Prenatal stress induces long term stress vulnerability, compromising stress response systems in the brain and impairing extinction of conditioned fear after adult stress.

Stress is a risk factor for the development of affective disorders, including depression, post-traumatic stress disorder, and other anxiety disorders. However, not all individuals who experience either chronic stress or traumatic acute stress develop such disorders. Thus, other factors must confer a vulnerability to stress, and exposure to early-life stress may be one such factor. In this study we examined prenatal stress (PNS) as a potential vulnerability factor that may produce stable changes in central stress response systems and susceptibility to develop fear- and anxiety-like behaviors after adult stress exposure. Pregnant Sprague-Dawley rats were immobilized for 1 h daily during the last week of pregnancy. Controls were unstressed. The male offspring were then studied as adults. As adults, PNS or control rats were first tested for shock-probe defensive burying behavior, then half from each group were exposed to a combined chronic plus acute prolonged stress (CAPS) treatment, consisting of chronic intermittent cold stress (4 °C, 6 h/d, 14 days) followed on day 15 by a single session of sequential acute stressors (social defeat, immobilization, cold swim). After CAPS or control treatment, different groups were tested for open field exploration, social interaction, or cued fear conditioning and extinction. Rats were sacrificed at least 5 days after behavioral testing for measurement of tyrosine hydroxylase (TH) and glucocorticoid receptor (GR) expression in specific brain regions, and plasma adrenocorticotropic hormone (ACTH) and corticosterone. Shock-probe burying, open field exploration and social interaction were unaffected by any treatment. However, PNS elevated basal corticosterone, decreased GR protein levels in hippocampus and prefrontal cortex, and decreased TH mRNA expression in noradrenergic neurons in the dorsal pons. Further, rats exposed to PNS plus CAPS showed attenuated extinction of cue-conditioned fear. These results suggest that PNS induces vulnerability to subsequent adult stress, resulting in an enhanced fear-like behavioral profile, and dysregulation of brain noradrenergic and hypothalamic-pituitary-adrenal axis (HPA) activity.

Cold adaptation as a life style strategy.

Humans evolved in Africa, where climate would have required cold adaptation. Modern humans have housing which separates them from the environment. Metabolic disorders are prevalent in populations with excess Western diet and lack of exercise. A thrifty genotype has been proposed as an explanation for susceptibility. An alternative explanation is that cold adaptation is absent. But the human genome is complex, and there are many variations possible in the metabolic pathways. Behaviour which uses the circadian rhythms of metabolism may be helpful for fitness.

Mass accuracy and sequence requirements for protein database searching.

To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein matches was examined by varying the mass error allowed in the search over a wide range (2-500 ppm). For the "clean" data obtained from apomyoglobin, very few masses were needed to return valid protein matches, and the mass error allowed in the search had little effect up to 500 ppm. However, in the case of alpha-lactalbumin more mass values were needed, and low mass errors increased the search specificity. Mass errors below 30 ppm were particularly useful in eliminating false protein matches when few mass values were used in the search. Collision-induced dissociation of an unassigned peak in the alpha-lactalbumin digest provided sufficient data to unambiguously identify the peak as a fragment from alpha-lactalbumin and eliminate a large number of spurious proteins found in the peptide mass search. The results show that even with a relatively high mass error (0.8 Da for mass differences between singly charged product ions), collision-induced dissociation can help identify proteins in cases where unfavorable digest conditions or modifications render digest peaks unidentifiable by a simple mass mapping search.

Distinguishing small molecular mass differences of proteins by mass spectrometry.

Electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry allows for high-resolution, accurate mass analysis of multiply charged ions of proteins. In the work described here, the ability of ESI-FTICR to distinguish small differences in molecular mass is evaluated. Ubiquitin was used as an internal mass calibration standard to measure the molecular mass of cytochrome c, myoglobin, and several carbonic anhydrase isoforms. Mass calibration was based on the tallest isotopic peak of each ubiquitin charge state. Ubiquitin performed well as an internal standard because its charge states covered the appropriate mass range, interference was minimal, and the tallest peak was easily identified. The peak masses of cytochrome c (12.5 kDa) and myoglobin (17 kDa) were measured to an accuracy of about 0.02 Da (<2ppm). However, errors of 1.0 Da were observed for some individual determinations because of the difficulty in identifying the tallest peak. When the technique was applied to bovine carbonic anhydrase II, even combining data from several charge states did not yield an unequivocal assignment of the tallest peak, resulting in a mass assignment of 29,023.7 or 29,024.7. Similarly, measurements of two isoforms with a mass difference of 1 Da, human carbonic anhydrase I, pI 6.0 and 6.6, yielded overlapping values for the mass of the tallest peak. However, these two isoforms were clearly distinguished by (a) identification of the tallest peak using a measurement of average mass as a guide and (b) comparison of the isotopic peak intensity patterns.

Captopril inhibits tumour growth in a xenograft model of human renal cell carcinoma.

The effect of captopril on tumour growth was examined in a xenograft model of human renal cell carcinoma (RCC). Inoculation of the human RCC cell line SN12K-1 (10(6) cells) under the left kidney capsule of severe combined immunodeficient (SCID) mice resulted in the growth of large tumours, with an increase in weight of the inoculated kidney of 3.69+/-1.63-fold (mean+/-s.d.) when compared with the contralateral normal kidney. In mice treated with captopril (19 mg kg(-1) day(-1) or 94 mg kg(-1) day(-1) administered in the drinking water), there was a significant dose-related reduction in tumour development; the tumour bearing kidneys weighed 1.9+/-0.42 and 1.55+/-0.42 times the normal kidneys, respectively (P< 0.05 compared with untreated animals). In vitro, captopril at clinically achievable doses (0.1-10 microM) had no significant effect on the incorporation of [3H]thymidine into SN12K-1 cells. Thus, this highly significant attenuation by captopril of in vivo tumour growth does not appear to be due to a direct effect on the proliferation of the tumour cells. Further studies are required to determine the mechanism of inhibition of tumour growth by captopril, in particular to evaluate the role of angiotensin II in this process.

Histologic and immunohistochemical responses after aortic valve allografts in the rat.

BACKGROUND: Human aortic valve allografts elicit a cellular and humoral immune response. It is not clear whether this is important in promoting valve damage. We investigated the changes in morphology, cell populations, and major histocompatibility complex antigen distribution in the rat aortic valve allograft. METHODS: Fresh heart valves from Lewis rats were transplanted into the abdominal aorta of DA rats. Valves from allografted, isografted, and presensitized recipient rats were examined serially with standard morphologic and immunohistochemical techniques. RESULTS: In comparison with isografts, the allografts were infiltrated and thickened by increased numbers of CD4+ and CD8+ lymphocytes, macrophages, and fibroblasts. Thickening of the valve wall and leaflet and the density of the cellular infiltrate was particularly evident after presensitization. Endothelial cells were frequently absent in presensitized allografts whereas isografts had intact endothelium. Cellular major histocompatibility complex class I and II antigens in the allograft were substantially increased. A long-term allograft showed dense fibrosis and disruption of the media with scattered persisting donor cells. CONCLUSIONS: The changes in these aortic valve allograft experiments are consistent with an allograft immune response and confirm that the response can damage aortic valve allograft tissue.

Ion-molecule reactions as probes of gas-phase structures of peptides and proteins.

A review with over 100 references describes the recent applications of ion-molecule reactions to the study of gas-phase protonated peptides and proteins. The topic is focused specifically on the proton transfer and hydrogen-deuterium exchange reactions of amino acids, peptides, and proteins. A brief background is given of the various methods used for assigning proton affinities and gas-phase basicities. The methods used for measuring the kinetics of deuterium incorporation of charged ion in the presence of a background pressure of deuterating reagents are also described. Ion-molecule reactions are used to determine, among other things, the gas-phase basicities and proton affinities of amino acids, peptides, and proteins, the sites of protonation, intra- and intermolecular interactions, and conformational differences and changes in gas-phase ionic species. Singly charged and multiply charged ions are both covered.

