Contrasting effects of an Mdm2 functional polymorphism on tumor phenotypes.

MDM2, an E3 ubiquitin ligase, is a potent inhibitor of the p53 tumor suppressor and is elevated in many human cancers that retain wild-type p53. MDM2 SNP309G is a functional polymorphism that results in elevated levels of MDM2 (due to enhanced SP1 binding to the MDM2 promoter) thus decreasing p53 activity. Mdm2SNP309G/G mice are more prone to spontaneous tumor formation than Mdm2SNP309T/T mice, providing direct evidence for the impact of this SNP in tumor development. We asked whether environmental factors impact SNP309G function and show that SNP309G cooperates with ionizing radiation to exacerbate tumor development. Surprisingly, ultraviolet B light or Benzo(a)pyrene exposure of skin shows that SNP309G allele actually protects against squamous cell carcinoma susceptibility. These contrasting differences led us to interrogate the mechanism by which Mdm2 SNP309 regulates tumor susceptibility in a tissue-specific manner. Although basal Mdm2 levels were significantly higher in most tissues in Mdm2SNP309G/G mice compared with Mdm2SNP309T/T mice, they were significantly lower in Mdm2SNP309G/G keratinocytes, the cell-type susceptible to squamous cell carcinoma. The assessment of potential transcriptional regulators in ENCODE ChIP-seq database identified transcriptional repressor E2F6 as a possible negative regulator of MDM2 expression. Our data show that E2F6 suppresses Mdm2 expression in cells harboring the SNP309G allele but not the SNP309T allele. Thus, Mdm2 SNP309G exhibits tissue-specific regulation and differentially impacts cancer risk.

Short communication: Jersey × Holstein crossbreds compared with pure Holsteins for body weight, body condition score, fertility, and survival during the first three lactations.

Crossbred cows (n=80) resulting from the use of Jersey (JE) semen on their pure Holstein (HO) dams were compared with pure HO cows (n=77) for body weight, body condition score, fertility, and survival during their first 3 lactations. Cows were in 2 research herds of the University of Minnesota and calved from September 2003 to June 2008. The JE × HO crossbred cows had significantly less body weight during the first (-56 kg), second (-67 kg), and third (-82 kg) lactations than pure HO cows. However, JE × HO cows had significantly greater body condition score during the first (2.94 vs. 2.84), second (2.97 vs. 2.84), and third (2.99 vs. 2.87) lactations than pure HO cows. For fertility, JE × HO cows had fewer days to first breeding during the first (-10.6d), second (-8.4d), and third (-12.3d) lactations than pure HO cows. Crossbred cows were not significantly different from pure HO cows for number of services during first lactation; however, JE × HO cows had significantly fewer services (2.2) than pure HO cows (2.7) during the second lactation. Also, JE × HO cows had significantly fewer days open than pure HO cows in the first (-24 d), second (-42 d), and third (-42 d) lactations. For survival, JE × HO cows were not significantly different from pure HO cows for percentage of cows calving a second time; however, a tendency existed for a higher percentage of JE × HO cows (63.8%) than pure HO cows (49.4%) to calve a third time, and a higher percentage of JE × HO cows calved a third time within 28, 34, and 40 mo of first calving than pure HO cows.

Short communication: Jersey × Holstein crossbreds compared with pure Holsteins for production, mastitis, and body measurements during the first 3 lactations.

Jersey (JE)×Holstein (HO) crossbred cows (n=76) were compared with pure HO cows (n=73) for 305-d milk, fat, and protein production, somatic cell score (SCS), clinical mastitis, lifetime production, and body measurements during their first 3 lactations. Cows were in 2 research herds at the University of Minnesota and calved from September 2003 to June 2008. Best prediction was used to determine actual production for 305-d lactations as well as lifetime production (to 1,220 d in the herd after first calving) from test-day observations. During first lactation, JE×HO cows and pure HO cows were not significantly different for fat plus protein production; however, JE×HO cows had significantly lower fat plus protein production during second (-25 kg) and third (-51 kg) lactation than pure HO cows. Nevertheless, JE×HO cows were not significantly different from pure HO cows for lifetime production or lifetime SCS. The JE×HO cows were not significantly different from pure HO cows for SCS and clinical mastitis during first and second lactations; however, JE×HO cows tended to have higher SCS (3.79) than pure HO cows (3.40), but significantly lower (-23.4%) clinical mastitis during third lactation. The JE×HO cows had significantly less hip height, smaller heart girth, less thurl width, and less pin width than pure HO cows during the first 3 lactations. Furthermore, JE×HO cows had significantly less udder clearance from the ground and significantly greater distance between the front teats than pure HO cows during their first 3 lactations.

