated by alpha-galactosylceramide, a glycolipid not expressed on mammalian cells. We have shown previously that GD3 can be cross-presented by CD1d to NKT cells and is the first tumor-derived glycolipid recognized by NKT cells. But the ability of NKT cells to modulate B-cell responses to tumor glycolipids that are themselves recognized by NKT cells has not been explored. METHODS: We tested whether NKT cells are required for antibody (Ab) responses to GD3. We immunized wild-type mice, mice deficient in invariant chain NKT cells (iNKT cells) and mice deficient in total NKT cells against GD3. Ab titer against GD3 was measured by ELISA. RESULTS: We found the IgM and IgG responses against GD3 were similar among the three strains of mice, including the IgG isotypes induced. Pre-expanded NKT cells to GD3 did not affect the anti-GD3 Ab response. DISCUSSION: We conclude that Ab responses to GD3 are independent of NKT cells and that strategies to manipulate NKT cells in vivo are not likely to enhance the anti-GD3 Ab response induced by vaccines.

Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

Pioglitazone improves the ability of learning and memory via activating ERK1/2 signaling pathway in the hippocampus of T2DM rats.

OBJECTIVE: To explore the correlation between effect of PIO (pioglitazone, PIO) on learning as well as memory and ERK1/2 (extracellular signal regulated kinase 1/2, ERK1/2) pathway in T2DM (type 2 diabetes mellitus, T2DM) rats, further to elucidate the potential mechanism of PIO in improvement of learning and memory. METHODS: 12-week-old male SD rats (number of 10 per group) were randomly divided into control group (CON), T2DM group (DM) and T2DM +PIO group (DM+PG). Rats in DM and DM+PG groups were given high fat diet for 20 weeks, then treated with Streptozotocin (27mg/kg) by intraperitoneal injection at 21week. After 72h, the FBG (fasting blood glucose, FBG) was greater than 7.0mmol/L can considered T2DM rats. DM+PG group was treated with PIO (10 mg·kg-1·d-1) by gavage daily. After Hyperinsulinemic-Euglycemic Clamp Study and Morris water maze test at 30-week, all of animals were sacrificed. The expressions of RKIP (Raf-1 kinase inhibitor protein, RKIP) and ERK1/2 in hippocampus were detected using Western Blot and real-time PCR. RESULTS: The FBG level: DM group (7.68±0.54mmol/L) was higher than CON group (5.35±0.63mmol/L) and DM+PG group (6.07±0.84mmol/L), the differences were considered statistically significant (P <0.05). Hyperinsulinemic-Euglycemic Clamp Studies: GIR (glucose infusion rate, GIR) of DM group (21.02±5.10 mg·kg-1·d-1) was less than CON group (27.64±3.87 mg·kg-1·d-1) and DM+PG group (26.04 ±5.41 mg·kg-1·d-1), the differences were considered statistically significant (P <0.05). Morris water maze training: The escape latencies and searching platform performance of DM group (24.54±5.02s) decreased significantly compared with CON group (16.73±4.02s) and DM+PG group (18.05±4.12s) (P <0.05). Changes of RKIP, ERK, p-ERK protein relative content in rat hippocampus: Compared with CON groupand DM+PG group, the relative content of RKIP in DM group remarkably increased (P<0.01); ERK protein levels were not considered statistically significant among the three groups (P>0.05); The relative content of p-ERK1/2 protein in CON group and DM+PG group rats dorsal were higher than those in group DM, the difference was considered statistically significant (P<0.01). Changes in hippocampus of rat RKIP and ERK gene relative content: Compared with CON group and DM+PG group, levels of RKIP mRNA in DM group were significantly increased (P<0.01); ERK mRNA levels were not considered statistically significant among the three groups (P>0.05). CONCLUSION: Activation of ERK1/2 signal transduction pathway via reducing RKIP in the hippocampus may be one of the mechanisms of PIO to improve the learning and memory of the T2DM rats.

Systemically expressed soluble Tie2 inhibits intraocular neovascularization.

