Acute brief heat stress in late gestation alters neonatal calf innate immune functions.

Heat stress, as one of the environmental stressors affecting the dairy industry, compromises the cow milk production, immune function, and reproductive system. However, few studies have looked at how prenatal heat stress (HS) affects the offspring. The objective of this study was to evaluate the effect of HS during late gestation on calf immunity. Calves were born to cows exposed to evaporative cooling (CT) or HS (cyclic 23-35°C) for 1 wk at 3 wk before calving. Both bull and heifer calves (CT, n=10; HS, n=10) were housed in similar environmental temperatures after birth. Both CT and HS calves received 3.78 L of pooled colostrum within 12 h after birth and were fed the same diet throughout the study. In addition to tumor necrosis factor α, IL-1ß, IL-1 receptor antagonist (IL-1RA), and toll-like receptor (TLR)2, and TLR4 mRNA expression, the expression of CD14(+) and CD18(+) cells, and DEC205(+) dendritic cells were determined in whole blood samples at d 0, 3, 7, 14, 21, and 28. The neutrophil to lymphocyte ratio, differential cell counts, and the hematocrit were also determined. During late gestation, the HS cows had greater respiration rates, rectal temperatures, and tended to spend more time standing compared with the CT cows. The HS calves had less expression of tumor necrosis factor-α and TLR2 and greater levels of IL-1ß, IL-1RA, and TLR4 compared with CT calves. The HS calves also had a greater percentage of CD18(+) cells compared with the CT calves. Additionally, a greater percentage of neutrophils and lesser percentage of lymphocytes were in the HS calves compared with the CT calves. The results indicate that biomarkers of calves' immunity are affected in the first several weeks after birth by HS in the dam during late gestation.

Dynamic associations between glucose and ecological momentary cognition in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic condition characterized by glucose fluctuations. Laboratory studies suggest that cognition is reduced when glucose is very low (hypoglycemia) and very high (hyperglycemia). Until recently, technological limitations prevented researchers from understanding how naturally-occurring glucose fluctuations impact cognitive fluctuations. This study leveraged advances in continuous glucose monitoring (CGM) and cognitive ecological momentary assessment (EMA) to characterize dynamic, within-person associations between glucose and cognition in naturalistic environments. Using CGM and EMA, we obtained intensive longitudinal measurements of glucose and cognition (processing speed, sustained attention) in 200 adults with T1D. First, we used hierarchical Bayesian modeling to estimate dynamic, within-person associations between glucose and cognition. Consistent with laboratory studies, we hypothesized that cognitive performance would be reduced at low and high glucose, reflecting cognitive vulnerability to glucose fluctuations. Second, we used data-driven lasso regression to identify clinical characteristics that predicted individual differences in cognitive vulnerability to glucose fluctuations. Large glucose fluctuations were associated with slower and less accurate processing speed, although slight glucose elevations (relative to person-level means) were associated with faster processing speed. Glucose fluctuations were not related to sustained attention. Seven clinical characteristics predicted individual differences in cognitive vulnerability to glucose fluctuations: age, time in hypoglycemia, lifetime severe hypoglycemic events, microvascular complications, glucose variability, fatigue, and neck circumference. Results establish the impact of glucose on processing speed in naturalistic environments, suggest that minimizing glucose fluctuations is important for optimizing processing speed, and identify several clinical characteristics that may exacerbate cognitive vulnerability to glucose fluctuations.

Expression of heat shock protein 70 is altered by age and diet at the level of transcription.

Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4- to 7-month-old) and old (22- to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5 degrees C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.

A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis.

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.

Crystallization of Sindbis virus and its nucleocapsid.

Crystals of Sindbis virus, which contains a lipid-bilayer membrane, have been grown using polyethylene glycol. The space group is R32, a = b = 640 A, c = 1520 A. The crystals are highly mosaic, and recorded diffraction is therefore restricted to spacings of about 30 A. The crystals show that the packing of glycoproteins E1 and E2 in the icosahedral outer shell is sufficiently precise that it permits regular and repeated interactions between virus particles in the lattice. Crystals of Sindbis nucleocapsids have also been grown. The limited diffraction data are consistent with close packing of nucleocapsids 404 A in diameter.

Expression and crystallization of a soluble form of Drosophila fasciclin III.

A truncated form of Drosophila fasciclin III has been engineered by site-directed mutagenesis. Secreted fasciclin III is expressed at 35 to 40 mg/l in insect cells with baculovirus carrying the recombinant gene. Single crystals of purified soluble fasciclin III have been grown by vapor diffusion versus polyethylene glycol 8000/sodium citrate at low pH. The space group is P6(1)22 or its enantiomorph P6(5)22, with unit cell dimensions a = b = 140 A, c = 260 A. Cryo-preserved crystals diffract to reciprocal lattice spacings beyond 3.0 A.

Impact of a cardiology data bank on physicians' prognostic estimates. Evidence that cardiology fellows change their estimates to become as accurate as the faculty.

