Short communication: Characterizing ovine serum stress biomarkers during endotoxemia.

Breeding stress-resilient livestock is a potential strategy to help mitigate the negative effect of environmental and pathogenic stressors. The hypothalamic-pituitary-adrenal axis and immune system are activated during stress events and release mediators into the circulation that help restore physiological homeostasis. The purpose of this study was to assess a comprehensive set of circulatory mediators released in response to an acute immune stress challenge to identify candidate biomarkers that can be used for the selection of stress-resilient animals. Fifteen female lambs were stress challenged with an intravenous bolus of lipopolysaccharide (LPS; 400 ng/kg), and blood was collected from the jugular vein at 0, 2, 4, and 6 h after LPS challenge to identify and monitor candidate stress biomarkers; temperature was also recorded over time. Biomarker responses were evaluated with a repeated-measures model to compare time points with baseline values. As expected, all sheep had a monophasic febrile response to LPS challenge, and cortisol increased and returned to baseline by 6 h. The cytokines tumor necrosis factor-α, IL-6, IFN-γ (proinflammatory), and IL-10 (anti-inflammatory) increased, but only tumor necrosis factor-α returned to baseline during the monitoring period. The cytokines IL-1α, IL-1ß, IL-17α (proinflammatory), and IL-4 (anti-inflammatory) did not respond to LPS challenge. All chemokines (CCL2, CCL3, CCL4, CXCL10, and IL-8) responded to LPS challenge; however, only CCL2, CCL3, CCL4, and CXCL10 increased over time, and only CCL3, CCL4, and CXCL10 returned to baseline during the monitoring period. MicroRNA (miR-145, miR-233, and miR-1246) also increased and remained elevated during the study. In summary, the LPS challenge induced a strong stress response in Rideau-Dorset sheep that could be monitored with a distinct profile of circulatory biomarkers.

Multiply charged thorium crystals for nuclear laser spectroscopy.

We have produced laser-cooled crystals of 232Th3+ in a linear rf Paul trap. This is the first time that a multiply charged ion has been laser cooled. Our work opens an avenue for excitation of the nuclear transition in a trapped, cold 229Th3+ ion. Laser excitation of nuclear states would establish a new bridge between atomic and nuclear physics, with the promise of new levels of metrological precision.

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.

Contribution of molecular chaperones to protein folding in the cytoplasm of prokaryotic and eukaryotic cells.

While it is clear that many unfolded proteins can attain their native state spontaneously in vitro, the efficiency of such folding is usually limited to conditions far removed from those encountered within cells. Two properties of the cellular environment are expected to enhance strongly the propensity of incompletely folded polypeptides to misfold and aggregate: the crowding effect caused by the high concentration of macromolecules, and the close proximity of nascent polypeptide chains emerging from polyribosomes. However, in the living cell, non-productive protein folding is in many, if not most, cases prevented by the action of a highly conserved set of proteins termed molecular chaperones. In the cytoplasm, the Hsp70 (heat-shock protein of 70 kDa) and chaperonin families of molecular chaperones appear to be the major contributors to efficient protein folding during both normal conditions and adverse conditions such as heat stress. Hsp70 chaperones recognize and shield short, hydrophobic peptide segments in the context of non-native polypeptides and probably promote folding by decreasing the concentration of aggregation-prone intermediates. In contrast, the chaperonins interact with and globally enclose collapsed folding intermediates in a central cavity where efficient folding can proceed in a protected environment. For a number of proteins, folding requires the co-ordinated action of both of these molecular chaperones.

In the queue for coronary artery bypass grafting: patients' perceptions of risk and 'maximal acceptable waiting time'.

OBJECTIVES: To elicit patients' maximal acceptable waiting times (MAWT) for non-urgent coronary artery bypass grafting (CABG), and to determine if MAWT is related to prior expectations of waiting times, symptom burden, expected relief, or perceived risks of myocardial infarction while waiting. METHODS: Seventy-two patients on an elective CABG waiting list chose between two hypothetical but plausible options: a 1-month wait with 2% risk of surgical mortality, and a 6-month wait with 1% risk of surgical mortality. Waiting time in the 6-month option was varied up if respondents chose the 6-month/lower risk option, and down if they chose the 1-month/higher risk option, until the MAWT switch point was reached. Patients also reported their expected waiting time, perceived risks of myocardial infarction while waiting, current function, expected functional improvement and the value of that improvement. RESULTS: Only 17 (24%) patients chose the 6-month/1% risk option, while 55 (76%) chose the 1-month/2% risk option. The median MAWT was 2 months; scores ranged from 1 to 12 months (with two outliers). Many perceived high cumulative risks of myocardial infarction if waiting for 1 (upper quartile, > or = 1.45%) or 6 (upper quartile, > or = 10%) months. However, MAWT scores were related only to expected waiting time (r = 0.47; P < 0.0001). CONCLUSIONS: Most patients reject waiting 6 months for elective CABG, even if offered along with a halving in surgical mortality (from 2% to 1%). Intolerance for further delay seems to be determined primarily by patients' attachment to their scheduled surgical dates. Many also have severely inflated perceptions of their risk of myocardial infarction in the queue. These results suggest a need for interventions to modify patients' inaccurate risk perceptions, particularly if a scheduled surgical date must be deferred.

