The canine baculum: the structure and mechanical properties of an unusual bone.

The baculum is an extraskeletal bone located in the penis of a few species in several orders of mammals such as carnivores, insectivores, rodents, bats and primates. This study aims to describe the structure, architecture and mechanical properties of the canine baculum. To this end canine bacula from castrated and uncastrated dogs were collected and examined by light microscopy, micro-computed tomography (microCT) scanning, histological staining, and mechanical testing. Their mineral density and mechanical properties were compared with those of a typical skeletal bone (the radius) in the same dog. Furthermore, a numerical model of a representative baculum was created and its mechanical performance analyzed using the finite element method, in order to try to elucidate its function. Examination of light microscopy images of transverse sections shows that the baculum consists of a typical sandwich structure, with two cortical plates separated, and joined, by loose cancellous bone. MicroCT scans reveal that the mineral density is lower in the baculum than in the radius, both in castrated as well as in uncastrated dogs, resulting in much lower stiffness. Castration was found to decrease the mineral density in both the baculum and the radius. The most likely function of the baculum of the dog is to stiffen the penis to assist intromission, and its much lower mineral density compared to that of the radius may be a mechanism designed to decrease the stiffness somewhat, and thus reduce the risk of fracture during copulation.

Inhibition of presenilin 1 expression is promoted by p53 and p21WAF-1 and results in apoptosis and tumor suppression.

Previously, we cloned a cDNA fragment, TSIP 2 (tumor suppressor inhibited pathway clone 2), that detects by northern blot analysis of M1-LTR6 cells a 3-kb mRNA downregulated during p53-induced apoptosis. Cloning the full-length TSIP 2 cDNA showed that it corresponds to the presenilin 1 (PS1) gene, in which mutations have been reported in early-onset familial Alzheimer's disease. Here we demonstrate that PS1 is downregulated in a series of model systems for p53-dependent and p53-independent apoptosis and tumor suppression. To investigate the biological relevance of this downregulation, we stably transfected U937 cells with antisense PS1 cDNA. The downregulation of PS1 in these U937 transfectants results in reduced growth with an increased fraction of the cells in apoptosis. When injected into mice homozygous for severe combined immunodeficiency disease (scid/scid mice), these cells show a suppression of their malignant phenotype. Our results indicate that PS1, initially identified in a neurodegenerative disease, may also be involved in the regulation of cancer-related pathways.

p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs.

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53(-/-)) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53(-/-) Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative 'real-time' reporter gene for cell therapy preclinical studies.

The c-fos proto-oncogene is a target for transactivation by the p53 tumor suppressor.

The p53 tumor suppressor gene is mutated in over 50% of human cancers, resulting in inactivation of the wild-type (wt) p53 protein. The most notable biochemical feature of p53 is its ability to act as a sequence-specific transcriptional activator. Through use of the suppression subtractive hybridization differential screening technique, we identified c-fos as a target for transcriptional stimulation by p53 in cells undergoing p53-mediated apoptosis. Overexpression of wt p53 induces c-fos mRNA and protein. Moreover, in vivo induction of c-fos in the thymus following whole-body exposure to ionizing radiation is p53 dependent. p53 responsiveness does not reside in the basal c-fos promoter. Rather, a distinct region within the c-fos gene first intron binds specifically to p53 and confers upon the c-fos promoter the ability to become transcriptionally activated by wt p53. Identification of c-fos as a specific target for transcriptional activation by p53 establishes a direct link between these two pivotal regulatory proteins and raises the possibility that c-fos contributes to some of the biological effects of p53.

Association of the C677T polymorphism in the MTHFR gene with breast and/or ovarian cancer risk in Jewish women.

The C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with reduced enzyme activity, hyperhomocysteinaemia and increased risk for atherosclerosis in homozygotes. We examined the frequency of this mutation and its association with disease pattern in 491 Jewish women with either sporadic (n = 355; 72%) or hereditary (n = 136; 28%) breast and/or ovarian cancer and in 69 asymptomatic BRCA1/2 mutation carriers, genotyped for the three predominant Jewish founder BRCA1/2 mutations (185delAG, 5382insC and 6174delT). 677T homozygotes were equally distributed among women with sporadic breast and/or ovarian cancer (71/355; 20.0%) and among BRCA1/2 mutation carriers (43/205; 21.0%) (P=non-significant). 677T homozygotes were equally distributed among women diagnosed with breast cancer prior to (22/122; 18.0%) and after 42 years of age (42/243; 17.3%). Among BRCA1/2 carriers, the rate of 677T homozygotes in manifesting cancer (32/136; 23.5%) and asymptomatic individuals (11/69; 15.9%) was not significantly different. The rate of 677T homozygotes (24/72; 33.3%) was higher (P=0.0026) among women with bilateral breast cancer and those with both breast and ovarian carcinoma than among those with unilateral breast cancer (64/365; 17.5%). Differences in morbidity (one versus multiple breast/ovarian tumours) are mainly attributed to 677T homozygosity and partly to BRCA1/2 mutations. Confirmation of these data, namely, that the 677T allele is significantly more common in cases of bilateral breast cancer or combined breast and ovarian cancer would have important clinical implications.