To determine whether physicians would be influenced by the prognostic information in a large coronary artery disease data bank, cardiology faculty and fellows made initial estimates of the prognoses of their patients and then made revised final estimates after seeing the outcome of matched patients (OMP) from the data bank. The faculty cardiologists' original estimates proved to be as accurate as those of the data bank's OMP, and the faculty revised their estimates minimally in response to the data bank's OMP. Conversely, the cardiology fellows' original estimates were less accurate than the data bank's OMP, and under all observed circumstances the fellows responded more to the data bank's OMP than did the faculty. As a result, the accuracy of the fellows' final estimates was similar to the accuracies of both the faculty cardiologists and the data bank's OMP. Computerized data banks seem more likely to have impact when their information is provided to physicians who are relatively inexperienced with the disease in question.

Expression of calbindin-D decreases with age in intestine and kidney.

The calbindins are Ca-binding proteins whose expression is regulated by 1,25-dihydroxyvitamin D3, the active metabolite of vitamin D3. The calbindins are found in high amounts in the proximal intestine (calbindin-D-9k) and the kidney (calbindin-D-28k), and they are thought to play a role in Ca transport by these tissues. Ca absorption by the intestine and perhaps the kidney declines with age, and this could be due to decreased expression of calbindin. Therefore, the expression of calbindins-D-9k and -D-28k was measured in F344 rats aged 2, 6, 13, and 24 months. mRNA levels were measured by dot blot hybridization to synthetic cDNA oligonucleotide probes, and protein levels were measured by enzyme-linked immunosorbent assay using specific antisera. Intestinal calbindin-D-9k mRNA decreased markedly between 2 and 6 months of age, but it then increased significantly between 13 and 24 months. Calbindin-D-9k protein paralleled the decrease in mRNA between 2 and 6 months, but continued to decline at 13 and 24 months despite the rise in mRNA. In the kidney, calbindin-D-28k mRNA declined between 2 and 13 months and then plateaued. Calbindin-D-28k protein followed a similar pattern. In the same studies expression of calmodulin by the intestine and kidney did not change with age. Plasma 1,25-dihydroxyvitamin-D3 correlated well with the expression of calbindin-D-9k in the intestine at 2 and 6 months of age and with the expression of calbindin-D-28k in the kidney at all ages. Decreased expression of calbindin-D with age may contribute to the age-related decrease in Ca transport in intestine and kidney.

Metabolic characteristics of aorta from spontaneously hypertensive and renal and deoxycorticosterone acetate-salt hypertensive rats.

The purpose of this study was to determine if any changes occurred in the basal and stimulated rates of oxygen consumption and lactate production of thoracic aortas from spontaneously hypertensive rats (SHR) and renal and deoxycorticosterone acetate (DOCA)-salt hypertensive rats, and, if so, whether these changes were similar in these three models of hypertension. Rings of thoracic aorta were placed in an isothermic (37 degrees C) muscle bath, and isometric tension development, oxygen consumption, and lactate production were measured. The results indicated that under basal conditions oxygen consumption, but not lactate production, was higher in aortas from all three hypertensive models; the elevation above control was greatest in the renal model (95%) and smallest in SHR (34%). On stimulation with 60 mM KCl, a significant increase in oxygen consumption above basal value occurred in all aorta samples (p less than 0.05); however, lactate production was increased above basal only in aortas from hypertensive animals. Only in aortas from renal and DOCA-salt models was the rate of oxygen consumption during stimulation significantly greater than that of their normotensive controls (p less than 0.05). Developed active stress in response to KCl was the same in all groups, and when the change in lactate production or oxygen consumption was expressed relative to the amount of active stress developed, no differences were observed. These results suggest that, 1) compared to values in aortas from normotensive animals, only the basal rate of oxygen consumption is higher; 2) this higher level of basal metabolic activity is not associated with an alteration in the metabolic cost of force development.(ABSTRACT TRUNCATED AT 250 WORDS)

Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats.

During reperfusion after ischemia, deleterious biochemical processes can be triggered that may antagonize the beneficial effects of reperfusion. Research into the understanding and treatment of reperfusion injury (RI) is an important objective in the new era of reperfusion therapy for stroke. To investigate RI, permanent and reversible unilateral middle cerebral artery/common carotid artery (MCA/CCA) occlusion (monitored by laser Doppler) of variable duration in Long-Evans (LE) and spontaneously hypertensive (SH) rats and unilateral MCA and bilateral CCA occlusion in selected LE rats was induced. In LE rats, infarct volume after 24 hours of permanent unilateral MCA/CCA occlusion was 31.1 +/- 34.6 mm3 and was only 28% of the infarct volume after 120 to 300 minutes of reversible occlusion plus 24 hours of reperfusion, indicating that 72% of the damage of ischemia/reperfusion is produced by RI. When reversible ischemia was prolonged to 480 and 1080 minutes, infarct volume was 39.6 mm3 and 16.6 mm3, respectively, being indistinguishable from the damage produced by permanent ischemia and significantly smaller than damage after 120 to 300 minutes of ischemia. Reperfusion injury was not seen in SH rats or with bilateral CCA occlusion in LE rats, in which perfusion is reduced more profoundly. Reperfusion injury was ameliorated by the protein synthesis inhibitor cycloheximide or spin-trap agent N-tert-butyl-alpha-phenylnitrone pretreatment.

