THE LOCALIZATION OF SPECTRIN ON THE INNER SURFACE OF HUMAN RED BLOOD CELL MEMBRANES BY FERRITIN-CONJUGATED ANTIBODIES

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and β-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.     

ELECTRON MICROSCOPY OF THE TISSUE MAST CELL

Electron microscopy was carried out on sections through tissue mast cells from the peritoneal fluid of rats and hamsters, either untreated, x-irradiated, or injected with toluidine blue, protamine sulfate, or stilbamidine. Mast cells from untreated animals have large nuclei and are filled with densely packed, cytoplasmic granules. The latter possess a distinct boundary and an internal structure which is reticular or vacuolar in nature. Between the granules are found elongate mitochondria and endoplasmic reticulum. Mitochondria often appear also in groups in granule-free areas adjacent to the nucleus. Nuclear and cell membranes of an apparent double nature are found. After treatment with toluidine blue, protamine sulfate, and stilbamidine the granules are surrounded by clear areas and are widely separated from one another; the endoplasmic reticulum is more conspicuous. The internal structure of the granule is unchanged. In the mast cells from x-irradiated animals there is an apparent coalescence of granules which is attented by a loss of intragranular structure. The findings are discussed in relation to other work on the structure and function of mast cells.     

GENETIC VARIATION IN THE SHEEP RED BLOOD CELL

1. There are 7 well-established red-cell antigen (blood group) loci. The R-O system has 3 phenotypes, R, O and i, identified by the ‘naturally occurring’ antibodies, anti-R and anti-O. The R and O substances are also present in soluble form in some body secretions. The expression of R and O is controlled by a dominant gene I, epistatic in effect, at an independent locus from that of R. The systems, A, C, M-L, B, D and X-2 are identified by means of ‘immune-type’ antibodies, and several of the loci have multiple alleles. An isoenzymic form of serum alkaline phosphatase is associated with the R-O system. The frequency for the genes at the various loci has been determined in a limited number of breeds. 2. Some sheep red cells have high K+ and low Na+ concentrations (HK type, or Key), others have low K+ and high Na⁺ concentrations (LK type or Kea). Two other rare forms exist; Key type which is HK but with lower than normal K+ values, and Kep type which has approximately equal Na⁺ and K⁺ concentrations. The red cells of foetuses and newborn lambs have high K+ levels irrespective of their potassium genotype. HK cells have 3–4 times greater (Na+-K+)-activated ATPase activity, a 3–4 times increased rate of active K+ transport and a larger number of ouabain-binding sites than LK cells. Antigen M is present on homozygous HK and heterozygous LK red cells, and antigen L is present on homozygous and heterozygous LK red cells. Sensitization of LK cells with anti-L stimulates active K+ transport and ATPase activity and exposes a larger number of ouabain-binding sites in these cells. Anti-M has no effect. The red cells of newborn lambs only show weak L and M antigen activity. It is postulated that L antigen inhibits cation transport in LK cells by masking the pump sites on the membrane. Immature red cells in LK-type sheep have a high rate of active K+ transport and yet have L antigen present. No satisfactory explanation for this has yet been advanced. There is no conclusive evidence that the potassium types have any significance from the point of view of adaptation or sheep breeding. The potassium-gene frequencies are known for a large number of breeds. 3. Two allelic genes, Hb⁴ and Hb^B control 3 haemoglobin phenotypes, A, AB, and B. Foetal haemoglobin (HbF) is present in foetuses and newborn lambs. Sheep with HbA also synthesize small amounts of another haemoglobin (HbC) and under conditions of severe anaemia, synthesis of HbC takes over from that of HbA. No change in HbB is observed in anaemia. A rare haemoglobin (HbD) has been found in 3 Yugoslavian sheep. Hbs A, B, C and F differ in their physicochemical properties; they share the same alpha chains but their non-alpha chains differ in a number of amino acids. HbD differs from HbA in one amino acid in the alpha chain. Certain genetic aspects are discussed. There is some evidence that sheep with HbA are less fertile than those with HbB. The gene frequencies for Hb are known for a large number of breeds. 4. Two isoenzymic forms of carbonic anhydrase are found in red-cell lysates and these are controlled by a pair of allelic autosomal genes, producing 3 phenotypes, CAF, CAFS and CAS. Only a few breeds have been studied but CA^F is apparently quite rare. 5. An unidentified protein, designated ‘X’ is present in electrophoretic separations of haemolysates from some sheep. Its presence is dominant to its absence. Polymorphism at this locus is present in all breeds so far studied. 6. A deficiency of reduced glutathione (GSH) in red cells is found in some sheep and is inherited as an autosomal recessive disorder. Sheep with this deficiency have lower red-cell K+ and Na^f concentrations than normal and it is suggested that the HK GSH-deficient sheep may be Ked type sheep. This deficiency has so far only been found with certainty in one breed of sheep. 7. In sheep twin chimeras, admixture of red-cell antigens, haemoglobin and ‘X’ protein types has been found. Various aspects of chimerism, which occurs only rarely in sheep, are discussed. 8. The significance of the genetic variation is discussed in the light of the physiology and immunology of the red cell and of the sheep itself.     

