Light and Scanning Electron Microscopy of Red Blood Cells From Humans and Animal Species Providing Insights into Molecular Cell Biology.

We reviewed the many discoveries in cell biology, made since the 17th century, which have been based on red blood cells (RBCs). The advances in molecular and structural biology in the past 40 years have enabled the discovery with these cells, most notably, of the first water channel protein (WCP) called today aquaporin1 (AQP1). The main aim of our work reviewed was to examine by light and electron microscopy a very wide range of RBCs from reptiles, birds, monotremes, marsupials and placentals, in order to estimate from these images the RBC cell volume and surface area. The diffusional water permeability of the RBC membrane from these species has further been measured with a nuclear magnetic resonance (NMR) spectroscopy technique. The significance of the observed permeability of RBCs to water and possible influences on the whole body are discussed.

Role of Aquaporins in the Physiological Functions of Mesenchymal Stem Cells.

Aquaporins (AQPs) are a family of membrane water channel proteins that control osmotically-driven water transport across cell membranes. Recent studies have focused on the assessment of fluid flux regulation in relation to the biological processes that maintain mesenchymal stem cell (MSC) physiology. In particular, AQPs seem to regulate MSC proliferation through rapid regulation of the cell volume. Furthermore, several reports have shown that AQPs play a crucial role in modulating MSC attachment to the extracellular matrix, their spread, and migration. Shedding light on how AQPs are able to regulate MSC physiological functions can increase our knowledge of their biological behaviours and improve their application in regenerative and reparative medicine.

Morphology and water permeability of red blood cells from green sea turtle (Chelonia mydas).

The morphology and diffusional water permeability (P d) of red blood cells (RBCs) from green sea turtle (GST) (Chelonia mydas) are presented for the first time. The RBCs had an ellipsoidal shape with full-axis lengths (diameters): D = 14.4 µm; d = 10.2 µm; h = 2.8 µm. The values of P d (cm s(-1)) were 5.1 × 10(-3) at 15 °C, 5.7 × 10(-3) at 20 °C, 6.3 × 10(-3) at 25 °C, 6.8 × 10(-3) at 30 °C, and 7.9 × 10(-3) at 37 °C (i.e., significantly higher than in human RBCs in which it was measured to be 4.2 × 10(-3) at 25 °C, 5.0 × 10(-3) at 30 °C, and 6.2 × 10(-3) at 37 °C). There was a lack of inhibition of P d of GST RBCs by p-chloromercuribenzoate (PCMB), a well-known inhibitor of the RBC water channel proteins (WCPs). The activation energy of water diffusion (E a,d) in GST RBCs was 15.0 ± 1.6 kJ mol(-1) which is lower than the E a,d for human RBCs (~25 kJ mol(-1)). These results indicate that in the membrane of GST RBCs, there were no WCPs that were inhibited by the mercurial reagent, while the lipid bilayer of this membrane is unusually permeable to water. This is likely to be a phylogenetically old trait, like that found in amphibians and even the later birds, all of which have nucleated erythrocytes; and it is also likely to be a result of the animal's adaptation to a herbivorous diet (algae and seagrasses).

Comparative studies of water permeability of red blood cells from humans and over 30 animal species: an overview of 20 years of collaboration with Philip Kuchel.

NMR measurements of the diffusional permeability of the human adult red blood cell (RBC) membrane to water (P(d)) and of the activation energy (E(a,d)) of the process furnished values of P(d) ~ 4 × 10(-3) cm/s at 25 °C and ~6.1 × 10(-3) cm/s at 37 °C, and E(a,d) ~ 26 kJ/mol. Comparative NMR measurements for other species showed: (1) monotremes (echidna and platypus), chicken, little penguin, and saltwater crocodile have the lowest P(d) values; (2) sheep, cow, and elephant have P(d) values lower than human P(d) values; (3) cat, horse, alpaca, and camel have P(d) values close to those of humans; (4) guinea pig, dog, dingo, agile wallaby, red-necked wallaby, Eastern grey kangaroo, and red kangaroo have P(d) values higher than those of humans; (5) mouse, rat, rabbit, and "small and medium size" marsupials have the highest values of P(d) (>8.0 × 10(-3) cm/s at 25 °C and >10.0 × 10(-3) cm/s at 37 °C). There are peculiarities of E(a,d) values for the RBCs from different species. The maximum inhibition of diffusional permeability of RBCs induced by incubation with p-chloromercuribenzene sulfonate varied between 0% (for the chicken and little penguin) to ~50% (for human, mouse, cat, sheep, horse, camel, and Indian elephant), and ~60-75% (for rat, guinea pig, rabbit, dog, alpaca, and all marsupials). These results indicate that no water channel proteins (WCPs) or aquaporins are present in the membrane of RBCs from monotremes (echidna, platypus), chicken, little penguin and saltwater crocodile whereas WCPs from the membranes of RBCs from marsupials have peculiarities.

