Willem J. de Grip

The proteome of red cell membranes and vesicles during storage in blood bank conditions

BACKGROUND During storage of red cells (RBCs) for transfusion, RBCs undergo a number of biochemical and morphologic changes. To be able to identify the mechanisms underlying these storage lesions, a proteomic analysis of the membranes of RBCs and their vesicles was performed during various periods of storage in blood bank conditions. STUDY DESIGN AND METHODS RBCs and vesicles were isolated from RBCs after various storage periods. The proteins of RBC membranes and vesicles were separated by gel electrophoresis and identified by a semiquantitative proteomic analysis. RESULTS Our findings confirm previous data, such as a storage-associated increase in hemoglobin binding to the membrane and aggregation and degradation of the integral membrane protein band 3, suggesting a remodeling of the RBC membrane during storage. Our data also show storage-dependent changes in the membrane association of proteasome and chaperone proteins, metabolic enzymes, small G proteins, and signal transduction proteins. Vesicles display similar changes in their protein composition during storage. CONCLUSION The results of this analysis indicate that the storage-related changes in the RBC membrane are the results of disturbance and/or acceleration of physiologic processes such as cellular aging, including vesicle formation. The latter may serve to remove damaged membrane patches that would otherwise lead to accelerated RBC removal. These data provide a framework for future studies toward the development of better storage conditions and a reduction of the side effects of RBC transfusion.

Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods

BACKGROUND: The use of fresh red blood cells (RBCs) is recommended for critically ill patients and patients undergoing surgery, although there is no conclusive evidence that this is beneficial. In this follow‐up study, the short‐term and the long‐term recovery of irradiated, leukoreduced RBCs transfused after either a short storage (SS) or a long storage (LS) period were compared. By consecutive transfusion of RBCs with a SS and LS period, a direct comparison of their survival within the same patient was possible.

Eye lens αA- and αB-crystallin: complex stability versus chaperone-like activity

Abstract The major lens protein α-crystallin is composed of two related types of subunits, αA- and αB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric αA-crystallin complexes perform better in preventing thermal aggregation of proteins, while αB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50°C αA- performs better than αB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of αA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of αA-crystallin’s secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of αA- and αB-crystallin, since regardless of the experimental temperature αB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of αA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of αB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric α-crystallin as a lens protein.

Large‐scale purification of functional recombinant human aquaporin‐2

The homotetrameric aquaporin‐2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre‐eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his‐tagged human AQP2 (HT‐AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT‐AQP2 could be obtained per liter of bioreactor culture. HT‐AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93±0.03×10−13 cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large‐scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Immunocytochemical evidence of molecular photoreceptor markers in cerebellar medulloblastomas

With the use of antisera against bovine retinal S‐antigen and bovine opsin the authors demonstrate that in cerebellar medulloblastomas certain tumor cells display immunocytochemical properties characteristic of retinal photoreceptors and pinealocytes. S‐antigen‐like and opsin‐like immunoreactions occur in nine of 28 medulloblastomas investigated. All tumors displaying S‐antigen‐like immunoreactive neoplastic cells also contain opsin‐like immunoreactive cells; however, the opsin‐like immunoreactive cells were less frequent than the S‐antigen‐like immunoreactive cells throughout all positive cases. The immunoreactive cells displayed several long processes. Generally, both S‐antigen and opsin‐like immunoreactive cells considerably vary in number among individual tumors. The results indicate that certain neoplastic cells of medulloblastoma are capable of expression of photoreceptor‐specific proteins and, thus, may be closely related to tumor cells of retinoblastoma and pineocytomas previously shown to bind antisera against retinal S‐antigen and opsin. No S‐antigen and opsin‐like immununoreaction was found in malignant teratomas and germinomas of the pineal gland, oat cell tumors, astrocytomas, ependymomas, oligodendrogliomas, glioblastomas, gangliogliomas, gangliocytoma, ganglioneuroblastomas, neuroblas‐tomas, and esthesioneuroblastoma.

Enzyme-linked immunosorbent assay for quantitative determination of the visual pigment rhodopsin in total-eye extracts.

A versatile, multispecies enzyme-linked immunosorbent assay for the rod visual pigment (rhod)opsin has been developed. For this quantitative inhibition assay a monospecific polyclonal antiserum is used which is elicited in rabbits against bovine rod outer-segment membranes. Detergent concentrations as high as 1.0% can be used in the assay with only a slight loss in sensitivity. The assay allows quantitative determination of the apoprotein opsin with a detection level of about 0.04 pmol per sample by using standards prepared by illumination of spectrophotometrically determined amounts of rhodopsin. The antiserum shows considerable cross-reactivity with opsin from several species (mouse, rat, quail, monkey and man). The high degree of monospecificity and cross-reactivity of the antiserum already allowed quantitation of opsin content in crude eye extracts of mouse, rat and quail with a sensitivity comparable to that of bovine opsin. Similar types of multispecies immunoassays for quantitation of highly conserved membrane proteins can be developed using the described approach, requiring only a monospecific antiserum elicited against an easily accessible species and crude tissue extracts both for coating and as a source of the inhibitory antigen.