Implantable micropump technologies for murine intracochlear infusions.

Due to the very small size of the mouse inner ear, 600 nL volume, developing effective, controlled infusion systems is quite challenging. Key technologies have been created to minimize both size and power for an implantable pump for murine intracochlear infusions. A method for coupling fine capillary tubing to microfluidic channels is presented which provides low volume, biocompatible interconnects withstanding pressures as high as 827 kPa (120 psi) and consuming less than 20 nL of volume exiting in-plane with the pump. Surface micromachined resistive bridges integrated into the flow channel for anemometry based flow rate measurement have been optimized for low power operation in the ultra-low flow rate regime. A process for creation of deformable diaphragms over pump chambers with simultaneous coating of the microfluidic channels has been developed allowing integration of a biocompatible fluid flow path. These advances represent enabling capabilities for a drug delivery system suitable for space constrained applications such as subcutaneous implantation in mice.

Birth traits of pure Holstein calves versus Montbeliarde-sired crossbred calves.

Pure Holstein calves and Montbeliarde-sired crossbred calves from multiparous Holstein dams were compared for gestation length, calf weight at birth, calving difficulty, and stillbirth in 2 research herds of the University of Minnesota. The Montbeliarde-sired calves from multiparous Holstein dams had significantly longer gestation lengths (283.2 d) than Holstein-sired calves from Holstein dams (278.4 d), and Montbeliarde-sired calves from multiparous Holstein dams had significantly greater calf weight at birth (48.3kg) compared with Holstein-sired calves from Holstein dams (43.3kg). However, calves sired by Montbeliarde bulls were not significantly different from calves sired by Holstein bulls for calving difficulty and stillbirth. In addition, Jersey x Holstein crossbred cows mated to Montbeliarde artificial insemination (AI) bulls were compared with pure Holstein cows mated to Holstein AI bulls for gestation length, calf weight at birth, calving difficulty, and stillbirth at their first 3 calvings. Gestation length was significantly longer for Jersey x Holstein cows bred to Montbeliarde bulls than for pure Holstein cows bred to Holstein bulls at first calving (280.3 versus 277.7 d) and second and third calving (282.2 versus 278.6 d); however, Jeresy x Holstein cows bred to Montbeliarde AI bulls were not significantly different from pure Holstein cows bred to Holstein AI bulls for calf weight at birth, calving difficulty, and stillbirth at the first 3 calvings.

Evaluation of Gram-positive rod surveillance for early anthrax detection.

Since 2003, Connecticut laboratories have reported Gram-positive rod (GPR) isolates detected within 32 h of inoculation from blood or cerebrospinal fluid. The objectives were to rapidly identify inhalational anthrax and unusual Clostridium spp. infections, and to establish round-the-clock laboratory reporting of potential indicators of bioterrorism. From 2003 to 2006, Connecticut's GPR surveillance system identified 1134 isolates, including 657 Bacillus spp. (none B. anthracis) and 241 Clostridium spp. Reporting completeness and timeliness improved to 93% and 92%, respectively. Baseline rates of Bacillus spp., Clostridium spp. and other GPR findings have been established and are stable. Thus far, no cases of anthrax and no unusual clusters of Clostridium spp. have been detected by the GPR surveillance system. This system would probably have confirmed the inhalational anthrax case in Pennsylvania in 2006 3 days sooner than traditional reporting. Using audits and ongoing evaluation, the system has evolved into a highly functional 24/7 laboratory telephone reporting system with almost complete reporting.

Crossbreds of Jersey x Holstein compared with pure Holsteins for body weight, body condition score, dry matter intake, and feed efficiency during the first one hundred fifty days of first lactation.