Neurofilament proteolysis after focal ischemia; when do cells die after experimental stroke?

To determine the occurrence and time-course of presumably irreversible subcellular damage after moderate focal ischemia, rats were subjected to 1, 3, 6, 9, or 24 hours of permanent unilateral middle cerebral and common carotid occlusion or 3 hours of reversible occlusion followed by 3, 6, or 21 hours of reperfusion. The topography and the extent of damage were analyzed with tetrazolium staining and immunoblot using an antibody capable of detecting breakdown of neurofilament. Neurofilament proteolysis began after 3 hours in the infarct core but was still incomplete in penumbral regions up to 9 hours. Similarly, tetrazolium-staining abnormalities were observed in the core of 50% of animals after 3 hours of ischemia. At 6 hours of permanent ischemia, infarct volume was maximal, and further prolongation of occlusion to 9 or 24 hours did not increase abnormal tetrazolium staining. In contrast to permanent ischemia and in agreement with the authors' previous demonstration of "reperfusion injury" in this model, prolongation of reperfusion from 3 hours to 6 and 21 hours after 3 hours of reversible occlusion gradually augmented infarct volume by 203% and 324%, respectively. Neurofilament proteolysis initiated approximately 3 hours after ischemia was quantitatively greatest in the core and extended during reperfusion to incorporate penumbra with a similar time course to that of tetrazolium abnormalities. These data demonstrate that, at least as measured by neurofilament breakdown and mitochondrial failure, extensive cellular damage is not present in penumbral regions for up to 9 hours, suggesting the potential for rescuing these regions by appropriate and timely neuroprotective strategies.

Interplay between the gamma isoform of PKC and calcineurin in regulation of vulnerability to focal cerebral ischemia.

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.

Immunohistochemical determination of calcium-calmodulin binding predicts neuronal damage after global ischemia.

Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+-CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+-CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p less than 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

An alternative method for the quantitation of neuronal damage after experimental middle cerebral artery occlusion in rats: analysis of behavioral deficit.

We tested the hypothesis that increasing durations of focal ischemia that have been shown to result in enlargement of cortical infarct will be associated with progression of behavioral dysfunction that can be measured by a battery of tests sufficiently sensitive and reproducible to detect a positive effect of pharmacotherapy. Untreated or N-methyl-D-aspartate receptor antagonist (CNS-1102)-treated spontaneously hypertensive rats underwent 45, 60, 90, or 120 min of tandem middle cerebral and common carotid artery occlusion followed by reperfusion. We then evaluated the extent of damage and its recovery for up to 21 days using nine behavioral tests aimed at analyzing strength, coordination, and bilateral asymmetry. Also using a graded bioassay that employs a curve-fitting computer program (ALLFIT) to correlate duration of ischemia with degree of behavioral dysfunction, we calculated the average of maximal behavioral dysfunction and duration of ischemia required to produce half-maximal behavioral dysfunction and compared these values in untreated controls with analogous values obtained from animals treated with CNS-1102. Three behavioral tests, forearm flex, tape (somatosensory neutralization), and foot-fault placing, were each separately and combined able to distinguish between the degrees of damage produced by increasing durations of ischemia. The behavioral abnormalities assessed using the tape test were reversible within a week, whereas those using forearm flex or foot-fault tests persisted for at least 21 days. CNS-1102 significantly reduced behavioral dysfunction measured by all three tests. This analysis of behavioral dysfunction represents a useful experimental model to grade efficacy of therapies aimed at protecting the brain from damage produced by acute stroke and might also be used to assess recovery from preexisting ischemic damage.

Ischemia-induced neuronal damage: a role for calcium/calmodulin-dependent protein kinase II.

Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.

Age-related alterations in the rodent brain cholinergic system and behavior.

Pre- and postsynaptic cholinergic markers were studied in various brain regions of mice and rats aged 6 to 30 months in an attempt to determine whether alterations in this transmitter system occur during the normal aging process. Reliable decreases in cholinergic receptor binding and choline acetyltransferase (CAT) activity were found in the cerebral cortex and corpus striatum. These alterations in the cholinergic system were typically more consistent and robust than changes involving glutamic acid decarboxylase, and enzyme marker for GABA neurons. No statistically significant changes in any markers were found in the hippocampus of either species. Significant age-related changes in retention of passive avoidance learning and locomotor activity were also observed in these same animals. These findings demonstrate that changes in the cholinergic system occur naturally in aged mice and rats and that both the loss of cholinergic receptors and decrease in cat activity may contribute to the motor and mental impairments that often accompany old age.