The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments

The B lymphocytes synthesizes two forms of IgM molecules during its development from a stem cell to a mature antibody-secreting plasma cell. The monomeric receptor IgM molecule is affixed to the plasma membrane and triggers the later stages of B cell differentiation, whereas the pentameric secreted IgM molecule is an effector of humoral immunity. The structural differences between membrane-bound and secreted IgM molecules are reflected in the differences between their heavy or mu chains. We have previously determined the complete amino acid sequence of a murine secreted mu (microsecond) chain. In this study, we have compared the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses. These studies demonstrate that the micron and microsecond chains are very similar throughout their VH, C mu 1, C mu 2, C mu 3 and C mu 4 domains. The micron and microsecond chains differ in the amino acid sequence of their C-terminal segments. These studies in conjunction with those carried out on the micron and microsecond mRNAs and the C mu gene suggest that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively. The amino acid sequence of the 41 residue C membrane terminal segment predicted from the corresponding micron mRNA is in agreement with all the protein studies reported in this paper.     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.     

Environmentally dependent erythrocyte membrane permeability as a source of error in the determination of hematocrit]

The volume of human red blood cells (RBC) was evaluated by means of the centrifugation method (hematocrit) and 131-J-labelled human serum albumin, respectively. Both of the methods yielded an identical volume of about 107 micron3 of the single RBC, provided the evaluation was performed in autologous plasma. Contrary to the 131-J-albumin method the results of which were found independent of various pretreatments of RBC, the centrifugation hematocrits of RBC previously washed with PBS and resuspended in PBS or saline protein media resulted in a mean cell volume of about 86 micron3. The decrease of the cell volume was associated with an efflux of K+ ions. If the RBC are centrifuged at 800 g instead of 15000 g, their volume will remain unchanged. The assessment of cytodeformability has shown, that RBC in PBS by loss of cell volume could enter a 2.3 micron micropipette completely. RBC in plasma, though traversing a 2.9 micron micropipette were incapable of entering a 2.3 micron channel completely. With pressures ranging from 300 to 350 mm H2O the processes of these cells undergo microspherulation.     

Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5-fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post-translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site-directed mutagenesis. However, whereas intracellular microseconds chains in IgM-secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down-regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.     

Eimeria from bats of the world: a new species in Tomopeas ravus from Peru

Two of 17 (12%) crevice bats, Tomopeas ravus (Chiroptera: Vespertilionidae), from Peru had coccidian oocysts in their feces when examined. Sporulated oocysts of Eimeria tomopea n. sp. are ellipsoid to subspheroid, 30.6 X 24.6 (26-34 X 20-28) micron with ovoid sporocysts 13.9 X 9.0 (12-15 X 8-10) micron. A micropyle and substieda body are absent, but polar bodies, oocyst and sporocyst residua, and small, indistinct Stieda bodies are present. The oocyst wall is thick, approximately equal to 1.5 micron and consists of 2 layers, the outermost being rough and mammillated. This is only the 13th eimerian to be described from bats worldwide. The currently recognized species of Eimeria are listed, and a possible New World-Old World phyletic split within the genus is described.     

Coccidia (Apicomplexa: Eimeriidae) from the subterranean rodent Ctenomys opimus Wagner (Ctenomyidae) from Bolivia, South America

Of 35 tuco-tucos (Ctenomys opimus) collected in Bolivia, South America, 31 (88%) had eimerian oocysts in their feces at the time they were examined. Eighteen (58%) of the 31 infected animals were concurrently infected with 2 or 3 eimerian species. Four species of Eimeria were recovered and are described as new species based on the characteristics of sporulated oocysts. Oocysts of Eimeria granifera n. sp. were ellipsoidal, 21.1 x 17.2 (15-26 x 11-20) micron with sporocysts ovoidal, 11.3 x 7.1 (8-14 x 5-9) micron. Oocysts of Eimeria montuosi n. sp. were spheroidal, 24.2 x 22.0 (21-28 x 18-25) micron with sporocysts ovoidal, 10.5 x 7.3 (8-14 x 6-9) micron. Oocysts of Eimeria opimi n. sp. were spheroidal to subspheroidal, 24.3 x 21.8 (18-29 x 15-26) micron with sporocysts ovoidal, 11.6 x 7.6 (10-13 x 6-9) micron. Oocysts of Eimeria oruroensis n. sp. were spheroidal to subspheroidal, 27.3 x 23.6 (23-32 x 20-28) micron with sporocysts ovoidal, 13.2 x 8.6 (10-16 x 8-11) micron.     