Jersey x Holstein crossbred (JxH) cows (n = 24) were compared with pure Holstein cows (n = 17) for body weight, body condition score, dry matter intake (DMI), and feed efficiency during the first 150 d of first lactation. Cows were housed in the University of Minnesota dairy facility at the St. Paul campus and calved from September 2004 to January 2005. The JxH cows were mated by artificial insemination with Montbeliarde bulls, and Holstein cows were mated by artificial insemination with Holstein bulls. Cows were weighed and body condition was scored every other week. Cows were individually fed a TMR twice daily, and feed refusals were measured once daily. The DMI of cows was measured daily and averaged across 7-d periods. Milk production and milk composition were from monthly Dairy Herd Improvement records. Best Prediction was used to calculate actual production (milk, fat, protein) for each cow from the 4th to 150th day of first lactation. The JxH cows had significantly less body weight (467 vs. 500 kg) and significantly higher body condition scores (2.90 vs. 2.76) than pure Holstein cows. The JxH cows had significantly less milk production (4,388 vs. 4,644 kg) during the 4th to 150th day of lactation than did pure Holstein cows. However, fat plus protein production during the first 150 d of lactation was not significantly different for JxH (302 kg) and Holstein (309 kg) cows. The JxH and pure Holstein cows did not differ significantly for daily DMI (22.0 vs. 22.7 kg, respectively), and the JxH (4.7%) and pure Holstein (4.5%) cows consumed similar DMI based on percentage of body weight. Consequently, feed efficiency for the 4th to 150th day of lactation did not differ for JxH and pure Holstein cows.

Transgenic expression of E2F3a causes DNA damage leading to ATM-dependent apoptosis.

Many early stage human tumors display markers of a DNA-damage response (DDR), including ataxia telangiectasia mutated (ATM) kinase activation. This suggests that DNA damage accumulates during the process of carcinogenesis and that the ATM-dependent response to this damage may function to suppress cancer progression. The E2F3a transcription factor plays an important role in regulating cell proliferation and is amplified in a subset of human cancers. Similar to human premalignant lesions, we find activated ATM and other markers of the DDR in the hyperplastic epidermis of transgenic mice expressing E2F3a through a keratin 5 (K5) promoter. Primary keratinocytes from K5 E2F3a transgenic mice contain increased levels of DNA breaks compared to wild-type cells. E2F3a overexpression also induced DNA damage in primary human fibroblasts that was inhibited by blocking DNA replication. The absence of ATM impaired apoptosis induced by E2F3a and treating K5 E2F3a transgenic mice with caffeine, an inhibitor of ATM and Rad3-related (ATR), promoted skin tumor development. These findings demonstrate that the deregulated expression of E2F3a causes DNA damage under physiological conditions and indicate that the ATM-dependent response to this damage is important for the induction of apoptosis and tumor suppression.

Crossbreds of Jersey x Holstein compared with pure Holsteins for production, fertility, and body and udder measurements during first lactation.

Jersey x Holstein crossbreds (JxH; n = 76) were compared with pure Holsteins (n = 73) for 305-d milk, fat, and protein production; conception rate; days open; proportion of cows pregnant within fixed intervals postpartum; and body and udder measurements during first lactation. Cows were housed at 2 research locations of the University of Minnesota and calved from September 2003 to May 2005. The JxH were mated to Montbeliarde sires, and Holstein cows were mated to Holstein sires. Best Prediction was used to determine actual production (milk, fat, and protein) for 305-d lactations with adjustment for age at calving, and records less than 305 d were projected to 305 d. The JxH (274 kg) and pure Holsteins (277 kg) were not significantly different for fat production, but JxH had significantly less milk (7,147 vs. 7,705 kg) and protein (223 vs. 238 kg) production than pure Holsteins. The JxH had significantly fewer days open than pure Holsteins (127 vs. 150 d). Also, a significantly greater proportion of JxH were pregnant at 150 and 180 d postpartum than pure Holsteins (75 vs. 59% and 77 vs. 61%, respectively). The JxH had significantly less body weight (60 kg) at calving, but significantly greater body condition (2.80 vs. 2.71). Furthermore, JxH had significantly less udder clearance from the ground to the bottom of the udder than pure Holsteins (47.7 vs. 54.6 cm), and greater distance between front teats (15.8 vs. 14.0 cm) than pure Holsteins during first lactation.

E2F1 suppresses skin carcinogenesis via the ARF-p53 pathway.

