Fv2 encodes a truncated form of the Stk receptor tyrosine kinase.

The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.

Neuroprotection by a caspase inhibitor in acute bacterial meningitis.

Half of the survivors of bacterial meningitis experience motor deficits, seizures, hearing loss or cognitive impairment, despite adequate bacterial killing by antibiotics. We demonstrate that the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (z-VAD-fmk) prevented hippocampal neuronal cell death and white blood cell influx into the cerebrospinal fluid compartment in experimental pneumococcal meningitis. Hippocampal neuronal death was due to apoptosis derived from the inflammatory response in the cerebrospinal fluid. Apoptosis was induced in vitro in human neurons by inflamed cerebrospinal fluid and was blocked by z-VAD-fmk. As apoptosis drives neuronal loss in pneumococcal meningitis, caspase inhibitors might provide a new therapeutic option directed specifically at reducing brain damage.

Tumor spectrum in ARF-deficient mice.

The p19ARF product of the INK4a/ARF locus is induced in response to potentially oncogenic hyperproliferative signals and activates p53 by interfering with its negative regulator, Mdm2. Mice lacking ARF are highly prone to tumor development, and in this study, 80% of these animals spontaneously developed tumors and died within their first year of life. Mice that were heterozygous for ARF also developed tumors after a longer latency, whereas their wild-type littermates did not. In heterozygotes, tumor formation was accompanied by loss of the residual ARF allele and/or lack of ARF mRNA expression, implying that ARF can act as a canonical "two-hit" tumor suppressor gene. Tumors occurred earlier in life in ARF-null animals that were neonatally irradiated or given dimethylbenzanthrene, and several animals treated with carcinogen simultaneously developed multiple forms of malignancy arising from distinct cell lineages. Although p53-null mice primarily develop lymphomas and fibrosarcomas, the frequency of these two tumor types was inverted in ARF-null animals, with undifferentiated sarcomas predominating in a 3:2 ratio; 28% of ARF-null animals developed carcinomas and tumors of the nervous system, which have been rarely observed in untreated p53-null mice. The longer latency of tumor formation in ARF-null versus p53-null mice, therefore, appears to enable a broader spectrum of tumors to emerge.

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.

High frequency of somatically acquired p53 mutations in small-cell lung cancer cell lines and tumors.

We analysed the p53 open reading frame (ORF) in 16 small-cell lung cancer (SCLC) cell lines by direct sequencing of cDNA/PCR products and in 20 SCLC tumors by chemical cleavage and single-strand conformation polymorphism analyses of genomic DNA/PCR products. Abnormalities of p53 were found in 16/16 cell lines (100%) and in 16/20 tumors (80%). In the SCLC cell lines, mutations (59% missense, 18% nonsense and 23% splicing) changing the coding sequence were dispersed between amino acids 68 and 342. In the tumor samples, while the mutations occurred predominantly in exons 5-8, other mutations were located outside these regions. G to T transversions were common, occurring in 32% of the cases. We found no p53 mutations in the corresponding normal tissue from 19 patients whose tumors had p53 lesions, indicating that the mutations were all somatically acquired. In analysing the clinical data of the patients we found no correlation between tumor response to therapy or survival and the location or type of mutations. We conclude from these data that: (1) p53 mutations are found in SCLC with high frequency; (2) p53 mutations in a significant fraction of cases generate cDNAs with nonsense or splicing mutations; and (3) to date, these mutations have all been somatically acquired events.

p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features.

We screened 77 non-small-cell lung cancer (NSCLC) cell lines for mutations of the p53 gene using a single-strand conformation polymorphism (SSCP) assay. We found that 57 cell lines (74%) had mutations of the p53 gene. Three cell lines had a deletion of the p53 gene. Of the remaining 54 cell lines, 49 cell lines were sequenced and 52 mutations were confirmed. In contrast to previously published p53 mutations in other human tumors, the p53 gene mutations in NSCLC were diverse with regard to the location and nature of the mutations. The region corresponding to codons 144-166, which is outside the evolutionarily conserved regions, was a frequent site of p53 gene mutations in NSCLC. The presence of a p53 gene mutation was not associated with age, sex, histological types, culture site, treatment intent, presence of prior cytotoxic treatment, neuroendocrine differentiation, median culture time or patient survival. The prevalence of p53 mutations in cell lines with ras mutations did not differ from that in cell lines without ras mutations. However, p53 gene mutations in NSCLC cell lines with ras mutations tended to cluster in exon 8, suggesting the presence of a functional domain of the p53 gene relating to interaction with the ras gene. We conclude that p53 and ras mutations are frequent and apparently independent genetic alterations which play different roles in the pathogenesis, progression and prognosis of NSCLC.

