Fluorometric measurement of urinary alpha-L-iduronidase activity. A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.


Isolation and characterization of a novel fucoganglioside of human erythrocyte membranes. A slow migrating monosialosyl ganglioside with blood group H activity was isolated from human erythrocyte membranes and identified as monosialosylceramide decasaccharide. Its structure was determined by degradation with exo- and endoglycosidases, by mass spectrometric characterization of a nonasaccharide liberated by endo-beta-galactosidase, and by methylation analysis and mass spectrometry of permethylated glycolipids before and after enzymatic degradation. The ganglioside contains two oligosaccharide chains branched through GlcNAcbeta1 leads to 6(GlcNAcbeta1 leads to 3) Gal structure, and the terminal Gal of the 1 leads to 6-linked oligosaccharide was fucosylated, whereas that of the 1 leads to 3-linked oligosaccharide was sialosylated as seen below.


Tibial fractures involving the ankle in children. The so-called triplane epiphyseal fracture. Triplane fractures of the distal end of the tibia in fifteen children (average age, thirteen years) represented 6 per cent of 237 consecutive epiphyseal fractures of the ankle. Thirteen children were treated by closed methods (including manipulation) and two had open reduction of the fractures. At an average of twenty-six months after injury, three of fourteen patients showed roentgenographic evidence of premature symmetrical epiphyseal closure with less than 0.5 centimeter of shortening and no angular deformity. Of twelve children examined clinically, three had a 5 to 10-degree external rotation deformity and one patient also had an articular incongruity due to inadequate reduction. In the five cases in which tomograms were used to the medial malleolus, and the anteromedial part of the epiphysis. The lateral fragment included the remainder of the epiphysis together with a piece of posterior metaphysis with attached fibula.


Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins? Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane-bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein-framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.


Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution. This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex-lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.


Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography. The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar absorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C. The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.


Capillary column gas chromatography--mass spectrometry in studies on rheumatoid arthritis. A method is described for determining the organic acid profile of synovial fluid. The method requires 50--100 microgram of sample and involves ion exchange for isolation of the acids followed by combined capillary column gas chromatography--mass spectrometry. Normal synovial fluid and synovial fluid from patients with rheumatoid arthritis show a similar qualitative pattern of acids (mainly fatty acids) resembling the pattern found in serum. The concentrations of the organic acids in the abnormal synovial fluids are 5--10 times higher than those in the fluid from the normal joints. In patients receiving treatment with gold thiomalate, free thiomalate was excreted in the urine. The release of this thiol upon gold therapy is of consequence for the further understanding of the mechanism of action of certain drugs against rheumatoid arthritis.


Use of UV scanning techniques in the identification of serum constituents separated by high-performance liquid chromatography. Serum constituents separated by the reversed-phase partition mode of high-performance liquid chromatography were identified by using a stopped-flow UV scanning technique. This method of peak identification eliminates post-chromatographic sample handling. Results from UV scanning were correlated with those obtained from absorbance ratios of reference compounds and the enzymatic peak-shift technique.


[Intraluminal duodenal diverticulum (author's transl)]. The authors report a case of intraluminal duodenal diverticulum and compare it with those reported previously in the literature. They emphasise the diagnostic methods, the pathological characteristics and consider the proper identification of the papilla of the ampulla of Vater very important since this structure is often adjacent to the diverticulum.


Comparison of sensitivity and specificity of purified lymphocyte and whole-blood in vitro lymphocyte stimulation assays in detection of Brucella abortus infection in cattle. A study was conducted to compare the sensitivity and specificity of purified lymphocyte and whole-blood in vitro lymphocyte stimulation assays in detection of Brucella abortus infection in cattle. Cattle used were infected with B. abortus field strains or strain 19. Peripheral blood was collected, and lymphocytes for the technique. The blood for the whole-blood lymphocyte stimulation assay was diluted 10-fold with RPMI 1640 medium (without additional serum supplement) and cultured. The two tests were run simultaneously, and B. abortus soluble antigen or concanavalin A was added to the cultures. The cultures were incubated for 6 days and assayed for [3H] thymidine incorporation into their DNA. Generally, cultures of the purified lymphocyte stimulation assay had higher counts per minute than those of the whole-blood lymphocyte stimulation assay, but the stimulation ratios for the two tests were comparable. The two assays were comparable in terms of their sensitivity and specificity as applied to detection of brucella infection in cattle.


