Primary vulvar Ewing's sarcoma/primitive neuroectodermal tumor in a post-menopausal woman: A case report

Ewing's sarcomas/peripheral primitive neuroectodermal tumors (ES/pPNETs) are high-grade malignant neoplasms rarely found outside the skeletal system. Only 12 cases of vulvar ES/pPNET have so far been reported, all involving children or women of child-bearing age. We describe the case of a 52-year-old woman who was admitted to our hospital for the local excision of a 4 cm vulvar mass, originally thought to be a Bartholin's gland cyst. It was subsequently found to consist of small round cells positive for anti-CD99 antibody, thus suggesting a diagnosis of ES/pPNET. The demonstration of EWSR1 gene translocations by means of fluorescent in situ hybridization excluded small-cell carcinoma, squamous cell carcinoma of the small type, Merkel cell carcinoma, and lymphoblastic lymphoma. After surgery, the patient received six cycles of polychemotherapy and radiotherapy; she is still alive and well after 1 year of follow-up. Our findings underline the crucial role of molecular biology techniques in the differential diagnosis of small round cell tumors in these unusual locations.     

Doxorubicin-loaded human serum albumin nanoparticles surface-modified with TNF-related apoptosis-inducing ligand and transferrin for targeting multiple tumor types

Human serum albumin (HSA) nanoparticles (NPs) surface modified with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and transferrin, and containing doxorubicin were designed and prepared. Surface amines of HSA were reversibly protected with dimethylmaleic anhydride (DMMA), and HSA-NPs were prepared using a desolvation technique. Furthermore, the surfaces of HSA-NPs were modified with thiolated TRAIL or transferrin using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The prepared TRAIL/transferrin plus doxorubicin HSA-NPs were characterized by TEM, FE-SEM, and particle size analysis, and their cytotoxic and apoptotic activities were evaluated in several cancer cell lines, namely, HCT 116, doxorubicin-resistant MCF-7, and CAPAN-1. In addition, the tumor-targeting abilities of NPs were assessed using an infrared imaging system in HCT 116-xenografted nu/nu mice. Results showed that the TRAIL/transferrin/doxorubicin HSA-NPs had remarkable cytotoxic and apoptotic activities in all cancer cells examined with a general or a drug-resistant character, and that these NPs had obvious synergistic cytotoxic effects particularly on CAPAN-1 cells. Moreover, these HSA-NPs were effectively localized to tumors in a HCT 116-xenografted nu/nu mouse over 32 h. The findings of this study suggest that the described TRAIL/transferrin/doxorubicin HSA-NPs are a useful targeting agent capable of killing different types of tumor cells in various tissue organs.     

Hyaluronic acid-modified hydrothermally synthesized iron oxide nanoparticles for targeted tumor MR imaging

We report a polyethyleneimine (PEI)-mediated approach to synthesizing hyaluronic acid (HA)-targeted magnetic iron oxide nanoparticles (Fe₃O₄ NPs) for in vivo targeted tumor magnetic resonance (MR) imaging applications. In this work, Fe₃O₄ NPs stabilized by PEI were first synthesized via a one-pot hydrothermal method. The formed PEI-stabilized Fe₃O₄ NPs were then modified with fluorescein isothiocyanate (FI) and HA with two different molecular weights to obtain two different Fe₃O₄ NPs (Fe₃O₄–PEI–FI–HA_6K and Fe₃O₄–PEI–FI–HA_31K NPs) with a size of 15–16 nm. The formed HA-modified multifunctional Fe₃O₄ NPs were characterized via different techniques. We show that the multifunctional Fe₃O₄ NPs are water-dispersible and colloidal stable in different aqueous media. In vitro cell viability and hemolysis studies reveal that the particles are quite cytocompatible and hemocompatible in the given concentration range. Furthermore, confocal microscopy and flow cytometry data demonstrate that HA-targeted Fe₃O₄ NPs are able to be uptaken specifically by cancer cells overexpressing CD44 receptors, and be used as efficient probes for targeted MR imaging of cancer cells in vitro and xenografted tumor models in vivo. With the tunable amine-based conjugation chemistry, the PEI-stabilized Fe₃O₄ NPs may be functionalized with other biological ligands or drugs for diagnosis and therapy of different biological systems.     