Nonlinear elastic-viscoplastic constitutive equations for aging facial tissues.

This paper reports on the initial stages of a project to simulate the nonlinear mechanical behavior of an aging human face. A cross-section of the facial structure is considered to consist of a multilayered composite of tissues with differing mechanical behavior. The constitutive properties of these tissues are incorporated into a finite element model of the three-dimensional facial geometry. Relatively short time (elastic-viscoplastic) behavior is governed by equations previously developed which are consistent with mechanical tests. The long time response is controlled by the aging elastic components of the tissues. An aging function is introduced which, in a simplified manner, captures the observed loss of stiffness of these aging elastic components due to the history of straining as well as other physiological and environmental influences. Calculations have been performed for 30 years of exposure to gravitational forces. Progressive gravimetric soft tissue descent is simulated, which is regarded as the main indication of facial aging. Results are presented for the deformations and stress distributions in the layers of the soft tissues.

The question of PANDAS in adults.

BACKGROUND: Pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS) are a well-defined cause of obsessive-compulsive disorder in children. However, they have not been described or fully investigated in adults newly diagnosed with obsessive-compulsive disorder. METHODS: We describe an adult with onset of obsessive-compulsive disorder at 25 years of age after a severe antibiotic-responsive pharyngitis. He was evaluated with multiple psychiatric rating scales for obsessive-compulsive disorder and Tourette's syndrome, as well as with serologic assays and radiologic studies. RESULTS: In all respects except age our patient fulfilled established criteria for PANDAS. Assays for antibodies to group A beta-hematolytic streptococci, serum D8,17 lymphocytes, antistriatal (neuronal) antibodies, and anticytoskeletal antibodies all supported the hypothesis that a poststreptococcal process was active. Magnetic resonance imaging was abnormal and is described. CONCLUSIONS: The findings suggest that this patient's illness is similar to PANDAS in presentation and that poststreptococcal disease may result in adult-onset obsessive-compulsive disorder.

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Mutations in the p53 gene in pulmonary blastomas: immunohistochemical and molecular studies.

Well-differentiated fetal adenocarcinomas and biphasic blastomas are types of lung cancer that contain glands that mimic the appearance of fetal lung. Biphasic blastomas also show a primitive embryonic stroma. Despite histological similarities leading these two tumors to be classified as pulmonary blastomas, they have distinct clinical and prognostic features. Little information is available on genetic changes in these tumors because they are rare; therefore, the authors studied nine biphasic blastomas and 12 well-differentiated fetal adenocarcinomas for the presence of mutations in the p53 gene. Mutations in the p53 gene are common in other lung cancers, and the type of mutation in the p53 gene can provide information about the original or inciting mutagens. The authors found five biphasic blastomas (42%) had mutations in the p53 gene by immunohistochemical and molecular analysis, whereas none of the well-differentiated fetal adenocarcinomas contained mutations. These results provide molecular support for the significance of distinguishing between well-differentiated fetal adenocarcinoma and biphasic blastoma histologically and identify several types of p53 gene mutations that occur in these tumors.

Identification of p53 mutations in archival prostate tumors. Sensitivity of an optimized single-strand conformational polymorphism (SSCP) assay.