The first discovered water channel protein, later called aquaporin 1: molecular characteristics, functions and medical implications.

After a decade of work on the water permeability of red blood cells (RBC) Benga group in Cluj-Napoca, Romania, discovered in 1985 the first water channel protein in the RBC membrane. The discovery was reported in publications in 1986 and reviewed in subsequent years. The same protein was purified by chance by Agre group in Baltimore, USA, in 1988, who called in 1991 the protein CHIP28 (CHannel forming Integral membrane Protein of 28 kDa), suggesting that it may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 the Agre group identified CHIP28's water transport property. One year later CHIP28 was named aquaporin 1, abbreviated as AQP1. In this review the molecular structure-function relationships of AQP1 are presented. In the natural or model membranes AQP1 is in the form of a homotetramer, however, each monomer has an independent water channel (pore). The three-dimensional structure of AQP1 is described, with a detailed description of the channel (pore), the molecular mechanisms of permeation through the channel of water molecules and exclusion of protons. The permeability of the pore to gases (CO(2), NH(3), NO, O(2)) and ions is also mentioned. I have also reviewed the functional roles and medical implications of AQP1 expressed in various organs and cells (microvascular endothelial cells, kidney, central nervous system, eye, lacrimal and salivary glands, respiratory apparatus, gastrointestinal tract, hepatobiliary compartments, female and male reproductive system, inner ear, skin). The role of AQP1 in cell migration and angiogenesis in relation with cancer, the genetics of AQP1 and mutations in human subjects are also mentioned. The role of AQP1 in red blood cells is discussed based on our comparative studies of water permeability in over 30 species.

On the definition, nomenclature and classification of water channel proteins (aquaporins and relatives).

A water channel protein (WCP) or a water channel can be defined as a transmembrane protein that has a specific three-dimensional structure with a pore that provides a pathway for water permeation across biological membranes. The pore is formed by two highly conserved regions in the amino acid sequence, called NPA boxes (or motifs) with three amino acid residues (asparagine-proline-alanine, NPA) and several surrounding amino acids. The NPA boxes have been called the "signature" sequence of WCPs. WCPs are a family of proteins belonging to the Membrane Intrinsic Proteins (MIPs) superfamily. In addition, in the MIP superfamily (with more than 1000 members) there are also proteins with no channel activity. The WCP family include three subfamilies: aquaporins, aquaglyceroporins and S-aquaporins. (1) The aquaporins (AQPs) are water selective or specific water channels, also named by various authors as "orthodox", "ordinary", "conventional", "classical", "pure", "normal", or "sensu strictu" aquaporins); (2) The aquaglyceroporins are permeable to water, but also to other small uncharged molecules, in particular glycerol; this family includes the glycerol facilitators, abbreviated as GlpFs, from glycerol permease facilitators. The "signature" sequence for aquaglyceroporins is the aspartic acid residue (D) in the second NPA box. (3) The third subfamily of WCPs have little conserved amino acid sequences around the NPA boxes, unclassifiable to the first two subfamilies. I recommend to use always for this subfamily the name S-aquaporins. They are also named "superaquaporins", "aquaporins with unusual (or deviated) NPA boxes", "subcellular aquaporins", or "sip-like aquaporins". I also recommend to use always the spelling aquaporin (not aquaporine), and, for various AQPs, the abbreviation AQP followed immediately by the number, (e.g. AQP1), with no space or--which might create confusions with "minus".

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVIII platypus (Ornithorhynchus anatinus) and saltwater crocodile (Crocodylus porosus).