The E2F1 transcription factor, which is deregulated in most human cancers by mutations in the p16-cyclin D-Rb pathway, has both oncogenic and tumor-suppressive properties. This is dramatically illustrated by the phenotype of an E2F1 transgenic mouse model that spontaneously develops tumors in the skin and other epithelial tissues but is resistant to papilloma formation when subjected to a two-stage carcinogenesis protocol. Here, this E2F1 transgenic model was used to further explore the tumor-suppressive property of E2F1. Transgenic expression of E2F1 was found to inhibit ras-driven skin carcinogenesis at the promotion stage independent of the type of promoting agent used. E2F1 transgenic epidermis displayed increased expression of p19(ARF), p53, and p21(Cip1). Inactivation of either p53 or Arf in E2F1 transgenic mice restored sensitivity to two-stage skin carcinogenesis. While Arf inactivation impaired tumor suppression and p21 induction by E2F1, it did not reduce the level of apoptosis observed in E2F1 transgenic mice. Based on these findings, we propose that E2F1 suppresses ras-driven skin carcinogenesis through a nonapoptotic mechanism involving ARF and p53.

Ruptured "giant" supratentorial dermoid cyst.

Intracranial dermoid and epidermoid cysts are rare lesions formed from the inclusion of ectodermal elements during neural tube closure. Although not entirely consistent, imaging characteristics on CT and MRI can aid differentiation of dermoids and epidermoids, as can age at presentation, location and tendency to rupture. The distinction between dermoid and epidermoid lesions is important prognostically and may impact on surgical management as a subtotally resected dermoid is less likely to recur than its epidermoid counterpart. The distinction of large dermoid lesions as "giant" adds little to information regarding the natural history or prognosis of these lesions and should be abandoned.

Comparison of feeding corn silages from leafy or conventional corn hybrids to lactating dairy cows.

Three corn hybrids (Pioneer 36F30, Mycogen TMF2450, and Mycogen TMF2404) were compared for yield and quality traits, and lactation performance and apparent digestibility by Holstein cows. The three corn silages were harvested at a target of 33 to 35% dry matter. Before harvest, six corn plants were randomly selected for plant fractionation. Grain-to-stover ratios were 0.92, 0.70, and 0.95 for the 36F30, TMF2450, and TMF2404 corn plants, respectively. Fifty-two multiparous Holstein cows were placed on a 120-d lactation trial after a 21-d covariate diet. Cows were blocked by calving date and randomly assigned within block to one of three dietary treatments, containing approximately 40% (dry matter basis) corn silage. Milk yield, milk components, and dry matter intake did not differ among dietary treatments. In vitro true and neutral detergent fiber digestibilities were numerically higher for TMF2404 than the other corn silage hybrids. Apparent total-tract crude protein and neutral detergent fiber digestibilities, as measured by acid insoluble ash, were higher for TMF2450 than the other two hybrids, but starch digestibility was not different between the corn silage dietary treatments. Although small differences in nutrient content and digestibility existed among corn silage hybrids, inclusion of these leafy hybrids in lactating cow diets at 40% of the dietary dry matter did not have a significant impact on lactation performance of dairy cattle.

Comparison of urine to sweat patch test results in court ordered testing.

A former cocaine and methamphetamine abuser was continuously monitored with both sweat patch and urine testing for approximately 6 months. Thirteen sweat patches were applied and collected, five were positive for cocaine and/or methamphetamine, but all the urine specimens collected were negative at the analytical cut-off levels. The high incidence of false positive sweat patch tests in relation to the sensitivity, specificity, and efficiency of the sweat patch assay is discussed. Possible mechanisms, which can lead to false positive results, are presented. The results of our study raise further questions about the preferential use of the sweat patch in detecting new episodes of drug use in formerly chronic drug users.

Tonic sympathetic support of metabolic rate is attenuated with age, sedentary lifestyle, and female sex in healthy adults.