To correlate molecular changes with clinical information in prostate tissue, it is necessary to have accurate methods for screening for mutations in clinically available specimens. We have refined the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere with SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samples are heterogeneous in containing tumor, normal tissue, and hyperplastic tissue. To address the first issue, the method included an initial selection of PCR products using exonuclease I, followed by a second-step selection using nested PCR. This step ensures adequate amplification of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue, in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restriction enzyme digestion of products prior to SSCP analysis. Detection of mutations in heterogeneous tissue was evaluated by determining the minimal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous clinical samples, we performed study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and two metastases) and detected abnormally migrating products in six of the prostate cancer specimens (four primaries and two metastases). In five of these samples, there was sufficient DNA to perform sequencing, which disclosed single-base change mutations in all five samples.

Benign brain stem lesions in pediatric patients with neurofibromatosis: case reports.

The symptoms and clinical courses of 4 patients with neurofibromatosis and lesions of the brain stem identifiable on computed tomographic and/or magnetic resonance imaging scans are described. Two patients underwent biopsy and both had low-grade astrocytomas with no evidence of anaplasia. Both received radiation and chemotherapy. The other 2 patients have been monitored without biopsy or treatment. Three patients are alive and clinically stable, having been followed up for an average of 4 years; neuroimaging studies have shown no change in their tumors. The fourth patient died of a supratentorial primitive neuroectodermal tumor. Imaging studies had shown no change in his brain stem lesion, which at autopsy was found to be a focal collection of fibrillary astrocytes. These data suggest that some patients with brain stem lesions and neurofibromatosis may have a prognosis distinctly different from that of the typical patient with a brain stem glioma. We recommend caution against aggressive operative and adjuvant therapy for brain stem lesions in patients with neurofibromatosis, unless progression of the lesion is documented clinically and/or by imaging.

Mechanical properties and microstructure of the superficial musculoaponeurotic system.

Because of the widespread reliance on SMAS tightening procedures in present-day face lift surgery, a study was undertaken to examine the physical properties and microscopic structure of both virginal (40 specimens) and reoperated (8 specimens) SMAS tissue. The findings could be of practical value to the surgeon and are reported herewith: First, the SMAS is a composite fibrofatty layer comprising collagen and elastic fibers interspersed with fat cells. Second, microscopic appearance shows a considerable amount of elastic fibers in close relationship to the collagen fibers. Third, on scanning electron microscopy, the collagen fibers in the virginal SMAS show a convoluted appearance similar to that found in the dermis. In the reexcised SMAS tissue, there is some evidence of parallelization of the collagen fibers as seen in the stretched dermis. Fourth, mechanical testing (Instron), i.e., a series of loading/unloading tests at various rates and amplitudes, and stress relaxation tests were performed on samples of preauricular skin and SMAS. These indicated definite viscoelastic properties for both sets of specimens, with the tendency of an increased stiffness and a reduction in viscoelastic effects on repeated working of the samples. Overall, the mechanical behavior of both tissues was somewhat similar, the viscoelastic effects in SMAS being less pronounced. A nonlinear viscoelastic model is under development to represent the behavior of both tissues. The implications of these results may help to explain the slackening effect observed in some postoperative patients.

Ultrasonographic determination, in vitro and in vivo, of canine gallbladder volume, using four volumetric formulas and stepwise-regression models.

Twelve resected canine gallbladders (in vitro) and the gallbladder in each of 14 dogs (in vivo) were ultrasonographically examined. Gallbladder volume was calculated from ultrasonographically measured geometric dimensions, using 4 volumetric model formulas: cone, ellipse, biplanar ellipse, and prolate ellipse. Calculated volume was compared with true gallbladder volume, as measured by water displacement. All examined models for calculation of gallbladder volume were closely associated with true gallbladder volume (P < 0.005), and all models provided accurate predictions of true gallbladder volume (r2 > 0.80). Calculated volumes can be corrected mathematically by use of the regression coefficient and constant for each model. Body weight was not significantly associated with gallbladder volume in any of the models considered. Use of ultrasonography to accurately measure gallbladder volume could be combined with synthetic cholecystokinin-stimulated gallbladder emptying to provide information about biliary function and patency in icteric animals. Such information could aid the clinical decision between surgical or medical treatment. Correction of calculated volumes would not be necessary in association with induced emptying studies, because volume change is more important than absolute volume.

Assessment of current techniques for determining tracheal luminal stenosis in dogs.