Serotonergic mechanisms are necessary for central respiratory chemoresponsiveness in situ

Evidence from in vivo and in vitro experiments conclude that serotonin (5-HT) neurons are involved in and play an important role in central respiratory CO₂/H⁺ chemosensitivity. This study was designed to assess the importance of 5-HT neurons and 5-HT receptor activation in the frequency and amplitude components of the hypercapnic response of the respiratory network in the unanesthetized perfused in situ juvenile rat brainstem preparation that exhibits patterns of phrenic nerve discharge similar to breathing in vivo. Exposure to a hypercapnic perfusate increased phrenic burst frequency and/or amplitude, the neural correlates of breathing frequency and tidal volume in vivo. Hypercapnic responses were also assessed during exposure to ketanserin (5-HT₂ receptor antagonist), and 8-OH-DPAT (inhibiting 5-HT neurons via 5-HT_1A autoreceptors). Neither of these drugs substantially altered baseline activity, however, both abolished hypercapnic responses of the respiratory network. These data illustrate that 5-HT neurons and 5-HT receptor activation are not required for respiratory rhythm generation per se, but are critical for CO₂ responses in situ, supporting the hypothesis that 5-HT neurons play an important role in central ventilatory chemosensitivity in vivo.     

FISH联合multiplex RT-PCR检测MLL基因重排的价值探讨

目的:采用荧光原位杂交(FISH)与逆转录多重巢式聚合酶链反应(multiplexRT-PCR)技术检测急性白血病中MLL基因重排的情况,分析两者联合应用的诊断价值。方法:对2008年1月~2011年5月在我院诊断为急性白血病的201例患者采用MLL双色断裂分离重排探针进行FISH检测,同时用multiplexRT-PCR技术检测11种较常见的MLL融合基因,观察MLL基因异常的检出率。所有患者均进行常规染色体核型分析(CCA),观察11q23重排率作为对照。结果:共有19例患者出现11q23／MLL基因重排,在急性髓细胞白血病(AML)中的检出13例(10.2%),急性淋巴细胞白血病(ALL)的检出6例(8.2%)。FISH联合multiplexRT-PCR对MLL基因重排的检出率为9.45%,CCA对11q23异常的检出率为5.47%。5例正常核型的患者和3例未涉及11号染色体异常的患者中FISH检出了1例MLL倒位和3例扩增信号,multiplexRT-PCR检出了7例dupMLL(11q23)重排。结论:FISH联合multiplexRT-PCR能提高MLL基因重排检出率。     

Human serum mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1): determination of levels in body fluids and identification of two forms in serum

We developed an ELISA for human serum MASP-1, a Cls-like serine protease which is known to function in C4 and C2 activation. We then determined MASP-1 levels in 1063 sera from normal Japanese subjects ranging in age from 3 to 100 years, as well as in certain body fluids using this assay. Individual serum MASP-1 levels ranged from 148 to 12–83 μg/ml, with a normal frequency distribution pattern. The arithmetic mean ± s.d. of MASP-1 levels in serum was 6–27 ± 185 μg/ml, whereas levels of MASP-1 in cerebrospinal fluid and in urine were almost undetectable. When the mean ± s.d. of serum MASP-1 was calculated for each age group (10 year range) and values were then compared, the age group consisting of 3–9-year-olds (7–54 ± l-39;μ/ml) was found to have the highest value. When MASP-1 was measured in cord blood, it was shown that levels were already as high as those of 3–9-year-olds. The serum MASP-1 level was found to be as strongly dependent on age as is the serum MBL level. MASP-1 and MBL are thought to play an active part in immunity in younger people. It was found that the serum level of MASP-1 was much higher than that of MBL, and the major portion of human serum MASP-1 appeared to exist in the circulation as a form unbound to MBL.     

A novel ropivacaine-loaded in situ forming implant prolongs the effect of local analgesia in rats

Introduction

Prolonged postoperative analgesia cannot be achieved by a single injection of local anesthetic solution. The objective of this study was to optimize the formulation of a ropivacaine hydrochloride (Ropi-HCl) loaded in situ forming implant (ISI) by addition of different co-solvents, and evaluate the in vitro release of Ropi-HCl, and the analgesic effect and toxicity of the optimized formulation in rats.