Partial sequencing of the rat steroidogenic acute regulatory protein message from immortalized granulosa cells: regulation by gonadotropins and isoproterenol.

Steroidogenic acute regulatory protein (StAR), a 30-kDa protein involved in the transport of cholesterol to inner mitochondrial membrane during stimulation of steroid hormone biosynthesis, has recently been cloned from human adrenals and MA-10 mouse Leydig tumor cells. We examined the regulation of StAR mRNA accumulation upon induction of steroidogenesis in immortalized rat granulosa cells. Granulosa cells were transfected with SV40 DNA alone (POGS5); with SV40 DNA and Ha-ras oncogene (POGRS1); with SV40 DNA, Ha-ras oncogene and LH/CG receptor (GLHR15) or with FSH receptor (GFSHR17) or with the beta 2-adrenergic receptor (G beta 2AR13) expression plasmids. Cells were cultured to confluency and then stimulated for 24 h with oFSH (4 nM), hCG (2.4 nM), isoproterenol (10 microM) or forskolin (50 microM). By quantitative RT-PCR, StAR mRNA was undetectable in non-steroidogenic cells (transfected with SV40 DNA alone, POGS5) either in the presence or in the absence of forskolin. In contrast, variable amount of the message was detected in all steroidogenic cell lines cotransfected with SV40 DNA and Ha-ras. Moreover, an increase in the StAR mRNA expression was evident in all steroidogenic cells upon stimulation with their respective agonists, concomitantly with enhanced progesterone production. The RT-PCR product was sequenced and the 379 base pairs of rat StAR were found to be 93% and 86% identical to mouse and human cDNA, respectively. The deduced 126 amino acid sequence was 95%, 88% and 88% identical to the mouse, human and bovine deduced protein sequences. We conclude that StAR message is expressed only in the steroidogenic rat granulosa cells and can be upregulated by FSH, hCG, isoproterenol and forskolin in the appropriate cell lines. In addition, we find that the rat StAR cDNA exhibit a high degree of homology with the mouse and human sequences.

Activation of p53 and its target genes p21(WAF1/Cip1) and PAG608/Wig-1 in ischemic preconditioning.

A brief, 3 min period of global forebrain ischemia in the rat, induced by bilateral common carotid occlusion combined with hypotension, confers resistance to hippocampal pyramidal neurons against a subsequent 10 min ischemia, which is normally lethal to these cells. The molecular mechanisms underlying this ischemic preconditioning, or tolerance, are poorly understood. The tumor suppressor p53 is a transcription factor implicated in neuronal death following various insults, including cerebral ischemia. p53 is activated in response to cellular stress, e.g. hypoxia and DNA damage. Using in situ hybridization, we investigated the hippocampal mRNA expression of p53, and two of its target genes, p21(WAF1/Cip1) and the recently cloned PAG608/Wig-1, in a two-vessel occlusion model of ischemic preconditioning. We also evaluated changes in the protein levels of p53 and PAG608/Wig-1 using immunohistochemistry. The mRNA levels of all three genes increased in the ischemia sensitive CA1 region both following 3 min (non-lethal) preconditioning and 10 min of (lethal) nonconditioned ischemia. In contrast, after 10 min of ischemia preconditioned by a 3 min ischemic insult 48 h earlier, no upregulation of these genes was detected in the CA1. Following 10 min of nonconditioned ischemia, increased neuronal immunostaining of p53 and PAG608/Wig-1 was observed in the hippocampus, which was less pronounced following 3 min of preconditioning ischemia and 10 min of preconditioned ischemia. Our results demonstrate that activation of p53 and its response genes p21(WAF1/Cip1) and PAG608/Wig-1 occurs in the brain following lethal as well as non-lethal ischemic insults, and that ischemic preconditioning markedly diminishes this activation.

The significance of family history for patients with carcinoma of the breast.