QUANTITATION OF HUMAN RED BLOOD CELL FIXATION BY GLUTARALDEHYDE

The uptake of glutaraldehyde by human red blood cells has been measured as a function of time by a freezing point osmometer. The rate of attachment of glutaraldehyde to the cell proteins is high over the first hour, declining to zero over a period of a few days. The number of glutaraldehyde molecules cross-linking with each hemoglobin molecule is of the order of 200, in reasonable agreement with the calculated number of attachment sites. The cell membrane is immediately highly permeable to glutaraldehyde. Selective permeability to ions is lost during fixation. Ionic equilibrium is obtained only after a few hours. An optimum fixation technique for shape preservation is suggested.     

ELECTRON MICROSCOPY OF THE VOLVOCACEAE AND ASTREPHOMENACEAE

Lang, Norma J. (U. Texas, Austin.) Electron microscopy of the Volvocaceae and Astrephomenaceae. Amer. Jour. Bot. 50(3): 280-300. Illus. 1963.—Clonal cultures of Gonium sociale, G. pectorale, Pandorina morum, Eudorina elegans, Eudorina sp., Volvulina steinii, V. pringsheimii, Platydorina caudata, Pleodorina illinoisensis, P. californica, Volvox aureus, V. tertius, V. globator, V. barberi, and Astrephomene gubernaculifera representing the Volvocaceae and Astrephomenaceae in the Volvocales were examined with the electron microscope and their ultrastructure compared. The ultrastructure of the various organelles is basically similar in the species studied and no increase in cellular complexity is found to accompany the evolutionary trends evidenced in the Volvocaceae. The ultrastructure of a colonial cell is basically that of Chlamydotnonas. A cytoplasmic membrane having a unit membrane structure encompasses a cell and is continuous with the double-membraned flagellar sheaths. The flagella contain the typical 9 + 2 fibril arrangement with the 2 axial fibrils terminating in a cylinder at the flagellar base and the 9 peripheral pairs continuing into the cytoplasm as a basal body. The organelles comprising the cytoplasm are: mitochondria with plate-like cristae; dictyosomes composed of stacks of agranular cisternae; small, rough or smooth-surfaced vesicles; an endoplasmic reticulum of granule-bearing and agranular tubules, lamellae and broad cisternae; vacuoles which are either contractile, contain fine granular and fibrillar material, or have dense contents probably representing polyphosphate; lipid bodies; and dense granules 100–150 A which have been called ribosomes. The finely granular nucleoplasm is surrounded by a porous, double-membraned nuclear envelope and contains a centric nucleolus composed of dense, spherical granules. The outer membrane of the nuclear envelope bears granules and may have granular extensions into the perinuclear cytoplasm. Each extension appears to encompass one or several dictyosomes and has been termed an “amplexus.” The amplexi are agranular on the surface contiguous to a dictyosome. A double-membraned chloroplast envelope is continuous around the single, cup-shaped chloroplast. The basic chloroplast units are discs closed at each end, occurring in stacks of varying number parallel to the envelope. The presumed proteinaceous matrix of the basal pyrenoid is penetrated by elongated, tubular elements which connect with the lamellar discs. Multiple rows of granules, associated with individual discs, form the anterior stigma within the chloroplast envelope. The colonial matrix is not a structureless, mucilaginous material uniting the cells in colonies, but it has rather a highly complex structure especially around the periphery of the colony and the flagellar channels. The apparent substitution of a fibrillar layer of the colonial matrix for the discrete compact cell wall, such as is found in Chlamydomonas, implies a greater degree of complexity in the evolution of these colonial genera than is generally assumed.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