Material and methods

Triacetin (TA), benzyl benzoate (BB) and polyethylene glycol 400 (PEG 400) were used as additives and added to the solvent of N-methyl-2-pyrrolidone (NMP). Drug release to the surface and inner structural properties of the formed implant were evaluated by scanning electron microscopy (SEM). The analgesic effect was determined by injection near the rat sciatic nerve.

Results

The solvent system added with TA or BB significantly decreased the burst release, whereas PEG 400 increased the Ropi-HCl burst release from the formulation. Over 70% of the incorporated Ropi-HCl was released from all formulations in 14 days in the in vitro assay. The SEM showed that the surface of NMP-BB formulation was less porous and more homogeneous, compared with the other formulations. Compared with Ropi-HCl injection, the optimized formulation (NMP-BB) significantly prolonged the analgesic effect in 48 h (p < 0.05), with a mild degree of motor block from 3 h to 12 h. Histological evaluation of the injection site revealed only mild inflammatory infiltration without obvious pathological nerve alterations.

Conclusions

The biodegradable Ropi-HCl-loaded ISI system with NMP-BB may prove to be an attractive and safe alternative for the delivery of parenteral local anesthetics to prolong pain relief.     

Regional adaptations in PSD-95, NGFI-A and secretogranin gene transcripts related to vulnerability to behavioral sensitization to amphetamine in the Roman rat strains

Genetically selected for high or low two-way active avoidance, Roman high-avoidance (RHA) and Roman low-avoidance (RLA) rats differ in their central dopaminergic activity, sensation/novelty- and substance-seeking profiles. These animals are, therefore, well suited to identify anatomical and neurochemical concomitants of behavioral sensitization, a phenomenon linked to addictive liability. We submitted inbred RHA (RHA-I), inbred RLA (RLA-I) and Sprague-Dawley-OFA (SD-OFA) rats to a sensitization regimen with amphetamine and studied the behavioral response to an amphetamine challenge after a 2-week withdrawal period. The expression patterns of nerve growth factor inducible clone A (NGFI-A), secretogranin, post-synaptic density protein of 95 Kd (PSD-95), prodynorphin and proenkephalin mRNA were also analyzed using in situ hybridization, after the challenge with amphetamine. RHA-I rats showed stronger sensitization than SD-OFA rats. RLA-I rats did not show sensitization but were hyper-reactive to amphetamine. Expression of behavioral sensitization in RHA-I rats activated secretogranin and PSD-95 mRNA in the nucleus accumbens core. On the other hand, high induction of NGFI-A mRNA in the central amygdala was observed in RLA-I rats when they experienced amphetamine for the first time in the challenge. Our results reveal that 1) the acute locomotor response to amphetamine does not predict vulnerability to behavioral sensitization and 2) differences in vulnerability to sensitization may involve distinctive cellular adaptations at particular brain locations which may be related to addictive vulnerability.     

The expression of GLTSCR2, a candidate tumor suppressor,is reduced in seborrheic keratosis compared to normal skin

Glioma tumor-suppressor candidate region gene 2 (GLTSCR2) is a recently identified nuclear protein that interacts with the tumor suppressor PTEN. GLTSCR2 regulates the stability of PTEN, and is therefore believed to have a tumor suppressive function. In a recent study, we demonstrated that GLTSCR2 often exhibits genetic alterations and down-regulation in glioblastoma specimens. However, GLTSCR2 expression levels in human tumors and its mechanism of tumor suppression remain largely unknown. We performed an immunohistochemical examination of GLTSCR2 expression in samples of seborrheic keratosis (SK, n=69), a common benign skin tumor, and normal skin (n=23), and assessed the relationship between GLTSCR2 expression and the patients' clinicopathologic factors. Our results showed that GLTSCR2 expression in SK was significantly lower than in normal skin (p<0.001), and this decreased expression of GLTSCR2 was associated with patient age (p=0.045). Using in situ hybridization analysis, we found that mRNA expression of GLTSCR2 was reduced in tumor cells compared to normal skin. This is the first study analyzing the expression of GLTSCR2, a putative tumor suppressor, in SK and normal skin. Our results show a down-regulation of this protein in SK, indicating that GLTSCR2 may have a protective effect on the development of SK.     