A dual vacuum chamber fourier transform mass spectrometer with rapidly interchangeable LSIMS, MALDI, and ESI sources: initial results with LSIMS and MALDI.

A design is presented involving two separate vacuum chambers to provide nearly simultaneous capabilities of liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) in an external source Fourier transform mass spectrometer. The instrument consists of two vacuum chambers, one with five stages of differential pumping for a combined LSIMS/MALDI source. The chamber dedicated to ESI was formerly a three-stage chamber with LSIMS and electron ionization. Two additional stages were added with the ESI source. LSIMS and MALDI have similar vacuum requirements and were moved to a newly built chamber with two stages of pumping. We present our first results obtained on the new vacuum chamber. Data presented for the MALDI source show that, with only two stages of pumping, and with shorter radio frequency-only quadrupole rods for ion injection, spectra comparable to those obtained on the formerly three-stage instrument can be obtained. Characterization of the MALDI source and data on linear, cyclic, and branched oligosaccharides are given. Finally, the design of the secon chamber is proposed as a low-cost prototype for an external source FTMS instrument.

Successful methods of pancreas transplantation in the rat using a cuff technique.

Two models of pancreas transplantation were examined in the rat with a view to choosing one for regular use in functional experiments. A cuff technique applied to the renal vessels of the recipient was used. The histology and endocrine functioning of whole pancreas-duodenal transplant (Tx) plus bladder drainage (drainage model), and the pancreatic duct ligated segmental pancreas Tx (ligated model) were examined. Syngeneic operations were performed using either Lewis or Wistar rats. Streptozotocin (STZ) 60 mg/kg intravenously (i.v.) was used to render the recipient rats diabetic. A cuff technique was used with both models to anastomose the grafts to the renal vessels instead of the conventional technique of hand suturing to the abdominal vessels. This allows a shorter warm ischaemia time for the donor pancreas and leaves the systemic circulation intact leading to a high success rate for both techniques. Operation survival rates were 93% (n = 30) and 90% (n = 10) in the ligated and drainage models, respectively. The recipients in both groups were normoglycaemic for > 100 days. Histological examination revealed atrophic exocrine tissue early in the ligated group but only two from the drainage group showed exocrine atrophy at > 100 days. There was no statistical difference in i.v. glucose tolerance tests with both models showing a normal pattern. Thus, endocrine function remained independent of exocrine function. Both models are quicker than the conventional techniques. The duct ligation model was a simpler transplant to perform, suggesting that this should be the technique of choice in future experiments.

Gas-phase hydrogen/deuterium exchange as a molecular probe for the interaction of methanol and protonated peptides.

The gas-phase hydrogen/deuterium (HID) exchange kinetics of several protonated amino acids and dipeptides under a background pressure of CH3OD were determined in an external source Fourier transform mass spectrometer. H/D exchange reactions occur even when the gas-phase basicity of the compound is significantly larger (> 20 kcal/mol) than methanol. In addition; greater deuterium incorporation is observed for compounds that have multiple sites of similar basicities. A mechanism is proposed that involves a structurally specific intermediate with extensive interaction between the protonated compound and methanol.

Leucocyte migration inhibition test in cadaveric renal transplantation.

The leucocyte migration inhibition test has been studied in a series of 192 renal allograft recipients. Seventy-seven patients showed no evidence of inhibition in the early post-transplant course, but 31 of these demonstrated clinical evidence of rejection, a false-negative rate of 16%. The remaining 115 recipients all demonstrated inhibition, with 13 of these showing no clinical evidence of rejection, a false-positive rate of 6.7%. Early antirejection therapy on the basis of inhibition did not result in improved kidney survival when compared with those recipients who did not receive specific therapy until there was clinical evidence of rejection. The leucocyte migration inhibition test did not detect changes attributable to humoral factors, which probably accounts for the high false-negative rate, and has not proved to be sufficiently reliable to be of value clinically as a single test. A combination of tests designed to detect both humoral and cellular factors responsible for rejection deserves further study.

