Food, energy, and the environment.

During the 2009 annual meeting of the Poultry Science Association, a symposium entitled "Global Views of New Agriculture: Food, Energy, and the Environment" was held that focused on several major issues affecting agriculture. Issues included future funding for basic agricultural research, sustainability, bioenergy, and their effects on global food markets. In many ways, a subtitle for the symposium could have been "Agriculture-Why What We Do Matters." It matters because of the fiscal and physical realities the planet will face in the coming decades relative to human population growth and the increasing demands to feed a hungry world. The challenges are daunting and the technologies to address them will require us to reevaluate the structure and policies we have established relative to agricultural research. In this case, change is all the more difficult because the traditional model of agricultural research has been so successful. One only needs to note the remarkable increases in productivity of the past half century of commodities such as corn and soybeans or feed efficiencies among broilers, laying hens, and turkeys to recognize the significant advancements that have been achieved. However, these historic gains have frequently required increased inputs, most notably fossil fuels. Food production in the future will likely be confronted with concerns involving energy, water, climate change, and the threat of agroterrorism. For example, we will need to develop crops that are more drought-resistant and more tolerant to a wider range of salinities as access to fresh water becomes more problematic. Animal agriculture will also need to adapt to diets composed of atypical feedstuffs. Whether future generations will inherit a world described by Paul Roberts in his books The End of Oil and The End of Food will be in part determined by the research model we adopt in the near term.

Elevated levels of inducible heat shock 70 proteins in human brain.

Differential expression of heat shock genes can modulate protein folding and stress-related cell death. There have been no comparisons of their levels of expression in animals and humans. Levels of expression of heat shock 70 genes in human brain were compared to levels in non-stressed and heat-stressed brain of rat. Levels of hsp70 proteins in human brain were 43-fold higher than in non-stressed rat brain and 14-fold higher than highest induced levels in brains of heat-shocked rats. Levels of constitutively synthesized hsc70 proteins were approximately 1.5-fold higher in human than in rat. Higher levels of hsp70 proteins in human brain may serve to protect brain cells against stress-related death or dysfunction throughout the lifespan.

Production of chick germline chimeras from fluorescence-activated cell-sorted gonocytes.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

Isolation of chicken primordial germ cells using fluorescence-activated cell sorting.

Presently, it is difficult to undertake germ line modification of the chicken with primordial germ cells (PGC) because it has been difficult to efficiently fractionate the PGC from the total somatic cell population. The objective of this study was to develop a method that allows isolation of an enriched population of viable PGC from embryonic blood and embryonic gonadal tissue. Blood was harvested from early chick embryos (stages 13 to 15), and cells were liberated from the gonads of stage 27 chick embryos. Subsequently, viable PGC were labeled with anti-stage-specific embryonic antigen-1 (SSEA-1), which was detected with goat-anti-mouse IgM-fluorescein isothiocyanate. Fluorescently labeled cells were sorted from the unlabeled cells using fluorescence-activated cell sorting (FACS), and the identities of the PGC were confirmed using periodic acid-Schiff (PAS) staining or anti-embryonic mouse antigen-1 (EMA-1) staining followed by microscopic evaluation. Finally, PGC were sorted from somatic cells of sex-identified embryos. Less than 0.1% of the blood cell population was collected as SSEA-1-positive cells. Similarly, approximately 2% of the gonadal cell population were collected as SSEA-1-positive cells. Therefore, fewer (-1,000 to 9,000) PGC were recovered from each isolate. Placing the sorted SSEA-1-positive cells on a glass slide from a microcentrifuge tube resulted in a recovery rate of 53 to 73% relative to the number detected by FACS. Furthermore, the proportions of sorted cells that stained with PAS or anti-EMA-1 following sorting were 92+/-4% PAS positive and 94+/-1% anti-EMA-1 positive. Finally, the sorted SSEA-1-positive cells were maintained in vitro to demonstrate their viability after sorting. It was demonstrated that it is possible to label blood and gonadal chicken PGC with SSEA-1 and subsequently to sort viable SSEA-1-positive PGC from somatic cells.