We recently demonstrated in young adult humans that the sympathetic nervous system contributes to the control of resting metabolic rate via tonic beta-adrenergic receptor stimulation. In the present follow-up study we determined the respective effects of age, habitual exercise status, and sex on this regulatory mechanism. Resting metabolic rate (ventilated hood, indirect calorimetry) was determined in 55 healthy sedentary or endurance exercise-trained adults, aged 18-35 or 60-75 yr (29 men and 26 women), before (baseline) and during the infusion of either a nonselective beta-adrenergic receptor antagonist (propranolol) or saline (control). Relative to baseline values, during beta-adrenergic receptor antagonism resting metabolic rate adjusted for fat-free mass was reduced to a lesser extent in older (mean +/- SE, -130 +/- 46 kJ/d) compared with young (-297 +/- 46) adults, sedentary (-151 +/- 50) compared with endurance exercise-trained (-268 +/- 46) adults, and women (-105 +/- 33) compared with men (-318 +/- 50; all P < 0.01). Reductions in resting metabolic rate during beta-adrenergic receptor antagonism were positively related to higher baseline resting metabolic rate and plasma catecholamine concentrations and negatively related to adiposity (all P < 0.05). Resting metabolic rate was unchanged in response to saline control in all groups. These results provide experimental support for the hypothesis that aging, sedentary living, and female sex are associated with attenuated sympathetic nervous system support of resting metabolic rate in healthy adult humans.

Myc lacks E2F1's ability to suppress skin carcinogenesis.

Myc and E2F1 can each stimulate proliferation, induce apoptosis, and contribute to oncogenic transformation. However, only E2F1 has been shown to have a tumor suppressive activity under some conditions. To examine the potential of Myc to suppress tumorigenesis under one of the conditions in which E2F1 functions to suppress tumorigenesis, transgenic mice expressing Myc under the control of a keratin 5 (K5) promoter were generated. Like K5 E2F1 transgenic mice, K5 Myc transgenic mice have hyperplastic and hyperproliferative epidermis and develop spontaneous tumors in the skin and oral epithelium. In addition, K5 Myc and K5 E2F1 transgenic mice both display aberrant, p53-dependent apoptosis in the epidermis. It has been demonstrated that deregulated expression of E2F1 in the epidermis of transgenic mice inhibits tumorigenesis in a two-stage skin carcinogenesis assay. In sharp contrast to those results, deregulated expression of Myc in the epidermis of transgenic mice resulted in an enhanced response to two-stage skin carcinogenesis. We conclude that while Myc and E2F1 have similar proliferative, apoptotic and oncogenic properties in mouse epidermis, Myc lacks E2F1's tumor suppressive property. This suggests that E2F1's unique ability to inhibit skin carcinogenesis is not simply a consequence of promoting p53-dependent apoptosis.

Drugs in postmortem adipose tissues: evidence of antemortem deposition.

INTRODUCTION: Drug concentration measured in postmortem adipose tissue may or may not reflect antemortem concentration. To examine the possibility of whether the presence of basic drugs in adipose tissue is the result of postmortem change, we examined: tissues with and without livor mortis, concentration gradients within the adipose layer, and the stability of drug concentrations during the postmortem period. CASE REPORTS: Five drug-related deaths with case histories and analytical data are presented. Adipose tissues with and without livor mortis from the thigh area of the same decedent were analyzed for cocaine. The cocaine concentration of the tissue exhibiting 4+ livor was equivalent to the concentration observed in tissue without livor. Analyses of cross sections of adipose tissues containing cocaine and methamphetamine disclosed that drug concentrations were equally distributed throughout the layer, from just beneath the dermis to directly above the muscle. When morphine and temazepam concentrations were measured in adipose tissues collected from similar sites, but at different times, from the same cadaver, they remained essentially the same over 3 days (approximately 80 h). CONCLUSIONS: Since concentrations were the same in areas with and without livor mortis, the possibility of redistribution into adipose from blood or vascular channels is eliminated. The absence of a concentration gradient within the adipose layer rules out diffusion or permeation from muscle into the adipose layer, and the failure of morphine or temazepam concentration to change over time indicates that drugs in the adipose tissue are stable during the postmortem interval. Our findings support the notion that drugs identified in postmortem adipose tissue are there because of antemortem deposition and not because of any postmortem change or event.

Deregulated expression of DP1 induces epidermal proliferation and enhances skin carcinogenesis.