Heteromorphism of corynebacteria. I. Macrocells]

A possibility of formation of macrocells (MaC) of diphtheria bacilli was expressed to a different degree; the extreme expression are giant and supergiant forms. Giant forms with section dimension of 3--5micron retained their capacity to disorderly septation with the resultant formation of microcells (MiC). Apparently some of the septa were not realized. Supergiant forms were revealed as a layer and its transverse section. The length of the latter reached 30micron with the transverse section of 2--3micron. The layer has festooned contours with shallow invaginations, but marked day-like cuttings into the body (in case of transverse layer section). Septation of supergiant form was abortive in character. Both forms had homogeneous cytoplasm surrounded by a cell wall. In the supergiant form the latter was morphologically defective in the deepest part of the invaginates. The ultrastructure of both bacterial forms pointed to their rapid growth; however, the supergiant form was likely to be doomed to degeneration and chaotic disintegration.     

Monkey photoreceptor calycal processes and interphotoreceptor matrix as observed by scanning electron microscopy.

Photoreceptors and the interphotoreceptor matrix of monkey retina were observed by scanning electron microscopy. Cone photoreceptors are easily distinguished from rod photoreceptors by their wide conical inner segments. The calycal processes of 50 rods and 50 cones were counted and measured. The calycal processes of cones were distinct, short, and uniform in diameter (0.1 micron). They were arranged equidistantly and, in most cases, were not continuous with longitudinal inner segment ridges, as previously suggested. In contrast to cones, rod calycal processes were fewer in number, were about one-fourth the number of the cones, were of variable length (0.7 micron to 3.0 micron), and tapered to a fine point at their distal ends. The interphotoreceptor matrix appeared spongelike, made up of anastomosing plates and strands filling the interphotoreceptor space. Other than an increased amount of matrix around cones, no structural difference between rod- and cone-associated interphotoreceptor matrix was observed.     

Bone marrow pre-B lymphocytes synthesize immunoglobulin mu chains of membrane type with different properties and intracellular pathways.

Mouse normal bone marrow pre-B lymphocytes synthesize only membrane mu chains (micron), as shown by mRNA studies and peptide analysis. The micron chains exist in two forms: free micron chains assembled into dimers, or L chain-bound micron chains present in IgM monomers (in the case of 'late pre-B cells', i.e., after productive L chain gene rearrangement). These two forms of molecules are very different in properties, fate and intracellular pathways. Free but not L chain-bound mu chains are highly susceptible to mild proteolysis, which degrades their entire Cmu 1 and VH domains. Free mu chains are rapidly degraded within the lysosomal compartment, which they reach via the cis, avoiding the trans, part of the Golgi complex. In contrast, as soon as mu chains bind to L chains, they are directed towards the 'trans' Golgi compartment, where they undergo terminal glycosylation, then to the cell surface, where they progressively accumulate. It is suggested that the conformation instability of the Cmu 1 and VH domains of the free mu chains plays a critical role in the intracellular targeting of these molecules, as compared with that of L chain-bound mu chains.     

The biometric analysis of bacteria in soil

The biometric analysis of bacterial cells in soil by light, fluorescence, and scanning electron microscopy showed that their average size is 0.8 micron in diameter, 1.4 microns in length, and 0.7 micron 3 in volume. In soil loci with enhanced microbiological activity (the rhizoplane of plants and the intestinal tract of soil invertebrates), the average size of bacterial cells was found to be 40% smaller than that of cells occurring in other parts of soil. It is the first experimental evidence showing that the metabolic activity of soil bacteria and their concentration and allometric parameters are related.     