Cell column chromatography: a new research tool to quantify cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

OBJECTIVE: The roles of individual types of cerebral cells in contributing to brain edema are undefined. The objective of this study was to determine the role of cerebral cell-column chromatography in quantifying cell volumes of individual cerebral cell lines, under chemically-induced anoxia/re-oxygenation (A/R). METHODS: Cerebral endothelial cells (4 experiments) or type II astrocytes (4 experiments) were cultured to confluence on microcarrier beads. A chromatographic cell-column of 1.5 cm height was filled with non-treated cell-covered beads. The column was perfused at 1 ml/min with a balanced perfusate for one hour (Baseline). The perfusate was then switched to that containing 5 mM thioglycolic acid for one hour (Anoxia). Then the column was perfused with the normal perfusate for another two hours (Re-oxygenation). The total free space in the column, reversely reflecting cell volumes, was determined by averaged transit time (TTa) of a non-permeable flow tracer blue dextran. Decreased TTa means that cells swell, and vice versa. RESULTS: TTa in endothelial cell columns increased with a peak at 60 minutes of re-oxygenation. TTa in astrocyte columns decreased with a nadir at 30 minutes of re-oxygenation. CONCLUSION: Cell column chromatography can be used to determine the cerebral cell volume changes following chemically-induced anoxia/re-oxygenation.

Ifenprodil treatment is associated with a down-regulation of brain aquaporin 4 following cardiac arrest in rats.

OBJECTIVES: Ifenprodil, a NMDA receptor polyamine site antagonist, reduces experimental cardiac arrest (CA)-elicited brain edema, which is associated with an up-regulation of aquaporin 4 (AQP4), a brain water-selective channel. However, the interacting roles of NMDA receptors and AQP4 in CA-elicited brain edema are unknown. The objective of this study was to test our hypothesis that ifenprodil treatment is associated with a down-regulation of brain AQP4. METHODS: Twenty-five rats were assigned to normal controls (group 1, n = 6) or subjected to eight minutes of asphyxial CA treated with placebo (group 2, n = 9) or ifenprodil (group 3, n = 10). Ifenprodil at 10 mg/kg or normal saline of equal volume was given intraperitoneally, 5 minutes before CA. The density of AQP4 protein and actin bands was scanned and expressed as the ratios of the optical density of AQP4 relative to that of actin. The ANOVA analysis was used to compare the group differences. RESULTS: The ratios of the optical density of AQP4 to that of actin were 0.88 +/- 0.06 in group 1, 1.11 +/- 0.08 in group 2 (p < 0.05 vs. group 1), and 0.78 +/- 0.04 in group 3 (p < 0.01 vs. group 2; NS vs. group 1). CONCLUSION: Ifenprodil given before CA is associated with a downregulation of brain AQP4 in rats.

Effects of vitamin C supplementation on plasma ascorbic acid and oxalate concentrations and meat quality in swine.

Two experiments were conducted to determine the effects of vitamin C supplementation 48 h before slaughter on plasma ascorbic acid and oxalate concentrations and its effect on pork quality. In Exp. 1, 16 pigs (87.8+/-2.13 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 1,000 mg/L; or 3) 2,000 mg/L supplemented in the drinking water for a 48-h period. This was then followed by an additional 48-h period without supplemental vitamin C. Vitamin C increased plasma ascorbic acid concentrations (11.6, 19.5, and 23.4 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P < 0.05) within 6 h of supplementation. Plasma ascorbic acid concentrations from treated pigs decreased and did not differ from those of control pigs (13.7, 18.2, and 18.6 microg/mL for 0, 1,000, and 2,000 mg/L of vitamin C; P = 0.30) within 2 h of ending supplementation. No differences in plasma ascorbic acid concentrations were found between the two levels of supplementation. Vitamin C did not affect plasma oxalate or cortisol; however, cortisol tended to increase quadratically (P = 0.077) with vitamin C after 96 h. In Exp. 2, 30 pigs (107.5+/-0.54 kg BW) were blocked by sex and weight and assigned randomly within block to one of three vitamin C treatments: 1) control; 2) 500 mg/L; or 3) 1,000 mg/L supplemented in the drinking water 48 h before slaughter. Pigs were slaughtered 4 to 5 h after vitamin C supplementation ended, and loin samples were collected for meat quality measurements. At the time of slaughter, no differences in plasma ascorbic acid or cortisol were observed, but oxalate tended (P = 0.074) to increase quadratically with increasing vitamin C. Muscle ascorbic acid at slaughter and lactic acid in muscle at 0 and 1.5 h after slaughter were not different; however, lactic acid increased (P = 0.048) quadratically at 24 h after slaughter. Vitamin C did not affect initial or ultimate pH. Initial fluid loss (P = 0.041), and fluid loss on d 4 (P = 0.014) and 8 (P = 0.076) of simulated retail display; L* on d 0 (P = 0.038), 4 (P = 0.010), and 8 (P = 0.051); a* on d 0 (P = 0.021); and b* on d 0 (P = 0.006), 4 (P = 0.035), and 8 (P = 0.017) were negatively affected in a quadratic manner when vitamin C was supplemented. Vitamin C tended (P = 0.086) to increase oxidation in chops on d 0, but not d 4 or 8. Results indicate that on-farm supplementation of vitamin C was generally not effective in improving pork quality, which may be related to timing relative to slaughter.