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from an aquatic monotreme, platypus (Ornithorhynchus anatinus), and an aquatic reptile, saltwater crocodile (Crocodylus porosus) were studied. The mean diameter of platypus RBCs was estimated by light microscopy and found to be approximately 6.3 microm. Pd was measured by using an Mn2+-doping 1H NMR (nuclear magnetic resonance) technique. The Pd (cm/s) values were relatively low: approximately 2.1 x 10(-3) at 25 degrees C, 2.5 x 10(-3) at 30 degrees C, 3.4 x 10(-3) at 37 degrees C and 4.5 at 42 degrees C for the platypus RBCs and approximately 2.8 x 10(-3) at 25 degrees C, 3.2 x 10(-3) at 30 degrees C, 4.5 x 10(-3) at 37 degrees C and 5.7 x 10(-3) at 42 degrees C for the crocodile RBCs. In parallel with the low water permeability, the Ea,d (activation energy of water diffusion) was relatively high, approximately 35 kJ/mol. These results suggest that "conventional" WCPs (water channel proteins), or AQPs (aquaporins), are probably absent from the plasma membranes of RBCs from both the platypus and the saltwater crocodile.

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XVI Dingo (Canis familiaris dingo) and dog (Canis familiaris).

As part of a programme of comparative measurements of Pd (diffusional water permeability) the RBCs (red blood cells) from dingo (Canis familiaris dingo) and greyhound dog (Canis familiaris) were studied. The morphologies of the dingo and greyhound RBCs [examined by light and SEM (scanning electron microscopy)] were found to be very similar, with regard to aspect ratio and size; the mean diameters were estimated to be the same (approximately 7.2 microm) for both dingo and greyhound RBCs. The water diffusional permeability was monitored by using an Mn2+-doping 1H NMR technique at 400 MHz. The Pd (cm/s) values of dingo and greyhound RBCs were similar: 6.5 x 10(-3) at 25 degrees C, 7.5 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C and 11.5 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing SH (sulfhydryl)-modifying reagent PCMBS (p-chloromercuribenzene sulfonate) was investigated. The maximal inhibition of dingo and greyhound RBCs was reached in 15-30 min at 37 degrees C with 2 mmol/l PCMBS. The values of maximal inhibition were in the range 72-74% when measured at 25 degrees C and 30 degrees C, and approximately 66% at 37 degrees C. The lowest value of Pd (corresponding to the basal permeability to water) was approximately 2-3 x 10(-3) cm/s in the temperature range 25-37 degrees C. The Ea,d (activation energy of water diffusion) was 25 kJ/mol for dingo RBC and 23 kJ/mol for greyhound RBCs. After incubation with PCMBS, the values of Ea,d increased, reaching 46-48 kJ/mol in the condition of maximal inhibition of water exchange. The electrophoretograms of membrane polypeptides of the dingo and greyhound RBCs were compared and seen to be very similar. We postulate that the RBC parameters reported in the present study are characteristic of all canine species and, in particular in the two cases presented here, these parameters have not been changed by the peculiar Australian habitat over the millennia (as in the case of the dingo) or over shorter time periods, decades or centuries (as in the case of the domestic greyhound).

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XV. Agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi).

The water diffusional permeability (P(d)) of red blood cells (RBC) from agile wallaby (Macropus agilis), red-necked wallaby (Macropus rufogriseus) and Goodfellow's tree kangaroo (Dendrolagus goodfellowi) was monitored using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. The P(d) (cm s(-1)) values of agile wallaby RBCs were 7.5 x 10(-3) at 25 degrees C, 9 x 10(-3) at 30 degrees C, 11 x 10(-3) at 37 degrees C, and 13 x 10(-3) at 42 degrees C. The inhibitory effect of a mercury-containing sulfhydryl (SH)-modifying reagent p-chloromercuribenzoate (PCMB) on agile wallaby RBCs was investigated. The maximal inhibition was reached in 90 min at 37 degrees C with 2 mmol L(-1) PCMB. The value of maximal inhibition was approximately 63% when measured at 25 degrees C, approximately 52% at 37 degrees C and approximately 45% at 42 degrees C. The lowest value of P(d) (corresponding to the basal permeability to water) was approximately 3 x 10(-3) cm s(-1) at 25 degrees C. For the RBCs from red-necked wallaby (M. rufogriseus) the values of P(d) (cm s(-1)) were 7 x 10(-3) at 25 degrees C, 8 x 10(-3) at 30 degrees C, 10 x 10(-3) at 37 degrees C, and 12 x 10(-3) at 42 degrees C. Higher values of P(d) (cm s(-1)) were found for the RBCs from Goodfellow's tree kangaroo (D. goodfellowi): 8.5 x 10(-3) at 25 degrees C, 10 x 10(-3) at 30 degrees C, 13 x 10(-3) at 37 degrees C, and 15 x 10(-3) at 42 degrees C. The mean values of the activation energy of water diffusion (E(a,d)) were approximately 25 kJ mol(-1) for RBCs from the agile wallaby and tree kangaroo, respectively, and approximately 23 kJ mol(-1) for RBCs from red-necked wallaby. The values of E(a,d) increased after exposure of agile wallaby RBCs to PCMB, reaching a value of approximately 43-46 kJ mol(-1) when the maximal inhibition of P(d) was achieved.