D2 and D4 dopamine receptor mRNA distribution in pyramidal neurons and GABAergic subpopulations in monkey prefrontal cortex: implications for schizophrenia treatment

D2 and D4 dopamine receptors play an important role in cognitive functions in the prefrontal cortex and they are involved in the pathophysiology of neuropsychiatric disorders such as schizophrenia. The eventual effect of dopamine upon pyramidal neurons in the prefrontal cortex depends on which receptors are expressed in the different neuronal populations. Parvalbumin and calbindin mark two subpopulations of cortical GABAergic interneurons that differently innervate pyramidal cells. Recent hypotheses about schizophrenia hold that the root of the illness is a dysfunction of parvalbumin chandelier cells that produces disinhibition of pyramidal cells. In the present work we report double in situ hybridization histochemistry experiments to determine the prevalence of D2 receptor mRNA and D4 receptor mRNA in glutamatergic neurons, GABAergic interneurons and both parvalbumin and calbindin GABAergic subpopulations in monkey prefrontal cortex layer V. We found that around 54% of glutamatergic neurons express D2 mRNA and 75% express D4 mRNA, while GAD-positive interneurons express around 34% and 47% respectively. Parvalbumin cells mainly expressed D4 mRNA (65%) and less D2 mRNA (15–20%). Finally, calbindin cells expressed both receptors in similar proportions (37%). We hypothesized that D4 receptor could be a complementary target in designing new antipsychotics, mainly because of its predominance in parvalbumin interneurons.     

Repeated microendoscopic discectomy for recurrent lumbar disk herniation

OBJECTIVES:

To explore the microendoscopic discectomy technique and inclusion criteria for the treatment of recurrent lumbar disc herniation and to supply feasible criteria and technical notes to avoid complications and to increase the therapeutic effect.

METHODS:

A consecutive series of 25 patients who underwent posterior microendoscopic discectomy for recurrent lumbar disc herniation were included. The inclusion criteria were as follows: no severe pain in the lumbar region, no lumbar instability observed by flexion-extension radiography and no intervertebral discitis or endplate damage observed by magnetic resonance imaging. All patients were diagnosed by clinical manifestations and imaging examinations.

RESULTS:

Follow-up visits were carried out in all cases. Complications, such as nerve injuries, were not observed. The follow-up outcomes were graded using the MacNab criteria. A grade of excellent was given to 12 patients, good to 12 patients and fair to 1 patient. A grade of excellent or good occurred in 96% of cases. One patient relapsed 3 months after surgery and then underwent lumbar interbody fusion and inner fixation. The numerical rating scale of preoperative leg pain was 7.4± 1.5, whereas it decreased to 2.1±0.8 at 7 days after surgery. The preoperative Oswestry disability index of lumbar function was 57.5±10.0, whereas it was 26.0±8.5 at 7 days after surgery.

CONCLUSION:

In these cases, microendoscopic discectomy was able to achieve satisfactory clinical results. Furthermore, it has advantages over other methods because of its smaller incision, reduced bleeding and more efficient recovery.     

Expression of stem cell factor (SCF), a KIT ligand, in gastrointestinal stromal tumors (GISTs): a potential marker for tumor proliferation

Gastrointestinal stromal tumors (GISTs) show a high incidence of gain-of-function mutations of the c-kit gene, which encodes a receptor tyrosine kinase KIT. This mutation is seen independently of metastasis and/or recurrence of tumors; thus, the factors involved in tumor proliferation rate and malignancy are still not known. Some mesenchymal and epithelial tumors have been reported to co-express KIT and its ligand, stem cell factor (SCF), for autonomous proliferation by the autocrine mechanism. The purpose of this study is to clarify whether GIST cells produce SCF, despite mutated KIT with constitutive activation. Immunohistochemically, we examined the co-expression of KIT and SCF in 36 GIST cases. All cases were found to be KIT-positive, and of these, 21 cases, including four recurrent or metastatic GISTs, showed co-expression of SCF. MIB-1 labeling index was significantly higher, and the average tumor size was larger in SCF-positive cases. By confocal microscopy, KIT was expressed on the cellular membrane, around which SCF was distributed less densely. Western blot analysis revealed that the membrane-bound SCF of 31 kDa was found to be approximately 10 times more abundant than the soluble SCF of 18.5 kDa, suggesting continuous KIT activation. These results indicate that proliferation of GIST cells can be caused not only by the gain-of-function mutation of c-kit, but also by the autocrine mechanism of the SCF/KIT system. Thus, SCF expression would be a useful marker for tumor proliferation.     