The virtual revolution: implications for academe.

The global expansion and acceptance of the Internet as been unprecedented. The emergence of the potential for distance learning (DL) has altered the way in which faculty, university administrators, and for-profit corporations view the educational process. In 1998, nearly 80% of public 4-yr institutions offered some DL courses. However, DL courses in agriculture and natural resources represented less than 1% of the total enrollment. Like any technology that ushers in a new era of change, DL has attracted enthusiastic supporters and detractors. Few view DL with neutrality. It is this divergence of opinion that has fueled the debate over the academic value of DL. A valid evaluation of the educational benefits or deficiencies of DL may require additional long-term studies. For some academic traditionalists, DL is viewed as the fusion of education and commerce and borders on the repugnant. Others embrace DL not only because it may provide a source of much needed revenue, but also because it allows for the low-cost delivery of information to a nontraditional pool of students. Well-funded, private, for-profit organizations and universities have developed a number of DL models. Some hybrid DL models exist in which public institutions have created independent for-profit corporations to develop and distribute their web-based courses. The question is not if DL will be a part of the educational landscape; it surely will. The challenge is to define the role DL can most effectively fulfill.

Improvement of poult performance following Bordetella avium challenge by administration of a novel oxy-halogen formulation.

The ability of a novel oxy-halogen formulation (OHF) to alter the development of bordetellosis (turkey coryza) in large white turkey poults was assessed. Bordetella avium (BA)-infected (1-day-of-age) and noninfected control poults received 0, 0.008%, or 0.016% of an OHF continuously in the drinking water. At 4, 7, 10, 14, and 17 days of age, reisolation of BA from infected poults was attempted. Infected poults receiving 0.016% OHF exhibited significantly lower cumulative BA reisolation rates (90%) when compared with infected poults receiving 0 (96.7%) or 0.008% OHF (100%). At 7, 14, and 17 days of age, infected poults in the OHF-treated groups were significantly heavier than those BA-challenged poults receiving control water. Feed utilization was significantly improved from hatch to 7 days of age in BA-infected poults receiving OHF when compared with infected poults receiving control water. Clinical symptoms were severe only in untreated, infected poults and were mild or absent in all others. Damage to the tracheal epithelium, as measured by scanning electron microscopy, paralleled the clinical signs. Tracheal epithelial damage was virtually eliminated by OHF administration in infected poults. These results suggest that OHF treatment ameliorates many of the symptoms frequently associated with bordetellosis in young turkeys.

Providing credentials to our graduates.

Provision of meaningful professional credentials to Poultry Science graduates requires the sharing of expertise among a number of departments. Increased access to specialized poultry courses will assist in assuring that all graduates have excellent technical credentials. Greater access could be brought about through distance learning materials developed on a regional basis. Professional credentials will protect the marketability of our graduates in a changing industry and provide them a starting point for continued professional development. All departments, large and small, can claim an essential niche in the instruction and examination process. Another benefit is that to maintain their professional status, graduates would periodically participate in seminars and short courses and be encouraged to maintain active memberships in associations such as the Poultry Science Association.

Educational opportunities and challenges in poultry science: impact of resource allocation and industry needs.