Water channel proteins (later called aquaporins) and relatives: past, present, and future.

Water channels or water channel proteins (WCPs) are transmembrane proteins that have a specific three-dimensional structure with a pore that can be permeated by water molecules. WCPs are large families (over 450 members) that are present in all kingdoms of life. The first WCP was discovered in the human red blood cell (RBC) membrane in 1980s. In 1990s other WCPs were discovered in plants, microorganisms, various animals, and humans; and it became obvious that the WCPs belong to the superfamily of major intrinsic proteins (MIPs, over 800 members). WCPs include three subfamilies: (a) aquaporins (AQPs), which are water specific (or selective water channels); (b) aquaglyceroporins (and glycerol facilitators), which are permeable to water and/or other small molecules; and (c) "superaquaporins" or subcellular AQPs. WCPs (and MIPs) have several structural characteristics which were better understood after the atomic structure of some MIPs was deciphered. The structure-function relationships of MIPs expressed in microorganisms (bacteria, archaea, yeast, and protozoa), plants, and some multicellular animal species [nematodes, insects, fishes, amphibians, mammals (and humans)] are described. A synthetic overview on the WCPs from RBCs from various species is provided. The physiological roles of WCPs in kidney, gastrointestinal system, respiratory apparatus, central nervous system, eye, adipose tissue, skin are described, and some implications of WCPs in various diseases are briefly presented. References of detailed reviews on each topic are given. This is the first review providing in a condensed form an overview of the whole WCP field that became in the last 20 years a very hot area of research in biochemistry and molecular cell biology, with wide and increasing implications.

The study of cystic fibrosis transmembrane conductance regulator gene mutations in a group of patients from Romania.

BACKGROUND: Cystic fibrosis (CF) is produced by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) gene. METHODS: One hundred twenty eight patients with CF were analysed for mutations in the CFTR gene in order to establish the frequency of CF mutations in the Romanian population. The chief methods of analysis were polymerase chain reaction (PCR) of DNA extracted from blood and electrophoresis of PCR products. RESULTS: The frequency of F508del in CF chromosomes from Romania is approximately 56.3%. Other frequent mutations noted are: G542X (3.9%), W1282X (2.3%), and CFTRdele2,3(21 kb)(1.6%); the remaining mutations have frequencies below 1%. CONCLUSIONS: We consider that the frequency of F508del in CF patients from Romania is higher than in previous reports, reaching 56.3%, probably owing to more rigorous selection of patients for genetic testing, allowing improved calculation of mutation frequencies.

Why does the mammalian red blood cell have aquaporins?

Aquaporins are now known to mediate the rapid exchange of water across the plasma membranes of diverse cell types. This exchange has been studied and kinetically characterized in red blood cells (erythrocytes; RBC) from many animal species. In recent years, a favoured method has been one based on NMR spectroscopy. Despite knowledge of their molecular structure the physiological raison d' etre of aquaporins in RBCs is still only speculated upon. Here, we present two hypotheses that account for the fact that the exchange of water is so fast in RBCs. The first is denoted the "oscillating sieve" hypothesis and it posits that known membrane undulations at frequencies up to 30 Hz with displacements up to 0.3 microm are energetically favoured by the high water permeability of the membrane. The second denoted the "water displacement" hypothesis is based on the known rapid exchange across the RBC membrane of ions such as Cl- and HCO3- and solutes such as glucose, all of whose molecular volumes are significantly greater than that of water. The ideas are generalizable to other cell types and organelles.

