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.

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.

Stratospheric hydroperoxyl measurements.

The hydroperoxyl radical (HO(2)) plays a key role in stratospheric chemistry through the HOx catalytic cycle of ozone destruction. Earlier measurements of stratospheric HO(2) have given mixed results; some measured mixing ratios greatly exceed theoretical predictions. Measurements of HO(2) have now been made with a balloon-borne farinfrared spectrometer. The measured daytime profile is in excellent agreement with theory up to 40 kilometers; above this level the measurements exceed theory by 30 percent, perhaps because of underprediction of ozone at these altitudes. The nighttime HO(2) profile is strongly depressed with respect to the daytime profile, in general agreement with theory.

Proportional release of norepinephrine and dopamine- -hydroxylase from sympathetic nerves.

Dopamine-beta-hydroxylase(DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, is localized in the vesicles containing catecholamine in sympathetic nerves. This enzyme is released with norepinephrine when the nerves to the guinea pig vas deferens are stimulated in vitro, and the amount of enzyme discharged increases as the length of stimulation periods increases. The amount of DBH released is proportional to the amount of norepinephrine released, and the ratio of norepinephrine to DBH discharged into the incubation medium is similar to that in the soluble portion of the contents of the synaptic vesicles from the vas deferens. These data are compatible with the release of the neurotransmitter norepinephrine and DBH from symnpathetic nerves by a process of exocytosis.

Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist.

The glucagon analog [l-N alpha-trinitrophenylhistidine, 12-homoarginine]-glucagon (THG) was examined for its ability to lower blood glucose concentrations in rats made diabetic with streptozotocin. In vitro, THG is a potent antagonist of glucagon activation of the hepatic adenylate cyclase assay system. Intravenous bolus injections of THG caused rapid decreases (20 to 35 percent) of short duration in blood glucose. Continuous infusion of low concentrations of the inhibitor led to larger sustained decreases in blood glucose (30 to 65 percent). These studies demonstrate that a glucagon receptor antagonist can substantially reduce blood glucose levels in diabetic animals without addition of exogenous insulin.

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.

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.

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.

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.

Glucagon response to hypoglycemia in sympathectomized man.

Hypoglycemia stimulates immunoreactive glucagon (IRG) secretion and increases the activity of the sympathetic nervous system. To ascertain if the augmented alpha cell activity evoked by glucopenia is mediated by the adrenergic nervous system, the glucagon response to insulin-induced hypoglycemia of five subjects with neurologically complete cervical transections resulting from trauma, thereby disrupting their hypothalamic sympathetic outflow, was compared to six healthy volunteers. In addition to clinical neurological evaluation, completeness of sympathectomy was verified by failure to raise plasma norepinephrine levels during hypoglycemia compared to the two- and threefold increase observed in controls. Total IRG response (IRG area above basal 0-90 min) and peak IRG levels achieved were the same in the quadriplegics and the controls. Although the glucagon rise tended to be slower, and the peak levels attained occurred later in the quadriplegic patients than in the controls, this response was appropriate for their sugar decline, which was slower and reached the nadir later than in the control subjects. These observations that the glucagon release during insulin-induced hypoglycemia is normal in subjects whose hypothalamic sympathetic outflow has been interrupted provide strong evidence that the sympathetic nervous system does not mediate the glucagon response to hypoglycemia.

A genetic analysis of the E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4.

The cellular transcription factor E2F appears to be a target for the regulatory action of the retinoblastoma tumor suppressor gene product. The recent isolation of the E2F1 cDNA clone, which encodes a polypeptide with properties characteristic of E2F, has now allowed a more detailed analysis of the regulation of E2F function by Rb as well as the Rb-related p107 protein and the adenovirus 19-kDa E4 gene product. Previous experiments have shown that each of these regulatory proteins can modulate the activity of cellular E2F. We find that each of these regulatory events can be mediated through the E2F1 product. Moreover, an examination of various E2F1 mutations reveals distinct specificities for these regulatory proteins. For instance, the ability of E4 to alter E2F1 function is dependent upon sequences within a putative leucine repeat of E2F1 as well as within the C-terminal acidic domain. In contrast, the leucine repeat element was not important for Rb- or p107-mediated inhibition of E2F1 activity. Although the C-terminal acidic domain of E2F1, previously shown to be important for Rb binding, appears to be a site for regulation of E2F1 by Rb and p107, point mutations within this region distinguish recognition by Rb and p107. These results underscore the complexity of E2F regulatory interactions and also demonstrate a qualitative distinction in the interactions of Rb and p107 with E2F1, perhaps reflective of functional differences.

E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo.

Loss of retinoblastoma (Rb) tumor suppressor function, as occurs in many cancers, leads to uncontrolled proliferation, an increased propensity to undergo apoptosis, and tumorigenesis. Rb negatively regulates multiple E2F transcription factors, but the role of the different E2F family members in manifesting the cellular response to Rb inactivation is unclear. To study the effect of deregulated E2F4 activity on cell growth control and tumorigenesis, transgenic mouse lines expressing the E2F4 gene under the control of a keratin 5 (K5) promoter were developed, and their phenotypes were compared to those of previously generated K5 E2F1 transgenic mice. In contrast to what has been observed in vitro, ectopically expressed E2F4 was found to localize to the nucleus and induce proliferation to an extent similar to that induced by E2F1 in transgenic tissue. Unlike E2F1, E2F4 does not induce apoptosis, and this correlates with the differential abilities of these two E2F species to stimulate p19(ARF) expression in vivo. To examine the role of E2F4 in tumor development, the mouse skin two-stage carcinogenesis model was utilized. Unlike E2F1 transgenic mice, E2F4 transgenic mice developed skin tumors with a decreased latency and increased incidence compared to those characteristics in wild-type controls. These findings demonstrate that while the effects of E2F1 and E2F4 on cell proliferation in vivo are similar, their apoptotic and oncogenic properties are quite different.