Because the number of Poultry Science departments in the U.S. has declined dramatically, and because scientist years and research funding for poultry, relative to other commodities, have also declined, a survey of poultry meat companies was conducted. Objectives of the survey were: to evaluate corporate concern over the status of Poultry Science departments, to categorize hiring patterns, to determine expectations for prerequisite skills of graduates, and to ascertain attitudes toward hiring of Associate-degreed students (A.S.). A two-page survey was distributed to corporate Vice Presidents or Directors of Human Resources of the 17 largest broiler and 10 largest turkey companies. When asked to gauge the difficulty they encountered in locating adequate numbers of Poultry Science graduates, 83% noted at least some difficulty. All respondents indicated concern over the loss of poultry programs in the U.S. and 44% noted "extreme" concern. There appears to be little resistance to hiring 2-yr A.S. degree graduates in Poultry Science. The relative scarcity of these programs is demonstrated by the fact that only one-third of the respondents had ever hired A.S. degree graduates. However, greater than 80% of the firms indicated they would hire these students. Finally, communication and business skills were more highly rated by human resources management than technical ability in Poultry Science. Given these results, academic programs must: develop curricula that reflect market-place expectations, enhance the efficiency of resource utilization, embrace new technologies that provide novel methods for information delivery, and reassess cooperative linkages among industrial and governmental organizations.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum.

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Developmental characterization of thymosin beta 4 and beta 10 expression in enriched neuronal cultures from rat cerebella.

The beta 4 and beta 10 thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin beta 4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin beta 10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the beta-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both beta-thymosins were initially expressed in granule cells, although expression, especially of thymosin beta 4, was truncated compared with the in vivo time course. As in vivo, thymosin beta 4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin beta 10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the beta-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating beta-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.

Heat-shock 70 messenger RNA levels in human brain: correlation with agonal fever.

Systematic review of antemortem clinical information on randomly selected Alzheimer disease (AD) patients revealed that approximately 40% of the patients had a recorded fever of > or = 39.2 degrees C at or near death. Using isolation and quantitation techniques appropriate for analysis of human brain mRNAs, we found that low levels of inducible heat-shock protein 70 (hsp70) mRNAs were present in cerebellum of afebrile AD patients and that mRNA levels were usually lower in two brain regions affected in AD, i.e., hippocampus and temporal cortex. Levels of hsp70 mRNAs were increased three- to 33-fold in cerebellum of febrile patients compared with levels in patients whose recorded temperatures were < or = 37.5 degrees C. Levels of hsp70 mRNAs were also increased in hippocampus and cortex of these febrile patients, but to a lesser extent than cerebellum. Heat-shock cognate 70 (hsc70) mRNAs were present at highest levels in afebrile cerebellum and were also present in the other brain regions. In cerebellum of patients with the highest temperatures, hsc70 mRNAs were induced severalfold over basal levels. Although there was a low and variable induction of hsc70 mRNAs in temporal cortex of these patients, there was no evidence for any induction in hippocampus. Increased heat-shock 70 mRNA levels did not correlate with hypoxia, coma, hypertension, hypoglycemia, seizures, or medication. These results indicate that a specific agonal stress, namely fever, can increase the levels of heat shock 70 mRNAs in AD brain; however, there is no evidence to suggest that affected regions of AD brain have higher overall levels of these mRNAs. Failure to obtain adequate agonal state information could result in inaccurately identifying short-term stress-related changes in postmortem brain as neuropathology characteristic of a chronic disease state.

Cell size and the heat-shock response in rat brain.

The expression of mRNAs encoding two members of the heat-shock protein 70 family, the constitutively-expressed heat-shock cognate (hsc70) mRNA and the strictly heat-inducible (hsp70) mRNA, was quantitated in cerebellar and hippocampal cells of rats 3 h after amphetamine-induced or heat-induced hyperthermia. Intracellular heat-shock mRNA levels in specific cell types were compared with those of total polyadenylic acid [poly(A)] mRNA or 18S rRNA in the same cell type. Levels of poly(A) mRNAs, 18S rRNAs, and hsc70 mRNAs were highest in large neurons and lowest in glia. hsp70 mRNAs were also present at highest levels in large neurons, suggesting that hsp70 mRNAs accumulated as rapidly in these cell types as they did in small neurons and glia. However, compared with levels of intracellular poly(A) mRNAs or levels of rRNAs, large neurons contained two- to 12-fold lower levels of hsp70 mRNAs than neurons of intermediate size and five- to 30-fold lower levels than glia. These results suggest that hsp70 mRNAs accumulated as rapidly in large neurons as in small neurons and glia, but that the large size of these neurons precluded intracellular hsp70 mRNA concentrations increasing as quickly. The susceptibility of large neurons to stress-induced cell death could be due, in part, to their inability to synthesize rapidly hsp70 in sufficient amounts to protect these cells from the initial molecular consequences of stress.