BACKGROUND: Since the risk of carcinoma of the breast is increased in women with a family history of the disease, new primary carcinomas of the breast may be increased after treatment. Women with several relatives with carcinoma of the breast are thought to be at higher risk of having a second primary carcinoma of the breast develop and mastectomy is more frequently recommended. STUDY DESIGN: The computerized registry of the Mount Sinai Medical Center Breast Service was used to identify 1,337 patients with complete information concerning family history. Three hundred fifty-nine patients with a family history of carcinoma of the breast were compared with women with no family history. RESULTS: Compared with patients with no family history of carcinoma of the breast, patients with a family history of carcinoma of the breast were significantly younger (54.0 versus 55.8 years of age, p < 0.01), were significantly more likely to have used oral contraceptives (26 versus 13 percent, p < 0.001), had significantly more ductal carcinoma in situ (10 versus 4 percent, p < 0.01), and were significantly more often treated with breast conservation (42 versus 31 percent, p < 0.001). Simultaneous contralateral carcinoma of the breast was diagnosed more frequently in patients with a family history (3 versus 1 percent, p < 0.025), but metachronous contralateral carcinomas were not increased. In comparing the two groups, there were no significant differences in proportion premenopausal, parity, use of postmenopausal hormones, tumor size, tumor differentiation, nodal involvement, TNM stage, estrogen receptor status, or use of adjuvant radiation, chemotherapy, or tamoxifen. Complete five-year follow-up evaluation for 748 patients, 179 with a family history, found no differences in local, distant, or disease-free survival rates for mastectomy or breast conservation in relation to family history. Outcome for patients with first-degree affected relatives and those with more than one affected relative was the same as those with no family history. CONCLUSIONS: These results indicate that women with a family history of carcinoma of the breast should be treated no differently than women with no family history.

Paranasal implants for correction of midface concavity.

Central midface concavity was corrected with the placement of porous polyethylene implants in the paranasal area. This simulated the effect of skeletal osteotomies and advancement without altering dental occlusion. Implants were placed to correct congenital, posttraumatic, and cleft-related skeletal midface retrusion in nine patients. In seven of the patients, paranasal augmentation was performed in conjunction with rhinoplasty. There have been no implant-related complications during a mean 33-month follow-up (range 5 to 83 months). Screw fixation of these implants ensures stable positioning and allows precise final contouring during surgery.

[Retinal injury induced by laser pointers].

Laser pointers originally designed for use during presentations are ubiquitous and are even sold as toys (such as pens or on key chains) in drug stores. Though reported as safe, the laser pointers still carry the risk of potential damage to the eye. We report a 16-year-old boy with bilateral retinal injury caused by 20-30 seconds of exposure to a laser pointing-device. Immediately thereafter, vision was blurred bilaterally and he noted a central red scotoma in each eye. Symptoms resolved spontaneously within 2 days but the retinal scars remained all during the 10 months of follow-up. It is clear from our report and 3 other publications that retinal damage can develop from misusing laser pointers. Laser hazards and safety should be stressed for the general public. We recommend that laser-pointers should not be available as toys to children and teenagers.

Myogenic program induction in mature fat tissue (with MyoD expression).

MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes.

Physician practices in the diagnosis and management of patients with pulmonary embolism.

To examine the diagnostic work-up and subsequent management of patients with pulmonary embolism, we retrospectively reviewed the medical records of 60 patients who had arteriographically proven pulmonary embolism. Ventilation-perfusion scans were utilized in 47 patients and were classified as showing a high (55.3%), moderate (14.9%), low (8.5%), or indeterminate (21.3%) probability of pulmonary embolism. Of the 33 patients treated with anticoagulation, complications occurred in 10 (30.3%). Of the 39 patients treated with inferior vena cava filters, complications occurred in 16 (41.0%) patients, with the most clinically significant complication being recurrent pulmonary embolism, found to occur in one (2.6%) patient. For patients treated with anticoagulation, heparin was started an average of 0.3 (+/- 0.5) days following the initial suspicion of a pulmonary embolism. The average time to reach a therapeutic partial thromboplastin time level was 2.0 (+/- 1.8) days, and warfarin was started an average of 6.2 (+/- 4.1) days after heparin was begun. Among the 60 patients, five (8.3%) died from the pulmonary embolism, and two of these had at least a five day delay before diagnosis was made by pulmonary arteriography. Since the ventilation-perfusion scan had a sensitivity of 70.2% and a false negative rate of 8.5%, it is not always sufficiently accurate in making final treatment decisions, so there should be less hesitation in attaining pulmonary arteriograms. Therapeutic partial thromboplastin time levels should be more aggressively sought. Warfarin should be started within the first few days of heparin therapy. Inferior vena cava filters should be utilized in patients who have a contraindication to anticoagulation therapy or a complication from anticoagulation therapy.

Pelvic radiation therapy as a risk factor for ischemic colitis complicating abdominal aortic reconstruction.