Subcellular distribution of [14C]5-hydroxytryptamine in the blood platelets of rabbits: effects of imipramine and and haloperidol

Long-term (10 days) administration of imipramine [20 mg/(kg X d)] to rabbits significantly increases the Km value (4.0 micron) of 5-hydroxytryptamine uptake in their platelets compared to those of saline- (0.7 micron) or haloperidol- (0.4 micron) treated rabbits. Administration of haloperidol inhibits the 5-hydroxytryptamine uptake non-competitively, and in vitro it had an ID50 value of 22 micron. Intravenous injections of [14C]5-hydroxytryptamine were given to the animals 1 h before blood collection. After isolation of platelets, their sonicates were subjected to 30-60% continuous sucrose gradient centrifugation. The subcellular distribution of [14C]5-hydroxytryptamine indicates that imipramine treatment, in contrast to the control and haloperidol treatment, led to a shift in the exogenous 5-hydroxytryptamine peak from within the granular zone (d 1.18) to the extragranular cytoplasm (d 1.15). Compared to control values, the imipramine treatment caused 63% inhibition in the platelet Na-K-ATPase activity.     

A quantitative histological study of the aortic nerve and the aortic body in the buffalo (Bubalus bubalis)

In the buffalo, the left aortic nerve ramifies in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis. The right aortic nerve ramifies over the dorsal and right aspects of the aorta ascendens near its origin. The histograms of myelinated fibres of both left and right aortic nerve are distinctly unimodal with peak around 4-6 micron (64.2-67.8%). The left aortic body is situated in the periarterial connective tissue between the ventral surface of the aortic arch and the truncus pulmonalis, while the right aortic body is located in the tunica adventitia of the dorsal and right aspects of the aorta ascendens near its origin. The greatest sagittal section area of the left aortic body is 0.102 +/- 0.009 mm2 and that of the right aortic body is 0.041 +/- 0.002 mm2. The organ is highly vascular. The mean size of the glomus cells from the left aortic body is 7.68 +/- 0.9 micron x 9.37 +/- 0.13 micron (short diameter x long diameter), whereas the corresponding value for the right aortic body is 7.84 +/- 0.14 micron x 9.86 +/- 0.21 micron; and their density values are (11,417 +/- 301.7)/mm2 and (9,839 +/- 213.3)/mm2 respectively.     

All-optical histology using ultrashort laser pulses

As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 microJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100 microm or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.     

Dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate.

The dependence of macromolecular composition and morphology of Streptomyces hygroscopicus on specific growth rate micron was investigated. The percentage of DNA on dry weight (%DNA) is constant, % protein is also nearly independent of micron whereas %RNA rises considerably with increasing micron, regarding mycelia grown in glucose-limited and ammonium-limited continuous cultures as well as in discontinuous cultures with various carbon sources. It is probable that the overall synthesis of DNA, RNA and protein is regulated in the mycelium-forming bacterium S. hygroscopicus by the same mechanisms found in unicellular bacteria like Escherichia coli because of the qualitatively similar dependence of %DNA, %RNA and %protein on micron. But differences exist in quantitative regard whereby %DNA, %RNA and %protein of S. hygroscopicus are much smaller at low micron and, with increasing micron, approach those of unicellular bacteria. The hypothesis about the increase of the hyphal regions showing high synthesis activity in S. hygroscopicus mycelia grown in glucose-limited continuous cultures with increasing micron -- derived from comparison of macromolecular composition of S. hygroscopicus and unicellular bacteria -- was confirmed autoradiographically with respect to protein synthesis. The increase of the part of mycelial regions showing high cytoplasmic activity results in an increase of mean hyphal diameter, of mean relative apical growth rate alpha and/or mean relative branching rate beta. Beta depends sigmoidally and alpha inverses sigmoidally on micron. Therefore, the morphology of the mycelium determined by alpha and beta also depends on micron. The hyphal growth unit L/N, the distance from apex to first branch Lp and the mean distance between neighbouring branches Ln decline with increasing micron and reach a minimum at micron = 0.32 (1/h). A further rise of micron is accompanied with an increase of L/N, Lp and Ln. This means that mycelia growing slowly or very quickly have a loose form whereas quickly growing mycelia are characterized by a more compact form. The complicated dependence of alpha, beta, L/N, Lp and Ln on micron indicates that the morphology is regulated by different mechanisms depending on the specific growth rate.     