E2F transcription factors have been implicated in several cellular processes, including proliferation, apoptosis, and oncogenic transformation. A functional E2F factor consists of a heterodimer containing an E2F polypeptide (E2F1-E2F6) and a DRTF1-polypeptide (DRTF1-polypeptide-1 (DP1) or DRTF1-polypeptide-2). It is the E2F subunit that supplies the transcriptional activation domain and the motif involved in binding to members of the retinoblastoma tumor suppressor family. The role of the DP subunit in regulating E2F-dependent activities is not completely understood. To examine the properties of DP1 in vivo, we generated transgenic mouse lines expressing DP1 under the control of a keratin 5 (K5) promoter. Overexpression of DP1 in basal layer keratinocytes caused mild hyperplasia and hyperproliferation of the epidermis but did not result in increased apoptosis or spontaneous tumor development. Coexpression of DP1 with E2F1 or E2F4 in the epidermis of bigenic mice modestly enhanced proliferation and apoptosis over the levels induced by E2F1 or E2F4 expression alone. In a two-stage chemical carcinogenesis assay, more and larger skin tumors developed in K5 DP1 transgenic mice than in nontransgenic mice. These findings show that in this in vivo model, deregulated expression of DP1 on its own induced proliferation and enhanced carcinogenesis.

Molecular cloning and characterization of a novel mouse epidermal differentiation gene and its promoter.

The transcription factor E2F1 is an important regulator of cell proliferation, apoptosis, and differentiation. A novel mouse gene (Eig3) was originally identified as up-regulated in E2F1-overexpressing keratinocytes by the rapid analysis of gene expression technique. An apparently full-length cDNA and a 2.8-kb genomic fragment containing the entire gene have been cloned. Sequence comparisons suggest that Eig3 is homologous to a human epidermal differentiation gene, XP5, and belongs to a family of at least 10 murine genes that are related to the small proline-rich genes involved in skin differentiation. Eig3 was expressed in adult mouse stomach and epidermis and overexpressed in keratinocytes transgenic for E2F1 or E2F4, but not in c-myc transgenics. Interestingly, Eig3 expression was highly increased in mouse skin papillomas but not in squamous carcinomas. Since there is no E2F consensus binding sequence in the promoter or first intron of Eig3, E2F regulation may be indirect.

Effects of pulsatile intravenous insulin therapy on the progression of diabetic nephropathy.

The purpose of this study was to assess the effects of pulsatile intravenous insulin therapy (PIVIT) on the progression of diabetic nephropathy in patients with type 1 diabetes mellitus (DM). This 18-month multicenter, prospective, controlled study involved 49 type 1 DM patients with nephropathy who were following the Diabetes Control and Complications Trial (DCCT) intensive therapy (IT) regimen. Of these, 26 patients formed the control group (C), which continued on IT, while 23 patients formed the treatment group (T) and underwent, in addition to IT, weekly PIVIT. Blood pressure in all patients was maintained below 140/90 mm Hg on antihypertensive medication, preferentially using angiotensin-converting enzyme (ACE) inhibitors. All study patients were seen in the clinic weekly for 18 months, had monthly glycohemoglobin (HbA1c), and every 3 months, 24-hour urinary protein excretion and creatinine clearance (CrCl) determinations. The HbA1c levels declined from 8.61% +/- 0.33% to 7.68% +/- 0.31% (P = .0028) in the T group and from 9.13% +/- 0.36% to 8.19% +/- 0.33% (P = .0015) in the C group during the study period. CrCl declined significantly in both groups, as expected, but the rate of CrCl decline in the T group (2.21 +/- 1.62 mL/min/yr) was significantly less than in the C group (7.69 +/- 1.88 mL/min/yr, P = .0343). We conclude that when PIVIT is added to IT in type 1 DM patients with overt nephropathy, it appears to markedly reduce the progression of diabetic nephropathy. The effect appears independent of ACE inhibitor therapy, blood pressure, or glycemic control.

cis-Dichloro(dimethyl sulfoxide-S)(2-methoxypyridine-N)platinum(II).

The title complex, [PtCl(2)(C(6)H(7)NO)(C(2)H(6)OS)], exhibits square-planar geometry. The plane of the pyridine ring makes a dihedral angle of 67.2 (3) degrees with the square plane of the metal center. The S-O bond is nearly aligned with the adjacent Pt-N bond, leaving the methyl groups of the dimethyl sulfoxide ligand to stagger the Pt-Cl bond.