Determination of immunoglobulin A concentrations in human nasal secretions with a serum immunoglobulin A standard. By quantitative immunodiffusion tests, nasal secretion immunoglobulin A was underestimated by approximately 5.6-fold when the serum 7S immunoglobulin A standard was employed.


Genetic control of retinal ganglion cell projections. We have assessed the effects of 15 pigmentation mutations on the development of retinal ganglion cell projections in mice in two ways: (1) by analyzing the pattern of innervation of the ipsilateral lateral geniculate nucleus as mapped in autoradiograms of brains of animals killed 12 days after intravitreal injection of 3H-proline into one eye and (2) by determining the ratio of axonally transported radioactive protein in the contralateral and ipsilateral optic tracts after similar intravitreal injections. Analysis of the ratio of transported protein in the two optic tracts provides a new and useful assay of the degree of decussation in experimental animals. The effects of the mutations on eye pigmentation, whole eye melanin content and relative tyrosinase activity also were examined. The degree of ipsilateral innervation generally correlates with the degree of pigmentation of the retinal pigment epithelium and with tyrosinase activity. However, discrepancies have been found in ch and ce mutants. In these animals the pigment epithelium is well pigmented, and the area of ipsilateral innervation in the lateral geniculate nucleus is extensive, despite a high ratio of label in contralateral to ipsilateral optic tracts and low tyrosinase activity. Furthermore, mice heterozygous for the c2J allele have pigmentation and optic projections that are normal even though tyrosinase is reduced to 40% of normal. The few anomalous results suggest that alternative or additional factors may control optic axon projections.


The medial geniculate body of the tree shrew, Tupaia glis. I. Cytoarchitecture and midbrain connections. In this study of the medial geniculate body in the tree shrew eight subdivisions are identified on the basis of differences recognized in Nissl-stained material. Experiments using the methods of anterograde and retrograde axonal transport and anterograde degeneration show that each subdivision has a unique pattern of connections with the midbrain. The ventral division of the medial geniculate body contains at least two subdivisions, the ventral nucleus and the caudomarginal nucleus. The ventral nucleus is characterized by densely-packed cells and receives topographically organized projections from the central nucleus of the inferior colliculus. The caudomarginal nucleus, on the other hand, receives its major midbrain projections from the medial nucleus in the inferior colliculus. In the dorsal division four subdivisions are distinguished. The suprageniculate nucleus contains large, loosely-packed cells and receives projections from the deep layers of the superior colliculus and from the midbrain tegmentum. The dorsal nucleus receives projections from the midbrain tegmentum. The deep dorsal and anterodorsal nuclei have neurons which resemble those in the dorsal nucleus. Both receive projections from the roof nucleus of the inferior colliculus but the deep dorsal nucleus receives an additional projection from the parabrachial tegmentum. The medial division has a rostral and a caudal subdivision. The ascending projections to the rostral nucleus are from the lateral zone in the inferior colliculus and from the spinal cord. The caudal nucleus contains cells with large somas and receives projections from most of the midbrain areas which project to the other subdivisions of the medial geniculate body.


The medial geniculate body of the tree shrew, Tupaia glis. II. Connections with the neocortex. In this study the temporal cortex of the tree shrew was subdivided on the basis of cytoarchitectonic criteria, and the connections of each subdivision with the thalamus and midbrain were analyzed with retrograde and anterograde techniques. The results indicate that, with one exception, each subdivision of the medial geniculate body projects to a separate cortical area. The primary auditory cortex receives projections from the ventral nucleus. Surrounding the primary cortex are at least five additional cytoarchitectonically distinct areas which receive projections from the remaining medial geniculate subdivisions. The evidence suggests that there is very little overlap in the projections from each of these geniculate subdivisions. An exception is the projection of the caudal nucleus of the medial division. This subdivision apparently projects to most, if not all, of the cortical target of the medial geniculate body. Although the cortical projections of the caudal nucleus overlap those of the other medial geniculate subdivisions, the laminar distribution of its terminations in cortex is different. The caudal nucleus projects primarily to layer VI whereas the other subdivisions of the medial geniculate body project primarily to layer IV and the adjacent part of layer III. Anterograde techniques were also used to study the projections from the cortex back to the thalamus and to the midbrain. The projections to the thalamus precisely reciprocate the thalamocortical connections. The projections to the midbrain are to the same areas which the preceding study (Oliver and Hall, '78) showed give rise to ascending projections to the medial geniculate body. An exception is the central nucleus of the inferior colliculus which apparently does not receive a projection from the temporal cortex.