Specific tissue expression and cellular localization of human apolipoprotein M as determined by in situ hybridization

Apolipoprotein M (apoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL), and in minor proportion in triglyceride-rich lipoprotein (TGRLP) and low-density lipoprotein (LDL). The gene coding for apoM has been detected in all mammal genomes. The function of apoM is unknown yet. In the present study, we demonstrated that apoM is exclusively expressed in a strong manner in adult liver and kidney, and is expressed weakly in fetal liver and kidney as detected with human multiple tissue expression array. Both immunohistochemical staining and apoM mRNA in situ hybridization demonstrated that apoM was exclusively expressed in hepatocytes in human liver and in tubular epithelial cells in human kidney. The present study helps to elucidate the pathophysiological functions of apoM in vivo.     

Reproducibility of three classification systems of ductal carcinoma in situ of the breast using a web-based survey

This study assessed the degree of diagnostic agreement among pathologists between three classification systems of ductal carcinoma in situ of the breast (DCIS). Thirteen pathologists received the same set of digitized images of microscopy of 43 DCIS cases and answered a questionnaire containing the criteria to compose the three classification systems studied: Holland, modified Lagios, and Van Nuys. A computer program was created, which organizes the information collected from each pathologist, supplying the histological grading of the cases within the three classification systems. The results were analyzed using percental agreement and the Kappa test. Diagnostic agreement for the three DCIS of the breast classification systems presented K values that varied from 0.27 to 0.37. Among the three classifications used, most agreement was for Van Nuys, showing a Kappa index of 0.37. These results matched the interobserver agreements, with Kappa indices varying from 0.13 to 0.64 for the Holland classification; 0.23 to 0.61 for the modified Lagios classification; and 0.23 to 0.74 for the Van Nuys classification. Pathologists specialized in breast pathology showed greater reproducibility for all the criteria evaluated. Comparing the three classification systems, diagnostic agreement and accuracy were rated higher for the classification of Van Nuys compared to modified Lagios and Holland.     

Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain

Rats repeatedly exposed to restraint show a reduced hypothalamic–pituitary–adrenal axis response upon restraint re-exposure. This hypothalamic–pituitary–adrenal axis response habituation to restraint does not generalize to other novel stressors and is associated with a decrease in stress-induced c-fos expression in a number of stress-reactive brain regions. We examined whether habituation to repeated restraint is also associated with adaptation of immediate early gene expression in brain regions that process and relay primary sensory information. These brain regions may not be expected to show gene expression adaptation to repeated restraint because of their necessary role in experience discrimination. Rats were divided into a repeated restraint group (five 1-hour daily restraint sessions) and an unstressed group (restraint naïve). On the sixth day rats from each group were either killed with no additional stress experience or at 15, 30 or 60 min during restraint. Immediate early gene expression (corticotrophin-releasing hormone heteronuclear RNA, c-fos mRNA, zif268 mRNA) was determined by in situ hybridization. A reduction in stress-induced hypothalamic–pituitary–adrenal axis hormone secretion (plasma corticosterone and adrenocorticotropic hormone) and immediate early gene expression levels in the paraventricular nucleus of the hypothalamus, the lateral septum and the orbital cortex was observed in repeated restraint as compared with restraint naïve animals. This reduction was already evident at 15 min of restraint. Unexpectedly, we also found in repeated restraint rats a reduction in restraint-induced c-fos expression in primary sensory-processing brain areas (primary somatosensory cortex, and ventroposteriomedial and dorsolateral geniculate nuclei of thalamus). The overall levels of hippocampal mineralocorticoid receptor heteronuclear RNA or glucocorticoid receptor mRNA were not decreased by repeated restraint, as may occur in response to severe chronic stress. We propose that repeated restraint leads to a systems-level adaptation whereby re-exposure to restraint elicits a rapid inhibitory modulation of primary sensory processing (i.e. sensory gating), thereby producing a widespread attenuation of the neural response to restraint.     