Selective postmortem degradation of inducible heat shock protein 70 (hsp70) mRNAs in rat brain.

1. Altered mRNA levels in postmortem brain tissue from persons with Alzheimer's disease (AD) or other neurological diseases are usually presumed to be characteristic of the disease state, even though both agonal state (the physiological state immediately premortem) and postmortem interval (PMI) (the time between death and harvesting the tissue) have the potential to affect levels of mRNAs measured in postmortem tissue. Although the possible effect of postmortem interval on mRNA levels has been more carefully evaluated than that of agonal state, many studies assume that all mRNAs have similar rates of degradation postmortem. 2. To determine the postmortem stability of inducible heat shock protein 70 (hsp70) mRNAs, themselves unstable in vivo at normal body temperature, rats were heat shocked in order to induce synthesis of the hsp70 mRNAs. hsp70 mRNA levels in cerebellum and cortex were then compared to those of their heat shock cognate 70 (hsc70) mRNAs, as well as to levels of 18S rRNAs, at 0 and at 24 hr postmortem. 3. Quantiation of northern blots after hybridization with an hsp70 mRNA-specific oligo probe indicated a massive loss of hsp70 mRNA signal in RNAs isolated from 24-hr postmortem brains; quantitation by slot-blot hybridization was 5- to 15-fold more efficient. Even using the latter technique, hsp70 mRNA levels were reduced by 59% in 24-hr-postmortem cerebellum and by 78% in cortex compared to mRNA levels in the same region of 0-hr-postmortem brain. There was little reduction postmortem in levels of the hsp70 mRNAs or of 18S rRNAs in either brain region. 4. In situ hybridization analysis indicated that hsp70 mRNAs were less abundant in all major classes of cerebellar cells after 24 hr postmortem and mRNAs had degraded severalfold more rapidly in neurons than in glia. There was no corresponding loss of intracellular 18S rRNA in any cell type. 5. We conclude from these results that the effect of postmortem interval on mRNA degradation must be carefully evaluated when analyzing levels of inducible hsp70 mRNAs, and perhaps other short-lived mRNAs, in human brain.

A harness and computer system to facilitate automated body temperature data collection in heat-stressed broilers.

An easy-to-use, low-cost system was developed that permitted nearly continuous, automated core body temperature (Tc) readings on 7-wk-old male broiler chickens via direct computer linkage to thermistor probes held in place by a specially designed harness. Elevated Tc was noted in heat stress studies following the replacement of expelled temperature probes in some hyperthermic birds. To demonstrate the usefulness of the data collection system described herein, three treatments with three to four birds per treatment were used to examine this observation. Birds were designated as handled only (HAN), handled to remove and replace the temperature probe (RPL), or left as nonhandled controls (CON). Treatments had no effect on subsequent Tc in experiments when the thermoregulatory capacity of the birds was not challenged. However, when the birds were sufficiently challenged, Tc of HAN and RPL birds increased within 4 min of the initiation of handling and remained above baseline for up to 45 min. The Tc of CON birds in that trial also increased, but to a smaller degree, within 5 min and remained above baseline for up to 20 min. This study indicates that Tc of hyperthermic birds can be superelevated by simulated manual placement of cloacal temperature probes and that fixed probes connected to an automated data monitoring and collection system is a relatively simple way to avoid this problem.

Anomalous binding of radiolabeled oligonucleotide probes to plaques and tangles in Alzheimer disease hippocampus.