The first water channel protein (later called aquaporin 1) was first discovered in Cluj-Napoca, Romania.

This invited review briefly outlines the importance of membrane water permeability, highlights the landmarks leading to the discovery of water channels. After a decade of systematic studies on water channels in human RBC Benga's group discovered in 1985 the presence and location of the water channel protein among the polypeptides migrating in the region of 35-60 kDa on the electrophoretogram of RBC membrane proteins. The work was extended and reviewed in several articles. In 1988, Agre and coworkers isolated a new protein from the RBC membrane, nick-named CHIP28 (channel-forming integral membrane protein of 28 kDa). However, in addition to the 28 kDa component, this protein had a 35-60 kDa glycosylated component, the one detected by the Benga's group. Only in 1992 Agre's group suggested that "it is likely that CHIP28 is a functional unit of membrane water channels". Half of the 2003 Nobel Prize in Chemistry was awarded to Peter Agre (Johns Hopkins University, Baltimore, USA) "for the discovery of water channels", actually the first water channel protein from the human red blood cell (RBC) membrane, known today as aquaporin 1 (AQP1). The seminal contributions from 1986 of the Benga's group were grossly overlooked by Peter Agre and by the Nobel Prize Committee. Thousands of science-related professionals from hundreds of academic and research units, as well as participants in several international scientific events, have signed as supporters of Benga; his priority is also mentioned in several comments on the 2003 Nobel Prize.

Birth of water channel proteins-the aquaporins.

If we compare aquaporin (as a proteic pathway for water permeation across biological membranes) with a child we can say that he had a very long gestation period. His possible existence was predicted for a long time (Overton in 1985, Stein and Danielli in 1956), some of his features (transport of water and its reversible inhibition) were assigned by Macey and Farmer in 1970, however this child was first detected by Benga and coworkers in 1986. We clearly demonstrated for the first time the presence and location of a water channel at the human RBC membrane among the polypeptides migrating in the region having 35-60 kDa on the electrophoretogram of RBC membranes, labeled with 203Hg-PCMBS in the conditions of specific inhibition of water diffusion; I suggested that a minor membrane protein that binds PCMBS is involved in water transport and also indicated the way in which the specific protein could be further characterized: by purification and reconstitution in liposomes. Our landmark papers in 1986 can be compared with the first detection of a child "in utero" by ultrasonography, since we discovered one of the essential components of the "aquaporin child" (a molecular weight of 35-60 kDa for the glycosylated component); we have also indicated the way to recognize him after birth (among other children of his group!): placing the isolated children in a certain environment and asking them to perform the same task (one should read: reconstitution studies in liposomes and measurement of water permeability), like aligning athletes for a running test. This was the only certain way to know that the child is really the fastest runner and not just one that is helping (by various means) another child to be fastest runner. A "new child" was observed in 1988 by Agre and coworkers, who identified a novel integral membrane protein in human RBCs having a non-glycosylated component of 28 kDa and a glycosylated component migrating as a diffuse band of 35-60 kDa; they suggested that the new protein (nick-named CHIP28 in 1991) may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 Agre and coworkers suggested that CHIP28 is a functional unit of membrane water channels; by reconstitution in liposomes it was demonstrated that CHIP28 is a water channel itself rather than a water channel regulator. In other words the child we first detected was recognized as having the predicted qualities only in 1992. In 1993 CHIP28 was renamed aquaporin 1. Looking in retrospect, asking the crucial question, when was the first water channel protein, aquaporin 1, discovered, a fair and clear cut answer would be: the first water channel protein, now called aquaporin 1, was identified or "seen" in situ in the human RBC membrane by Benga and coworkers in 1986. It was again "seen" when it was by chance purified by Agre and coworkers in 1988 and was again identified when its main feature, the water transport property was found by Agre and coworkers in 1992. If a comparison with the discovery of The New World of America is made, the first man who has "seen" a part, very small indeed, of The New Land was Columbus; later, others, including Amerigo Vespucci (from whom the name derived), have better "seen" a larger part of the new Continent and in the subsequent years many explorers discovered the complexity of the Americas!