The influence of uric acid treatments on liver glutathione system prevent oxidative damages in experimental autoimmune encephalomyelitis mice

Recently we reported that antioxidant system in brain and spinal cord in experimental autoimmune encephalomyelitis (EAE) mice is mainly affected at early stages of the disease [M. Zargari, A. Allameh, M.H. Sanati, T. Tiraihi, S.H. Lavasani, O. Emadyan, Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice, Neurosci. Lett. 412 (2007), 24–28]. The aim of the present study was to investigate the role of uric acid (UA) on antioxidant system in liver and plasma of EAE mice. EAE was induced in C57/BL6 mice (n = 60), followed by i.p. administration of UA (10 mg/kg BW) in 30 mice at three distinct clinical stages (A: prior to onset, B: after onset, C: after development of EAE). Livers were removed and processed for measurement of lipid peroxidation products, reduced glutathione (GSH), and glutathione S-transferase (GST) and total antioxidant capacity of plasma (FRAP). The results showed that lipid peroxidation products in liver of EAE mice was increased significantly (∼85%) as compared to normal. UA administration to EAE mice caused a significant suppression of liver lipid peroxidation products (∼45%) at early stages (A and B). There was an inverse relationship between lipid peroxidation and cellular GSH in liver. GSH was significantly depleted in mice liver during the EAE progression, but it was recovered (∼29%) when UA was injected before the onset of the disease (groups A and B). Plasma total antioxidant capacity was significantly decreased during the development of EAE, however it was subsided in mice treated with UA as compared to the corresponding controls (21%) in groups A and B. Elevated liver GST as a result of EAE induction was reversed in mice treated with UA particularly in groups A and B. These results indicate that hepatic glutathione system, particularly GST plays a major role in modulation of oxidative damages to central nervous system (CNS) during EAE induction. The positive response of antioxidant system to UA administration in EAE mice was corroborated with improvement of clinical manifestation of the animals.     

Delayed epithelial ingrowth caused by viral conjunctivitis after LASIK

To report a patient who presented with epithelial ingrowth caused by viral keratoconjunctivitis 3 months after LASIK surgery. A 41-year-old man presented with decreased visual acuity in the right eye, which had developed about 3 weeks before. He had undergone LASIK surgery 3 months prior without complications. Two months after the surgery, he was treated for viral conjunctivitis. During the treatment period, filamentary keratitis developed, and a therapeutic bandage contact lens was applied for 2 weeks. Upon presentation, examination revealed a corrected visual acuity of 20/100 and irregular epithelial sheets under the edematous flap. The flap was lifted, and the in-grown epithelium was removed. The flap was repositioned with double continuous 10-0 nylon sutures. Post-operatively, the patient developed a mild diffuse lamellar keratitis that resolved rapidly with topical corticosteroid treatment. At 2 months, the corrected visual acuity was 20/20 without interface opacities. As the patient showed no complications prior to viral conjunctivitis, we suspect that the viral infection caused edema of the corneal flap, which caused epithelial ingrowth under the flap. Patients who have viral conjunctivitis after LASIK surgery should be examined carefully and managed with consideration of flap complications.     

Mouse mammary tumor virus derived from wild mice does not target Notch-4 protooncogene for the development of mammary tumors in inbred mice

The colony of wild mice, named Jyg, has been shown to express an exogenous mouse mammary tumor virus (Jyg-MMTV). This virus induces mammary tumors in its natural host at a high incidence (≈ 80%) resulting from insertion mutations in Notch-4 (43%), Wnt-1 (26%), and Fgf-3 (13%). Since the activation of Notch-4 is not common in mammary tumors of standard laboratory strains of mice infected with various MMTV strains, we examined the consequences of Jyg-MMTV infection in BALB/c and C57BL/6 mice. The results show that Jyg-MMTV induces mammary tumors in both mouse strains, but the incidence of mammary tumors in BALB/c mice is greater than in C57BL/6 mice. Surprisingly, however, none of the 75 mammary tumors, analyzed both by Southern and Northern hybridizations, showed insertion mutations in or expression of Notch-4. In contrast, both Wnt-1 and Fgf-3 were found to be involved in these tumors. Our findings may suggest, among other possibilities, the existence of a structural difference(s) between laboratory and wild mice at the Notch-4 locus that regulates the integration of Jyg-MMTV proviral DNA.