Ischemic colitis is an infrequent but potentially devastating complication of abdominal aortic reconstruction. Identification of patients with predisposing risk factors for the development of ischemic colitis can guide intraoperative measures to preserve or restore colonic blood flow during aortic surgery. Previous radiation therapy for pelvic malignancy may be one such predisposing risk factor. Two cases are presented in which ischemic colitis complicated abdominal aortic reconstruction in the setting of previous pelvic irradiation. In the months after radiation therapy for prostate cancer, one patient underwent infrarenal abdominal aortic aneurysm repair. Ischemic infarction of the sigmoid colon developed acutely after surgery and required emergent sigmoid colectomy. The second patient underwent reconstruction of an infrarenal abdominal aortic aneurysm after having had radiation therapy for a bladder tumor. Despite an initial satisfactory result, the patient's abdominal pain and diarrhea progressively worsened and he eventually required sigmoid colectomy for severe ischemic colitis. In both of these patients, the inferior mesenteric arteries were patent and had not been reimplanted. The association of pelvic radiation therapy with ischemic colitis after aortic reconstruction should focus attention to the operative details for maintaining the colonic circulation in these patients. Reimplantation of the inferior mesenteric artery in particular may prevent both the acute and the insidious variants of this complication in patients who undergo aortic surgery and decrease the incidence of this complication in patients with a history of radiation therapy to the pelvis.

Use of urokinase during secondary ischemia in experimental skin flaps.

The ability of urokinase to salvage experimental flaps after a secondary ischemic insult was investigated in a rat model. Unilateral abdominal island skin flaps based on the superficial inferior epigastric vessels were raised and subjected to either 4 or 6 hours of primary ischemia followed by 12 hours of reperfusion and varying lengths of secondary ischemia. At the conclusion of secondary ischemia, the flaps were perfused with either lactated Ringer's solution or urokinase. One group of flaps served as a control and received no postischemic perfusion washout. The secondary critical ischemia time at which 50% of the flaps failed clinically was greater for flaps perfused with urokinase. Furthermore, the survival rates for all flaps perfused with urokinase were significantly greater than either control flaps or flaps perfused with lactated Ringer's solution (p < 0.05). Flap survival decreased significantly in all groups with increasing primary and/or secondary ischemia time (p < 0.05).

Numerous candidate plasticity-related genes revealed by differential cDNA cloning.

Plasticity is a property of the nervous system that allows it to modify its response to an altered input. This capacity for change suggests that there are molecular mechanisms in neurons that can couple stimuli to long-term alterations in phenotype. Neuronal excitation elicits rapid transcriptional activation of several immediate-early genes, for example c-fos, c-jun and zif268. Many immediate-early genes encode transcription factors that control expression of downstream genes whose products are believed to bring about long-term plastic changes. Here we use a highly sensitive differential complementary DNA cloning procedure to identify genes that may participate in long-term plasticity. We cloned 52 cDNAs of genes induced by the glutamate analogue kainate in the hippocampus dentate gyrus. The number of these candidate plasticity-related genes (CPGs) is estimated to be 500-1,000. One of the cloned CPGs (16C8), encoding a protease inhibitor, is induced by a stimulus producing long-term potentiation and during dentate gyrus development; a second, cpg1, is dependent on activation of the NMDA (N-methyl-D-aspartate) receptor for induction and encodes a new small, dentate-gyrus-specific protein. Seventeen of the cloned CPGs encode known proteins, including six suggesting that strong neuronal activation leads to de novo synthesis of vesicular and other synaptic components.

Enhancement of fluorescein perfusion in experimental skin flaps following postischemic washout with iloprost, urokinase, verapamil, and University of Wisconsin solution.

The enhancement of blood flow in experimental skin flaps following postischemic perfusion washout was investigated in rats. Unilateral island skin flaps based on the superficial epigastric vessels were raised and subjected to 6 hr of primary ischemia. Group 1 was designated as a control and did not undergo postischemic perfusion washout. In the remaining rats, postischemic washout was performed with one of five agents: Group 2--lactated Ringer's solution; Group 3--University of Wisconsin solution, an organ preservation medium; Group 4--verapamil, a calcium channel blocker; Group 5--urokinase, a thrombolytic agent; Group 6--iloprost, a stable prostacyclin analog. Two hours following perfusion washout, fluorometric analysis revealed a statistically significant enhancement of blood flow in Groups 4, 5, and 6, compared to Groups 2 and 3 (p < 0.05). Furthermore, a significant increase in skin surface fluorescence was demonstrated in all the flaps that underwent perfusion washout, compared to the control flaps (p < 0.05). By analyzing skin surface fluorescence, the enhancement of nutritive blood flow in flaps, following postischemic perfusion washout, was evaluated. This is the first study in which the above pharmacologic agents were compared in a quantitative manner.