Retinal and choroidal neovascularization are the most frequent causes of severe and progressive vision loss. Studies have demonstrated that Tie2, an endothelial-specific receptor tyrosine kinase, plays a key role in angiogenesis. In this study, we determined whether adenovirus-mediated gene delivery of extracellular domain of the Tie2 receptor (ExTek) could inhibit experimental retinal and choroidal neovascularization. Immunofluorescence histochemistry with a monoclonal antibody to human Tie2 showed that Tie2 expression is prominent around and within the base of newly formed blood vessels of retinal and choroidal neovascular lesions. A single intramuscular injection of adenovirus expressing ExTek genes achieved plasma levels of ExTek exceeding 500 microg/ml in mice for 10 days (in neonates) and 7 days (in adults). This treatment inhibited retinal neovascularization by 47% (p < 0.05) in a murine model of ischemia-induced retinopathy. The same treatment reduced the incidence and extent of sodium fluorescein leakage from choroidal neovascular lesions by 52% (p < 0.05) and 36% (p < 0.01), respectively, in a laser-induced murine choroidal neovascularization model. The same mice showed a 45% (p < 0.001) reduction of integrated area of the choroidal neovascularization. These findings indicate that Tie2 signaling is a common component of the angiogenic pathway in both retinal and choroidal neovascularization, providing a potentially useful target in the treatment of intraocular neovascular diseases.

Specificity of the nick-closing activity of bacteriophage T4 DNA ligase.

Bacteriophage T4 DNA ligase effectively joins two adjacent, short synthetic oligodeoxyribonucleotides (oligos), as guided by complementary oligo, plasmid and genomic DNA templates. When a single bp mismatch exists at either side of the ligation junction, the efficiency of the enzyme to ligate the two oligos decreases. Mismatch ligation is approximately five-fold greater if the mismatch occurs at the 3' side rather than at the 5' side of the junction. During mismatch ligation the 5' adenylate of the 3' oligo accumulates in the reaction. The level of the adenylate formation correlates closely with the level of the mismatch ligation. Both mismatch ligation and adenylate formation are suppressed at elevated temperatures and in the presence of 200 mM NaCl or 2-5 mM spermidine. The apparent Km for the oligo template in the absence of salt is 0.05 microM, whereas the Km increases to 0.2 microM in the presence of 200 mM of NaCl. In this report, we demonstrate these properties of T4 DNA ligase for oligo pairs complementary to the beta-globin gene at the sequence surrounding the single bp mutation responsible for sickle-cell anemia. Because of the highly specific nature of the nick-closing reaction, ligation of short oligos with DNA ligase can be used to distinguish two DNA templates differing by a single nucleotide.

Glial environment in the developing superior colliculus of hamsters in relation to the timing of retinal axon ingrowth.

We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In contrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time retinotectal projections are being fully established. These raphe glia may be involved in maintaining the laterality of the retinotectal projection.

Abnormalities in the export and fate of recent thymic emigrants in diabetes-prone BB/W rats.

Abnormalities in postthymic T cell development in the BB/W rat model of autoimmune insulin-dependent diabetes mellitus (IDDM) result in part from a lymphopenia (lyp) gene defect. To better characterize these abnormalities, the phenotypes of T cells from diabetes-prone (DP) and diabetes-resistant (DR) coisogenic rats were analyzed by multiparameter flow immunocytometry (FCM). Marked decreases in the numbers of Thy1- RT6+ T cells, most of which are CD8+, were documented in DP rats by live-gating. Conversely, an approximately 3-fold increase was observed in the percentage of Thy1+ RT6- T cells, which normally serve as the precursors of both Thy1- RT6+ and Thy1- RT6- T cell subsets in rats. These results suggested that, at a minimum, an arrest in maturation of the Thy1+ precursors of RT6+ T cells occurs postthymically in DP rats. To determine more precisely the stage(s) in T cell development at which lymphopenia occurs, the export and fate of recent thymic emigrants (RTE's) and their immediate descendants in DP rats was traced after intrathymic (i.t.) labelling with fluorescein isothiocyanate (FITC). The results showed that in DP, as compared with DR, rats: 1) 5-fold fewer RTE's are exported from the thymus per 24 hr; 2) more than 80% of the RTE's are CD4+; 3) most of the immediate descendants of RTE's disappear from the peripheral lymphoid tissues within one week after export from the thymus; and 4) few of the descendants of the RTE's that do survive differentiate into RT6+ T cells. Staining with propidium iodide revealed that a significantly higher proportion of Thy1+ T cells in DP than in DR rats are in cycle (S/G2/M), thereby accounting for their disproportionately high numbers relative to RTE's. These results indicate that, in addition to defective thymic export, most of the immediate descendants of RTE's in DP rats undergo non-productive proliferation and death at the time (3-7 days postthymic) at which their counterparts in DR rats differentiate into Thy1- RT6+ T cells. The resulting deficiency of immunoregulatory T cells, acting in concert with defective intrathymic selection of effector T cell precursors, appears to conspire to markedly enhance the predisposition of DP rats to autoimmunity.