Effect of Carboxyl Mutations on Functional-Properties of Bovine Rhodopsin

Bovine rod rhodopsin and membrane-carboxyl group mutants are expressed using the recombinant baculovirus expression system. Biosynthesis of wild-type and the mutant D83N is normal. The mutations E122L and E134D/R affect glycosylation and translocation. After regeneration, purification and reconstitution in retina lipids a wild-type photosensitive pigment with spectral and photolytic properties identical to native bovine rod rhodopsin is generated. Only the mutations D83N and E122L affect the spectral properties and then only slightly. All mutations induce a shift in the Meta I<==>Meta II equilibrium towards Meta I (E134D/R) or Meta II (D83N, E122L). FT-IR analysis shows that the mutation E134D/R does not significantly affect the carboxyl-vibration region but, in particular in the case of E134R, affects secondary structural changes upon Meta II formation. E122L also has an effect on secondary structural changes and in addition eliminates a negative band at 1728 cm-1. The mutation D83N removes a pair of negative/positive bands from the carboxyl-vibration region, indicating that Asp83 stays protonated upon formation of Meta II but undergoes a change in hydrogen bonding.

Immunoassay of rod visual pigment (opsin) in the eyes of rds mutant mice lacking receptor outer segments

In 020/A mice, homozygous for the retinal degeneration slow (rds) gene, the photoreceptor cells fail to develop outer segments, and in the absorption spectra of retinal extracts the rhodopsin peak is lacking. Application of an enzyme-linked immunoassay using antisera against bovine opsin shows, however, that opsin is present in the homozygous mutant retina (0.010 nmol/eye) at 3% of the level of the normal retina (0.38 nmol/eye) of Balb/c mice. In the retina of heterozygous mice the opsin level (0.19 nmol/eye) is about half of the normal. Detection of opsin in the rds mutant retina demonstrates the functional basis for the reported electroretinographic response and light-mediated reduction in cyclic nucleotide levels in this mutant.

Development and degeneration of retina in rds mutant mice: immunoassay of the rod visual pigment rhodopsin

Development and loss of photoreceptor cells in mice, afflicted by the rds (retinal degeneration slow) gene, was analyzed by measuring the ocular visual pigment content as rhodopsin (spectroscopy) and opsin (immunoassay). With regard to the postnatal age, where opsin was just detectable, and to the initial rate of opsin synthesis, the mutants did not strongly deviate from the normal animals. The final maximal visual pigment level was, however, about half of normal for the heterozygous mutants and about 3% of normal for the homozygous mutants, both in the pigmented and in the albino strain. In the pigmented normal or heterozygous mutant the (rhod)opsin levels remain stable up to at least 1 year of age. For the corresponding albino animals this was only observed up to 9 months of age. Thereafter the level declines. In the homozygous mutants, maximal opsin levels were observed at about 3 weeks postnatal. Subsequently, this level gradually declined to about 40% in the pigmented and about 15% in the albino mutant. The results indicate that the rds gene does not directly affect the biosynthetic pathways of opsin. The physiological effect of the rds gene is aggravated by photodamage for which the albino animal is particularly susceptible.

Identification of circadian brain photoreceptors mediating photic entrainment of behavioural rhythms in lizards

We have shown previously that in ruin lizards (Podarcis sicula) the ablation of all known photoreceptive structures (lateral eyes, pineal and parietal eye) in the same individual animal does not prevent entrainment of their circadian locomotor rhythms to light. The present study was aimed at identifying the circadian brain photoreceptors mediating entrainment. For this purpose, we looked for opsin expression in the brain by means of immunocytochemistry. Using anti‐cone‐opsin antiserum CERN 874 we have localized photoreceptors in the periventricular area of hypothalamus, near the third cerebral ventricle. We also cloned a brain opsin cDNA that, on the basis of the deduced amino acid sequence, appears to belong to the RH2 class of cone‐opsins. We named the cloned cone‐opsin Ps‐RH2. To examine whether brain cone‐opsins mediate photic entrainment of circadian locomotor rhythms, we performed post‐transcriptional inactivation experiments by injecting an expression eukaryotic vector transcribing the antisense cone‐opsin Ps‐RH2 mRNA in the third cerebral ventricle of pinealectomized–retinectomized lizards previously entrained to a light–dark (LD) cycle. Injections of the antisense construct abolished photic entrainment of circadian locomotor rhythms of pinealectomized–retinectomized lizards to the LD cycle for 6–9 days. CERN 874 completely failed to label cells within the periventricular area of hypothalamus of brains injected with antisense construct. Thus, abolishment of photic entrainment is due to inactivation of endogenous brain cone‐opsins mRNA. The present results demonstrate for the first time in a vertebrate that brain cone‐opsins are part of a true circadian brain photoreceptor participating in photic entrainment of behavioural rhythms.