Effects of phase separating systems on lyophilized hemoglobin

Polymer liquid-liquid two-phase systems offer a unique opportunity to study the mechanisms of protein stabilization during freezing and freeze-drying. Fourier transform infrared spectroscopy was used to monitor the structural integrity of recombinant hemoglobin frozen and lyophilized in the separated phases of a polyethylene glycol (PEG)-dextran system. Protein in each phase of an equilibrated biphasic PEG-dextran system experiences similar levels of structural protection against freezing stresses despite large differences in polymer concentration. This result further demonstrates previous suggestions that proteins are protected during freezing by the preferential exclusion mechanism. There are, however, distinct differences in the level of structural protection that polymers in equilibrium phases provide to proteins during lyophilization, emphasizing that the mechanisms of protein protection during freezing and drying are fundamentally different. In addition, we provide evidence that phase separation per se occurring during the course of the lyophilization cycle can be detrimental to the structural stability of a protein.     

Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus macaques

We investigated whether enumeration of lymphokine-secreting T cells can be used as a quantitative measure to determine the immunogenicity of foreign proteins in rhesus monkeys. In addition, it was assessed whether this approach can supplement and/or substitute for the well-established lymphoproliferation assay. Two candidate vaccine proteins (e.g., HIV-1 gp120 and HSV-2gD) were used as model antigens for immunization. PBMCs from immunized animals were antigenically stimulated and evaluated on their proliferative capacity and lymphokine release at the single cell level. The experiments showed a close quantitative correlation between antigen-triggered proliferative responses and the antigen-induced generation of IL-2 and IFN-gamma producing cells (pc). IL-4pc were found to appear relatively late after the initiation of antigen exposure. The data indicate that ELISPOT assays provide valuable tools for the assessment of the antigenicity of foreign proteins in vivo.     

Prenatal MR diagnosis of a thick corpus callosum

Fetal sonography revealed cerebral, facial, and genitourinary abnormalities, prompting MR at 33 weeks' gestational age. Cerebral MR confirmed a thickened corpus callosum and showed open sylvian fissures, abnormal gyri in the frontal lobes, and presumed neuronal heterotopias. An abortion was performed at 34 weeks' gestational age, and pathologic findings corresponded well to the MR manifestations.     

Pericardiectomy for pericardial constriction

Constrictive pericarditis is a pathologic condition that may lead to significant morbidity. Definitive management of constrictive pericarditis requires pericardiectomy. A retrospective review of pericardiectomy for constrictive pericarditis at the Ochsner Clinic was undertaken. Twenty-one patients (17 male, four female) underwent pericardiectomy for constrictive pericarditis between January 1969 and June 1994. Ages ranged from 15 to 66 years (mean 41.5 years). Pedal edema, dyspnea, fatigue, and chest pain were the most common symptoms. Fifteen patients had important comorbidities. Preoperative New York Heart Association (NYHA) class was I (2), II (8), III (6), IV (5). Mean preoperative catheterization data, available in 17 patients (81%), demonstrated elevated intracardiac pressures (right atrial 17.4 mm Hg, right ventricular end-diastolic 22.4 mm Hg, pulmonary artery 26.2 mm Hg, pulmonary capillary wedge 20.2 mm Hg, left ventricular end-diastolic 20.1 mm Hg). A total pericardiectomy was performed in nine patients (sternotomy 8, thoracotomy 1). Pericardiectomy limited anteriorly to the phrenic nerves was performed in 11 patients (sternotomy 9, thoracotomy 2). One partial pericardiectomy was performed through a sternotomy. Cardiopulmonary bypass was used in six patients (29%). Mean hospital stay was 12 days (preoperative 4.2, postoperative 7.67). All patients achieved NYHA Class I postoperatively. Sixteen patients were discharged in sinus rhythm. No early mortality (<30 days), or major postoperative complications were observed. Pericardiectomy for pericardial constriction can be performed safely low morbidity and mortality and can favorably impact the natural history of this debilitating condition.     

Impaired priming on the general knowledge task in amnesia.

The status of priming on the general knowledge test was examined in amnesia. Twenty amnesic and 20 control participants studied words (e.g., CHEETAH) under semantic and nonsemantic encoding conditions and attempted to answer general knowledge questions (e.g., "What is the fastest animal on earth"?) under implicit and explicit retrieval instructions. The measure of memory was how many more test questions participants answered correctly using studied than nonstudied words. Amnesic patients showed impaired memory under implicit and explicit retrieval instructions. Control participants showed equal memory under implicit and explicit retrieval instructions, a result indicating that they engaged in explicit retrieval in both instruction conditions. General-knowledge priming appears to involve explicit retrieval that depends on medial-temporal and diencephalic regions damaged in amnesia. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis

Retinoic acid (RA) has been implicated as a signaling molecule for the morphogenesis of some tissues and organs. The morphogenesis of the rat testis occurs relatively late in development, culminating in puberty. Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular Ra-binding protein (CRABP) and cellular RA-binding protein II (CRABP-II). Both CRABP and CRABP-II are present at various sites in the developing mouse embryo. Here we report the developmental expression and localization of CRABP and CRABP-II in rat testis. Northern blot analysis of CRABP-II demonstrated the highest messenger RNA expression on day 4 (the earliest time point assayed by this technique), decreasing thereafter until day 20, when it became undetectable. Western blot analysis, begun on day 19 of fetal development, indicated that high levels of protein expression in the testis already existed at that time. CRABP messenger RNA expression reached its highest levels between postnatal days 16-20 and decreased thereafter. Immunolocalization revealed that CRABP-II was confined to the fetal population of Leydig and Sertoli cells. We observed that CRABP-II was expressed in certain cells that synthesized retinoic acid in the uterus and ovary (unpublished). The expression of CRABP-II in Sertoli cells and fetal Leydig cells suggested that these cells may well be the site of RA synthesis in the developing testis. CRABP was localized to gonocytes in earlier stages and spermatogonia later, where it was clearly excluded from the nucleus, indicating that the role of CRABP may be to protect these cells from the effects of RA. The reported expression of CRABP-II in embryonal tissues, which are RA responsive and undergoing morphogenesis, coupled with CRABP-II expression in the testis at a critical morphogenic stage suggest that RA may play a prominent role in the morphogenesis of the testis.     

Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy

Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- charge-separated state. Fe-removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (kQ) from the intermediate acceptor H- to the primary acceptor QA of (200 ps)-1 vs (3-6 ns)-1 were observed. Correlation of the transient EPR and optical results shows that the observed slow kQ rate in Fe-removed RCs is H-subunit-independent, and, in some cases, independent of Fe-site occupancy as Zn2+ substitution does not ensure retention of the native kQ. In addition, shifts in the optical spectrum of P865 and differences in the high-field region of the Q-band ESP spectrum for Fe-removed RCs with slow kQ indicate possible structural changes near P865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where kQ is at least 15-fold slower at room temperature than the (200 ps)-1 rate observed for native Fe-containing RCs have different relative amplitudes and small g-value shifts compared to the spectra of Zn-RCs which have a kQ unchanged from native RCs. These differences reflect the trends in polarization predicted from the sequential electron transfer polarization (SETP) model [Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al. (1996) Chem. Phys. Lett. 253, 293-298]. Thus, SETP modeling of these highly resolved ESP spectra obtained with well-characterized proteins will provide definitive information about any light-induced structural changes of P865, H, and QA that occur upon formation of the P865+QA- charge-separated state.