Several reports indicate that Alzheimer disease (AD) brain contains elevated levels of heat shock 70 proteins. To determine the cellular localization of the heat shock 70 mRNAs, specific oligonucleotide probes were in situ hybridized to AD and control brains. When oligonucleotides were in situ hybridized to brain sections with no AD neuropathology, hybridization was cell-specific and prior ribonuclease (RNase) treatment of adjacent sections resulted in no hybridization signal. However, in situ hybridization to AD hippocampus resulted in heavy grain deposition over senile plaques and neurofibrillary tangles. Despite altering a number of experimental variables, we observed a similar pattern of grain deposition with most of the oligonucleotides tested, including one oligonucleotide specific for glutamic acid decarboxylase mRNA. In situ hybridization with either an RNA probe for glutamic acid decarboxylase or an oligonucleotide probe specific for 18S rRNA did not show this pattern of grain deposition. In control studies a sense hsc70 oligonucleotide showed no grain deposition in either cerebellum or hippocampus. Sections from AD hippocampus pretreated with RNase prior to in situ hybridization demonstrated enhanced grain deposition with the majority of probes tested. Anomalous in situ hybridization to AD hippocampus was usually eliminated by removing formamide from the posthybridization washes, although post-RNase sticking often remained intense. These findings indicate that artifactual probe binding to senile plaques and neurofibrillary tangles may complicate the analysis of in situ hybridization studies using oligonucleotide probes to determine mRNA distribution in AD brain.

Alterations in actin-binding beta-thymosin expression accompany neuronal differentiation and migration in rat cerebellum.

The beta 4- and beta 10-thymosins, recently identified as actin monomer-sequestering proteins, are developmentally regulated in brain. Using specific mRNA and protein probes, we have used in situ hybridization and immunohistochemical techniques to investigate the distribution of the beta-thymosin mRNAs and their proteins in developing rat cerebellum. Early in postnatal development, both beta-thymosin mRNAs were expressed at highest levels in the postmitotic, premigratory granule cells of the external granular layer; expression diminished as granule cells migrated to and differentiated within the developing internal granular layer. In addition, both beta-thymosin proteins were present in bundles of cerebellar afferent fibers in the white matter at this time. Throughout the maturation period, both proteins were present in elongating parallel fibers in the upper portion of the molecular layer. Later in cerebellar development, thymosin beta 4, but not thymosin beta 10, was expressed in Golgi epithelial cells and Bergmann processes. Thymosin beta 4 was expressed in a small population of cells with microglial morphology scattered throughout the gray and white matter. Thymosin beta 10 was detected in an even smaller population of glia. Expression of thymosin beta 4 and thymosin beta 10 in premigratory granule cells and in growing neuronal processes is consistent with the possibility that both beta-thymosins are involved in the dynamics of actin polymerization during migration and process extension of neurons.

Relative bioavailability of L-ascorbyl-2-polyphosphate in broiler chickens.

Broiler chick diets and drinking water were supplemented with two sources of vitamin C: crystalline L-ascorbic acid (AsA) or L-ascorbyl-2-polyphosphate (APP) to provide 0, 25, 50, 100, 200, 400, 800, 1,600, and 3,200 ppm (mg/kg) AsA. The bioavailability of APP relative to AsA, as estimated by the change in plasma AsA concentration, was evaluated during 24-h periods of supplementation. When provided in the feed, no differences in dietary AsA content were attributed to vitamin source. In contrast, APP administration at 25 and 50 ppm, resulted in higher (P < .001) AsA values in drinking water when compared with AsA supplementation. Plasma AsA values were elevated (P < .05) above baseline when either AsA or APP were supplemented in the feed or water at a level of 400 ppm or greater. Plasma AsA concentrations, following supplementation of the diets, were higher (P < .05) in AsA-treated (800 ppm) chicks when compared with APP-supplemented chicks. During water supplementation, AsA (800 ppm) and APP (3,200 ppm) administration resulted in higher plasma AsA values when compared with their alternate vitamin source. At all other levels of water supplementation, no differences in plasma AsA were associated with vitamin source. The absence of a consistent difference in plasma AsA, relative to vitamin source, suggests that the isolated differences observed may be due to chance. It was concluded that APP was of similar bioavailability to that of AsA, as estimated by the ability to elevate plasma AsA concentrations in broiler chicks.