OBJECTIVE: To assess the accuracy of current antemortem and postmortem techniques for determining tracheal luminal stenosis. ANIMALS: 15 dogs. PROCEDURE: Percentage of tracheal luminal stenosis (PTLS) was determined by 6 methods, using measurements obtained by radiography, tracheoscopy, and necropsy after selected tracheostomy techniques were performed. To calculate PTLS, dorsoventral tracheal diameter was measured from preoperative and postoperative lateral cervical radiographic views. Preoperative or normal tracheal segments adjacent to the stenotic area were used to obtain normal tracheal diameter measurements. Planimetrically determined cross-sectional area (CSA), obtained from pre- and postoperative tracheoscopic photographs, was used to calculate PTLS. The CSA of tracheal specimens obtained at necropsy was determined, using the formula for an ellipse. Percentage of luminal stenosis was calculated, using CSA of the stenotic site and of segments craniad and caudad to the site obtained at necropsy or at surgery. All methods were compared with the control method of planimetrically determined CSA of sections obtained at necropsy of the tracheostomy and segments craniad and caudad to the site. RESULTS: Correlation was poor for radiographic and tracheoscopic techniques (r = 0.146 to 0.458, P > 0.05) The formula for an ellipse accurately predicted PTLS when measurements obtained at surgery (r = 0.516, P = 0.049) or segments craniad and caudad (r = 0.853, P < 0.001) to the site were used. CONCLUSION: Antemortem methods of assessing PTLS did not correlate with control planimetric methods. Methods using CSA determined by tracheal diameter were weakly correlated to control planimetric techniques. CLINICAL RELEVANCE: Accurate measurement of the degree of tracheal stenosis cannot be made in clinical patients using current techniques.

Use of streptokinase in four dogs with thrombosis.

Four dogs with thrombosis were referred for diagnostic testing and were subsequently treated by the use of streptokinase. The range of duration of clinical signs associated with thrombosis was 6 to 120 days. Causes of thrombosis were heart disease (1 dog), protein-losing nephropathy and hyperadrenocorticism (1), hyperadrenocorticism (1), and idiopathic (1). Possible factors that predisposed dogs to hypercoagulability included hypertension (2 dogs) and diabetes mellitus (1). All dogs were treated for underlying disease by use of supportive care. The first dog was treated with a loading dose of 250,000 U of streptokinase, i.v., with a subsequent maintenance dosage of 100,000 U/h, i.v., and also was treated with anticoagulant. The subsequent 3 dogs were treated with a loading dose of 90,000 U of streptokinase, i.v., and maintenance dosage of 45,000 U/ h, i.v., at various intervals. These dogs also were treated with anticoagulant. Three dogs had minor hemorrhage as an adverse effect to streptokinase infusion, but they did not require treatment for the hemorrhage. Complete resolution of the thrombus was observed in 3 dogs, and partial resolution of the thrombus was observed in the other dog. In all dogs, partial or complete resolution of clinical signs associated with thrombosis was seen. Streptokinase may be an effective treatment for dogs with thrombosis.

Abdominal vascular sonography.

This is an introductory article on abdominal vascular ultrasound in dogs. An overview of the hemodynamics of venous and arterial blood flow and Doppler principles, spectral analysis, and velocity waveforms is given. The anatomic and Doppler features of major abdominal vessels that can be examined routinely with ultrasonography are discussed. Select cases of vascular pathology affecting various abdominal vessels in the dog and cat are described.

Updates in interventional ultrasonography.

Interventional ultrasonography, including diagnostic, therapeutic, and intraoperative interventions, is a rapidly developing field in both human and veterinary medicine. Growing expertise and significant improvements in the technology have contributed to the innovation of numerous ultrasound-assisted procedures. Ultrasound-guided biopsy and fine-needle aspiration are now commonly performed. New techniques and the indications for other procedures such as cavity drainage, indwelling catheter placement, and intraoperative procedures are presented.

Ultrasonography.

The basic principles of diagnostic ultrasonography are discussed. Normal and abnormal ultrasonographic appearances of the major organs are presented in tables and with illustrations. A description of the current status of neurosonology with illustrations of hydrocephalus is also presented.