Caulobacter crescentus flagellar filament has a right-handed helical form

Caulobacter crescentus flagellar filaments were examined for their shape and handedness. Contour length, wavelength and height of the helical filaments were 1.34 +/- 0.14 micron, 1.08 +/- 0.05 micron and 0.27 +/- 0.04 micron, respectively. Together with the value of the filament diameter, 14 +/- 1.5 nm, the parameters of the curvature (alpha) and twist (phi) were calculated as 3.9(%) for alpha and 0.026 (rad) for phi, which are similar to those of the curly I filament of Salmonella typhimurium. Dark-field light microscopic analysis revealed that the C. crescentus wild-type filament possesses a right-handed helical form. Given the result that C. crescentus cells normally swim forward, in the opposite direction to a polar flagellum, it is likely that C. crescentus swims by rotation of a right-handed curly shaped flagellum in a clockwise sense, whereas S. typhimurium and Escherichia coli swim by rotation of left-handed normal type flagella in a counterclockwise sense.     

Reconstruction of a type-B configuration monkey Sertoli cell: size, shape, and configurational and specialized cell-to-cell relationships

A model of a stage-V monkey Sertoli cell was reconstructed from electron micrographs taken of semiserial sections. The configuration (type-B) was one in which spermatids were positioned near the lumen, their heads occupying shallow cylindrical recesses at the apical portion of the Sertoli cell. The cell volume was calculated to be 4,100 micron3, the surface area 2,400.68 micron2, and the surface-to-volume ratio 0.58:1. The reconstructed cell extended from the basal lamina to the tubular lumen and was generally of the tall columnar type although its surface contour was highly irregular. The dimensions of the cell [centripetal (68.46 micron), circumferential (18.40 micron), and longitudinal (21.63 micron)] were determined and cell surfaces designated. Relative and absolute surface areas of the reconstructed cell which faced other Sertoli cells, germ cells, basal lamina, and tubular lumen were calculated. Junctions and surface specializations were enumerated, catalogued, and depicted on diagrams of the cell surface. Where appropriate, type-A rat and type-B monkey Sertoli cells were compared and discussed. Morphometry was utilized to analyze the relative surface areas of germ cells adjoining the reconstructed cell to determine the percentage of their surface facing cellular and acellular elements, and these data were compared to data obtained for the rat.     

Rate of DNA replication and size of replicons in human diploid cells]

The rate of DNA replication and the distances between initiation sites (size of replicons) have been studied in human cultured fibroblasts. The modified Huberman and Riggs technique of DNA fiber autoradiography has been used: the pulse-labelled regions were analysed in DNA fibers preliminarily labelled along the whole length. This enabled us: a) to analyse the arrangement of replicons along the length of labelled DNA fibers with the lengths of 200-750 micron, reaching 2700 micron in some cases; b) to select only single DNA molecules for the analysis. This technique decreases the danger of a mistake when minor labelled regions belonging to different DNA molecules are referred to the same one. The rate of DNA replication varies from 0.2 to 1.2 micron/min, the average of 0.6 micron/min. This conforms with findings of other authors. The distances between initiation sites vary from 15 to 140 micron with the modal interval of 50-60 micron. This value is twice higher than those obtained by other authors. The possible reasons for such difference are discussed.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia

The cochleae of chick embryos of 8 days of incubation until hatching (21 days) were examined by scanning electron microscopy. Unlike what one would expect from the literature, the total number of hair cells per cochlea (10,405 +/- 529) is already determined and visible in a 10-day embryo and the growth of the cochlea is a result of the growth in size and surface area of the hair cells. We also find that the hair cells differentiate simultaneously throughout the cochlea and have followed the differentiation of individual hair cells throughout development. During development we find that the total number, hexagonal packing, and orientation of the stereocilia in each hair cell is determined early and accurately (9- to 10-day embryos). The stereocilia then begin to elongate in all the cells of the cochlea at approximately 0.5 micron/day. By Day 12 the tallest stereocilia in each cell are 1.5-1.8 micron long, the mature length for cells at the proximal end of the cochlea. At this point all stereocilia cease elongating, but those along the inferior edge gradually increase in width from 0.11 micron to maximally 0.19 micron in 17-day embryos. When the stereocilia on the inferior edge reach their mature width, widening ceases and the elongation of stereocilia in the distal hair cells begins again. When these stereocilia have attained their mature lengths, they stop growing. Thus elongation and widening of stereocilia are separated in time. During this period, 11 to 13 days, the shape of the tufts at the proximal end of the cochlea changes. This occurs because stereocilia in the front of each tuft are absorbed while others at the sides appear de novo. This rearrangement converts a circular bundle of stereocilia to a rectangular bundle.