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.

Relationship between phenotypes of cell-function differentiation and pathobiological behavior of gastric carcinomas.

AIM: To reveal the correlation between the functional differentiation phenotypes of gastric carcinoma cells and the invasion and metastasis by a new way of cell-function classification. METHODS: Surgically resected specimens of 361 gastric carcinomas(GC) were investigated with enzyme-, mucin-, and tumor-related marker immunohistochemistry. According to the direction of cell-function differentiation, stomach carcinomas were divided into five functionally differentiated types. RESULTS: (1) Absorptive function differentiation type (AFDT): there were 82 (22.7%) patients including 76 (92.7%) aged 45 years. Sixty-nine (84.1%) cases belonged to the intestinal type. Thirty-eight (46.3%) expressed CD44v6 and 9 (13.6%) of 66 male patients developed liver metastasis. The 5-year survival rate of patients in this group (58.5%) was higher than those with the other types (P<0.01). (2) Mucin secreting function differentiation type (MSFDT): 54 (15%) cases. Fifty-three (98.1%) tumors had penetrated the serosa, 12 (22.2%) expressed ER and 22 (40.7%) expressed CD44v6. The postoperative 5-year survival rate was 28.6%. (3) Absorptive and mucin-producing function differentiation type (AMPFDT): there were 180 (49.9%) cases, including 31 (17.2%) aged younger than 45 years. The tumor was more common in women (62, 34.4%,) and expressed more frequently estrogen receptors (ER) (129, 81.7%) than other types (P<0.01). Ovary metastasis was found in 12 (19.4%) out of 62 female subjects. The patients with this type GC had the lowest 5-year survival rate (24.7%) among all types. (4) Specific function differentiation type (SFDT): 13 (3.6%) cases. Nine (69.2%) tumors of this type derived from APUD system, the other 4 (30.7%) were of different histological differentiation. Sixty per cent of the patients survived at least five years. (5) Non-function differentiation type (NFDT): 32 (8.9%) cases. Nineteen (59.4%) cases had lymph node metastases but no one with liver or ovary metastasis. The 5-year survival rate was 28.1%. CONCLUSION: This new cell-function classification of GC is helpful in indicating the characteristics of invasion and metastasis of GC with different cell-function differentiation phenotypes. Further study is needed to disclose the correlation between the cell-functional differentiation phenotypes and the relevant genotypes and the biological behavior of gastric carcinoma.

Computed tomography and magnetic resonance imaging of diffuse pigmented villonodular synovitis: report of a case.

We describe the computed tomographic (CT) and magnetic resonance imaging (MRI) features of a surgically proven case of diffuse pigmented villonodular synovitis (PVNS) of the knee in a 34-year-old woman. A complex mass consisting of solid and multicystic components was clearly demonstrated by CT and MRI. The solid part showed homogeneous hypodense attenuation relative to adjacent muscles on CT scans, but it showed inhomogeneous signal intensity on spin echo T1- and T2-weighted images (WI). The solid component enhanced homogeneously on CT scans but heterogeneously on MR images. Multiseptated enhancement of the cystic component on both CT and MR images were displayed. All of the above features were better demonstrated on MRI. Multiple marked hypointense round and patchy areas, and also a few areas isointense to subcutaneous fat within the lesion were also found on T1WI, proton density WI and T2WI. These characteristic MRI features of PVNS correlated well with its histologic structures: depositions of hemosiderin and fat in the proliferative synovial villi and bloody cystic content. These features may help to distinguish PVNS from other disease entities arising from the synovium.