The stoichiometry and stability of the NADP complexes with manganese(II) ions as studied by electron paramagnetic resonance.

Magnetic resonance techniques have been applied to study the stability of the complexes formed between Mn(II) ions and NADP in aqueous solutions at a pH of 7.5 and 20 degrees C. The electron paramagnetic resonance (epr) data indicate that at low Mn(II) ion concentrations ([Mn(II)] less than 1 mM; [NADP] approximately 5 mM), a 1:1 complex is formed with an apparent stability constant K1 = 370 +/- 50 M-1 at an ionic strength of 0.22 in the presence of 0.20 M Cl-. At high Mn(II) ion concentrations, a Mn(II)2-NADP species, with an apparent stability constant K2 = 54 +/- 17 M-1, is present in significant amounts. When the epr data are corrected for the presence of the MnCl+ ion, the analysis of the new Scatchard plot yields stability constants for the two sites of K1 = 640 +/- 90 M-1 and K2 = 88 +/- 13 M-1, respectively. The presence of two metal ion binding sites on the NADP molecule has not been observed previously, and previous workers have always analyzed their data in terms of the 1:1 Mn(II)-NADP complex. An epr temperature study of K1 yields a value of delta H equal to 1.3 +/- 0.2 kcal/mol (1 cal = 4.187 J).

Stereoacuity in mentally retarded and intellectually normal adolescents.

Stereoacuity was measured in 30 intellectually normal and 30 mentally retarded adolescents, matched for age and sex, using the Titmus stereo test to provide nasalward and temporalward target disparities. The intellectually normal group had significantly better stereoacuity for both types of disparity. No statistically significant differences between nasal and temporal disparities were found within groups. A learning effect is shown for the mentally retarded.

In-vitro studies of gallstone dissolution: the effect of added heparin in bile salt solutions.

The dissolving effect of four bile salt solutions (sodium cholate, sodium taurocholate, sodium deoxycholate and sodium chenodeoxycholate) upon gallstones was tested in an in-vitro preparation, using 226 stones from 38 patients. The effect of each solution was measured by recording weight loss in the gallstone at the end of a ten-day period of immersion in the bile salt solution. Sodium deoxycholate and sodium chenodeoxycholate produced the greatest average weight loss in the groups of stones tested with pure bile salt solutions, but the addition of heparin to solutions of sodium cholate and sodium deoxycholate produced a significant increase in weight loss in these solutions. This effect of heparin in the presence of bile salts, in comparison with the failure of heparinized saline to induce weight loss in gallstones, is discussed. Sodium chenodeoxycholate cannot be recommended for clinical use on the grounds of its toxicity, and in view of the possible toxicity of sodium deoxycholate it is concluded that a combination of sodium cholate with heparin is the optimum solution for the dissolution of retained intraduct calculi in vivo.

In vitro studies of gallstone dissolution using bile salt solutions and heparinized saline.

A controlled in vitro study of gallstone dissolution has been carried out using a model designed to simulate the in vivo situation in the bile ducts. Sodium taurocholate, sodium cholate, heparinized saline and physiological saline were infused for 10 days over 81 stones from 32 patients and changes in weight and structure were recorded. The bile salt solutions caused weight loss in 81-5 and 85-7 per cent respectively of the stones treated, but the two saline solutions caused weight gains in 74-5 and 88 per cent respectively. Fragmentation occurred in 18-5% of stones treated with sodium taurocholate, in 25% of those treated with sodium cholate and in 11-8 per cent of those treated with heparinized saline. Small stones lost a lower absolute amount of weight than large stones but this represented a greater proportion of their initial weight. These investigations confirm the advantages of a dynamic in vitro model to study gallstone dissolution. Bile salt solutions infused into the bile ducts may clear retained stones by causing reduction in stone weight or fragmentation or both, but heparinized saline appears to be unsuitable for gallstone dissolution. Larger stones may require longer periods of infusion.