Characterisation of several Hsp70 interacting proteins from mammalian organelles.

Since both the spectrum and characteristics of in vivo substrates with affinity for Hsp70 members are largely unknown, we have investigated the range and type of mammalian organellar proteins which selectively interact with immobilised Escherichia coli Hsp70 (DnaK). Amongst a subset of organellar proteins selectively retained on DnaK, the major constituents represent unstable proteins and subunits of oligomeric proteins. The interactions with DnaK were diminished in the presence of mt-Hsp70 and BiP, while the complexes formed with DnaK were dissociated in the presence of K+ and GrpE-like co-chaperones, suggesting that these organellar proteins constitute general Hsp70 substrates. Protein sequence analysis identified the major DnaK interacting constituents as the mitochondrial transcription factor A, the alpha- (but not the beta-) subunit of succinyl CoA synthetase, mitochondrial 2,4-dienoyl CoA reductase, endoplasmic reticulum cyclophilin-B, peroxisomal multifunctional enzyme and a previously undescribed peroxisomal protein suspected to represent an isoform of 2,4-dienoyl CoA reductase. The selective retention of these fully synthesised proteins on Hsp70 most likely reflects the function of this molecular chaperone in protein biogenesis, but additionally, could extend the known functions of Hsp70 to include modulating the activities of certain proteins or enzymes which are important in cellular homeostasis.

Evidence for the existence of distinct mammalian cytosolic, microsomal, and two mitochondrial GrpE-like proteins, the Co-chaperones of specific Hsp70 members.

We previously reported the cDNA cloning and characterization of a mammalian mitochondrial GrpE protein ( approximately 21 kDa, mt-GrpE#1) and now provide evidence for the presence of distinct cytosolic ( approximately 40 kDa), microsomal ( approximately 50 kDa), and additional mitochondrial ( approximately 22 kDa, mt-GrpE#2) GrpE-like members. While a cytosolic GrpE-like protein has recently been identified, the demonstration of both a microsomal and a second mitochondrial GrpE-like member represents the first in any biological system. Investigation of the microsomal and two mitochondrial GrpE-like proteins revealed that they bound specifically to Escherichia coli DnaK, and the complexes formed were not disrupted in the presence of 0.5 M salt but were readily dissociated in the presence of 5 mM ATP. The functional integrity of mt-GrpE#1 and #2 was verified by their ability to specifically interact with and stimulate the ATPase activity of mammalian mitochondrial Hsp70 (mt-Hsp70). Analysis of the cDNA sequences encoding the two mammalian mitochondrial GrpE-like proteins revealed approximately 47% positional identity at the amino acid level, the presence of a highly conserved mitochondrial leader sequence, and putative destabilization elements within the 3'-untranslated region of the mt-GrpE#2 transcript which are not present in the mt-GrpE#1 transcript. A constitutive expression of both mitochondrial GrpE-like transcripts in 22 distinct mouse tissues was observed but possible different post-transcriptional regulation of the mt-GrpE#1 and #2 transcripts may confer a different expression pattern of the encoded proteins.

Selective induction of mitochondrial chaperones in response to loss of the mitochondrial genome.

Molecular chaperones are known to play key roles in the synthesis, transport and folding of nuclear-encoded mitochondrial proteins and of proteins encoded by mitochondrial DNA. Although the regulation of heat-shock genes has been the subject of considerable investigation, regulation of the genes encoding mitochondrial chaperones is not well defined. We have found that stress applied specifically to the mitochondria of mammalian cells is capable of eliciting an organelle-specific, molecular chaperone response. Using the loss of mitochondrial DNA as a means of producing a specific mitochondrial stress, we show by Western-blot analysis that mtDNA-less (rho 0) rat hepatoma cells show an increase in the steady-state levels of chaperonin 60 (cpn 60) and chaperonin 10 (cpn 10). Nuclear transcription assays show that the upregulation of these chaperones is due to transcriptional activation. There was no effect on the inducible cytosolic Hsp 70, Hsp 72, nor on mtHsp 70 in rho 0 cells, leading us to concluded that stress applied selectively to mitochondria elicits a specific molecular chaperone response. Heat stress was able to provide an additional induction of cpn 60 and cpn 10 above that obtained for the rho 0 state alone, indicating that these genes have separate regulatory elements for the specific mitochondrial and general stress responses. Since the mitochondrial-specific chaperones are encoded by nuclear DNA, there must be a mechanism for molecular communication between the mitochondrion and nucleus and this system can address how stress is communicated between these organelles.

Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation.

Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.

Characterization of novel hsp70 in mammalian cells.

A new protein detected in heat resistant mutants of a murine fibrosarcoma has been identified as a member of the hsp70 family. The protein is similar to the constitutive hsp70 of the parent cells with regard to its antibody cross-reactivity, its ability to bind to an ATP affinity column and partial amino acid sequencing. It is present in addition to, and in similar amounts to, the constitutive isoform of the heat resistant mutant cells and the parent cell line. The lack of either of two post-translational modifications common to other hsps, phosphorylation and ADP-ribosylation, and the demonstration of mRNA for the novel protein suggest that it is a separate gene product and not a post-translational modification of the constitutive hsp70. To our knowledge, this protein has not been described in other systems and may be important in the expression of the heat resistant phenotype of these cells. The in vivo phosphorylation patterns also show that hsp90 and hsp28 are heavily phosphorylated in the heat resistant mutant, but that two other stress proteins reported to be phosphorylated under some conditions are not phosphorylated in these cells.

Prospective comparison of the effects of Occucoat, Viscoat, and Healon on intraocular pressure and endothelial cell loss.

We compared the effect of Occucoat (2% hydroxypropylmethyl-cellulose), Viscoat (sodium hyaluronate-chondroitin sulfate), and Healon (sodium hyaluronate) on postoperative intraocular pressure (IOP) and endothelial cell damage. One hundred fourteen patients having planned extracapsular cataract extraction with posterior chamber lens implantation using a viscomaterial were prospectively randomized into one of five groups. Group I received Occucoat which was removed from the anterior chamber at the conclusion of surgery. Group II received Occucoat which was not removed (retained). Group III received Viscoat which was removed, Group IV received Viscoat which was retained, and Group V received Healon which was removed. No prophylactic ocular hypotensive medications were given. Intraocular pressure was measured at four hours, 24 hours, one week, one month, three months, and one year postoperatively. Compared to preoperative IOP, all groups had a significant IOP increase at four hours. All but the Viscoat removed group (Group III) showed a statistically significant increase at 24 hours postoperatively (P less than .05). No group had a significant increase at one week or later. Specular microscopy showed no significant difference in cell loss between any of the groups at three months or within each group when compared to preoperative cell counts (P greater than .1).

Unexpected dystocia secondary to a fetal sacrococcygeal teratoma: a successful outcome.

Sacrococcygeal teratoma is a rare cause of dystocia. With recent advances in perinatal care, particularly the increasing use of maternal ultrasound, it is unlikely that unexpected dystocia secondary to this tumor will be seen by many physicians. Recent reports of this type of dystocia are rare and infant mortality secondary to it is high. However, if and when encountered, such dystocia need not imply a bad prognosis for either mother or infant. This is a report of successful management of one case as well as a presentation of recently reported cases and recommendations for management.

Primary tumours of the thymus in the rat.

The features of 192 primary thymic tumours occurring in the rat are described. Of these neoplasms, 170 were classified as benign thymomas, one as a benign fibrous histiocytoma, 20 as various types of malignant thymoma, 3 lympho-epithelioma-like carcinomas, one mixed small cell undifferentiated-squamous cell carcinoma, one sarcoma-like carcinoma, 4 undifferentiated carcinomas, 11 squamous cell carcinomas and the one remaining tumour as a carcinoid. A mouse, anti-epithelial, monoclonal antibody, lu-5, was used to confirm the epithelial nature of the malignant thymomas, and neuron-specific enolase to confirm the diagnosis of carcinoid. The tumours showed many features in common with those reported in man.

Rat endomyocardial disease: a neural origin?

Of 58 cardiac lesions in rats from a Sprague-Dawley-derived strain, representing different stages of endomyocardial disease including 10 cases progressing to tumors, most stained positively for S-100 protein, while duplicate sections of selected cases also reacted positively for neuron-specific enolase. The results demonstrate the probable neural origin of these lesions.