The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

E2F1 has both oncogenic and tumor-suppressive properties in a transgenic model.

Using a transgenic mouse model expressing the E2F1 gene under the control of a keratin 5 (K5) promoter, we previously demonstrated that increased E2F1 activity can promote tumorigenesis by cooperating with either a v-Ha-ras transgene to induce benign skin papillomas or p53 deficiency to induce spontaneous skin carcinomas. We now report that as K5 E2F1 transgenic mice age, they are predisposed to develop spontaneous tumors in a variety of K5-expressing tissues, including the skin, vagina, forestomach, and odontogenic epithelium. On the other hand, K5 E2F1 transgenic mice are found to be resistant to skin tumor development following a two-stage carcinogenesis protocol. Additional experiments suggest that this tumor-suppressive effect of E2F1 occurs at the promotion stage and may involve the induction of apoptosis. These findings demonstrate that increased E2F1 activity can either promote or inhibit tumorigenesis, dependent upon the experimental context.

E2F-1 gene transfer enhances invasiveness of human head and neck carcinoma cell lines.

The transcription factor E2F-1, a downstream regulator of the p16-cyclinD-Rb pathway, is required for cell cycle progression. Evidence shows that overexpression of E2F-1 can either promote or inhibit development of tumors, depending on tissue or experimental conditions. To study whether the E2F-1 gene plays a role in tumor progression, the expression of E2F-1 protein was evaluated in 10 human head and neck squamous cell carcinoma cell lines using Western blot analysis. In addition, the invasive ability of these cell lines was determined by evaluating the penetration of cell lines into the tracheal wall in an in vivo invasion assay using deepithelialized tracheas transplanted into the s.c. tissue of Scid mice. This study showed that the aggressive cell lines had higher expression of E2F-1 than the less invasive cell lines. To evaluate the hypothesis that E2F-1 enhances invasiveness, we selected two cell lines, SCC9 and SCC12, for a gene transfer experiment. These cell lines exhibited low invasive ability with low expression of E2F-1. Two stable clones with overexpression of transfected E2F-1 gene and two clones with their respective vector-alone control were selected from each cell line for in vivo invasion evaluation. The clones containing the transfected E2F-1 gene had significantly higher invasive ability than their respective vector-alone clones. Flow cytometry showed that parental, transfected E2F-1, and vector-alone cells had a similar proliferation pattern under normal culture conditions. Nevertheless, transfected E2F-1 cells exhibited a higher portion of cells in S phase than the control cells after serum-starvation and refeeding. The results indicated that overexpression of E2F-1 plays a positive role in cell cycle reentry from quiescence and is associated with increased in vivo invasiveness.

Regulation of E2F-1 gene expression by p130 (Rb2) and D-type cyclin kinase activity.

Previous experiments have demonstrated that the regulation of E2F-1 transcription factor activity is critical for the maintenance of normal cell proliferation control. Regulation of E2F-1 is accomplished through at least two mechanisms: posttranslational regulation by binding proteins such as Rb and transcriptional regulation of the E2F-1 gene. The E2F-1 gene promoter has recently been isolated to examine this latter aspect of E2F-1 regulation. Preliminary studies demonstrate that the E2F-1 promoter is under E2F-dependent negative control during the cell growth response, being transcriptionally repressed through E2F sites in G0 and early G1. We now demonstrate that the presence of an E2F DNA-binding complex containing the Rb-related p130 protein (Rb2) correlates with E2F-1 gene repression and that overexpression of p130 inhibits transcription from the E2F-1 promoter. Moreover, D-type cyclin-dependent kinase activity specifically activates the E2F-1 promoter by relieving E2F-mediated repression but is inhibited by coexpression of the cdk4 and cdk6 inhibitor p16 (CDKN2, MTS1, INK4). Taken together, these findings suggest that E2F-1 gene expression is controlled during cell cycle progression by a regulatory network involving at least one oncogene (cyclin D1) and several potential tumor suppressor genes.

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.

Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development.

In cell culture studies, overexpression of the E2F1 transcription factor has been shown to stimulate proliferation, induce apoptosis, and cooperate with an activated ras gene to oncogenically transform primary rodent cells. To study the effect of increased E2F1 activity on epithelial growth and tumorigenesis in vivo, transgenic mice expressing E2F1 under the control of a keratin 5 (K5) promoter were generated. Expression of E2F1 in the epidermis results in hyperplasia but does not inhibit terminal differentiation. In a transgenic line expressing high levels of E2F1, mice have decreased hair growth likely as a result of aberrant apoptosis in developing hair follicles. Coexpression of a cyclin D1 transgene with E2F1 augments epidermal hyperplasia and further disrupts hair follicle development suggesting that hypophosphorylated Rb antagonizes the proliferative and apoptotic-promoting activities of E2F1. Finally, the E2F1 transgene is found to cooperate with a v-Ha-ras transgene to induce skin tumors in double transgenic animals. These findings confirm that many of the activities ascribed to E2F1 through in vitro studies can be reproduced in vivo and demonstrate for the first time that deregulated E2F activity can contribute to tumor development.