家鸡联会复合体的亚显微结构分析

本文以表面铺展——硝酸银染色技术,对家鸡的联会复合体(Syneptonemal Complex,SC)作亚显微结构分析。根据对10个精母细胞和10个卵母细胞SC的测量结果,绘制组型图。发现雌雄家鸡的常染色体的SC组型相同。在精母细胞中,性染色体(ZZ)的行为与常染色体相似。在卵母细胞中,性染色体ZW的长度不同,长轴为Z,短轴为W,两者之间只有部分配对,形成SC。从早粗线期到晚粗线期,由同源配对调整为非同源配对。另外,在一只雌鸡中,第一次观察到,有些细胞的常染色体能正常配对,而性染色体完全不配对的现象。     

白唇鹿血象值,红细胞形态和心脏重的测定

作者通过对42只白唇鹿的血样和14只鹿心脏重的测定。结果表明:白唇鹿红细胞系统的血象值有随海拔升高而增多的趋势。红细胞形态呈双面凹陷的轮胎形,侧面观为哑铃状,其直径为4.368±0.1926微米。不同海拔高度的鹿心脏重的比值为25.0％—33.3％,差异不显著。对适应低氧环境的机理,本文作了讨论。     

24种红细胞血型抗原在黑龙江省赫哲族人群中的分布

对３代均为赫哲族的黑龙江省同江县街津口乡和八叉乡的９４人进行了１０个红细胞血型系统（ＡＢＯ、Ｒｈ、ＭＮＳｓ、Ｄｕｆｆｙ、Ｋｉｄｄ、Ｌｕｔｈｅｒａｎ、Ｄｉｅｇｏ、Ｐ、Ｋｅｌｌ、Ｌｅｗｉｓ）共２３种抗原分布的调查,结果显示,基因频率ｑ（０．２２２３）〉ｐ（０．１６９２）,ｍ（０．６０１１）〉ｎ（０．３９８９）,为我国北方人群特点。其它基因频率为,ｒ＝０．６０８５;Ｓ＝０．０６９１,ｓ＝０．９３０９,Ｍ     

THE AGGLUTINATION OF RED BLOOD CELLS IN THE PRESENCE OF BLOOD SERA

1. The addition of blood serum displaces the optimum for agglutination of red blood cells in a salt-free medium to the reaction characteristic of flocculation of the serum euglobulin. 2. This effect is not due merely to a mechanical entanglement of the cells by the precipitating euglobulin, since at reactions at which the latter is soluble it protects the cells from the agglutination which occurs in its absence. 3. A combination of some sort appears therefore to take place between sheep cells and sheep, rabbit, and guinea pig serum euglobulin, and involves a condensation of the serum protein upon the surface of the red cell. 4. At the optimal point for agglutination of persensitized cells both mid- and end-piece of complement combine with the cells. 5. Agglutination is closely related to an optimal H ion concentration in the suspending fluid, and probably of the cell membrane, and not to a definite reaction in the interior of the cell.     

ELECTRON MICROSCOPY OF PHIALIDES AND CONIDIOGENESIS IN TRICHODERMA SATURNISPORUM

Each phialide had a thick-walled neck region located immediately below a light microscopically inconspicuous collarette. The thickened wall of the phialide neck was multilaminate, with layers of different electron transmission properties. A developmental stage in the formation of the first conidial initial was observed. Conidial initials blew out through the thickened neck region, increased in size, and were eventually delimited by centripetally developing septa. Mature, winged conidia had an electron-opaque outer wall layer and an electron-transparent inner wall layer. The wing was formed by separation of these outer and inner wall layers and buckling or wrinkling of the outer layer. As early as they could be discerned, conidial initials had developed the electron-opaque wall layer which characterized mature conidia. Each conidium-delimiting septum became bilayered; the upper layer formed part of the conidial base, and the lower layer became a portion of the wall of the next conidial initial. Phialides lacked an electron-opaque wall layer, and they possessed areas of abundant rough endoplasmic reticulum, as well as free ribosomes. Lipid globules were also abundant, especially in conidia. The distinction between phialides and annellides was questioned.