Patterns of retinal terminations and laminar organization of the lateral geniculate nucleus of primates. Autoradiographic tracing procedures have been used to study the organization of retinogeniculate axons in seven primates, i.e., four species of New World monkeys, one species of Old World monkeys and two species of prosimians. These data suggest that the basic primate pattern of geniculate lamination consists of two parvocellular layers, two magnocellular layers, and two poorly developed and highly variable superficial (S) layers which are ventrally located. Ocular input to each member of each of the three pairs differs. In the macaque, the squirrel, and the saki monkey, the parvocellular layers subdivide and interdigitate into four leaflets so as to give the appearance of four parvocellular "layers." These leaflets are much less extensive in the owl and marmoset monkeys. In some individual macaque monkeys, there is further splitting of the parvocellular leaflets into subleaflets, giving the appearance of six parvocellular "layers." The prosimians (galago and slow loris) have two additional layers that are not found in pithecoid primates, and only one superficial layer is apparent. The two additional layers are termed "koniocellular" since they consist of very small cells. Finally, New and Old World monkeys have both ipsilateral and contralateral retinal input to the interlaminar zones. We conclude that the basic pattern of lateral geniculate organization is six layers, but not the traditional six. Prosimians have evolved two additional layers, the koniocellular layers, and have possibly lost one superficial layer. Both New World and Old World monkeys have elaborated the parvocellular layers by forming leaflets to varying extents. With the possible exception of the single S layer in prosimians, layers form pairs that are similar in cell types, but different in ocular input.


The efferent connections of the anterior hypothalamic area of the rat, cat and monkey. The general morphology and topographic relations of the anterior hypothalamic area (AHA) in the rat, cat and squirrel monkey have been described, and its efferent connections analyzed autoradiographically, after small injections of 3H-labeled amino acids into, or around, the area. In all three species the AHA is rather poorly separated from the surrounding preoptic and hypothalamic areas and nuclei but shows three distinct cellular condensations, located rostrally, centrally, and posterodorsally. Closely associated with the AHA are the retrochiasmatic area, the anterior periventricular nucleus and the scattered neurons usually referred to as the accessory supraoptic nucleus. The AHA has primarily short connections to the adjoining medial preoptic area, the lateral hypothalamic area, the periventricular nucleus, the dorsomedial nucleus, and to the "capsule" of the ventromedial nucleus. However, it also has certain more distant projections, rostrally to a narrow zone centered in the ventral part of the lateral septal nucleus, and caudally to the dorsal premammillary nuclei, the posterior hypothalamic area and the central gray. There is some evidence to suggest that the various subdivisions of the AHA have different efferent connections. Thus the posterodorsal cell condensation appears to give rise to the bilateral projection to the dorsal premammillary nuclei, while the projections to the septum, the posterior hypothalamic area and the central gray seem to have their origin in the central condensation. Similarly, the retrochiasmatic area sends its efferents through the ventral supraoptic commissure to the amygdala, the anterior periventricular nucleus contributes to the periventricular fiber system and to the external lamina of the median eminence, and the accessory supraoptic neurons project to the internal lamina of the median eminence.


The identification of relay neurons in the dorsal lateral geniculate nucleus of monkeys using horseradish peroxidase. Intraaxonal retrograde transport of the protein horseradish peroxidase (HRP) was used to identify relay neurons in the dorsal lateral geniculate nucleus (LGN) of owl (Aotus trivirgatus) and rhesus (Macaca mulatta) monkeys. In both species, from 94.1-98.6% of the neurons within columns extending through both parvocellular and magnocellular layers were labeled following injection of HRP into striate cortex. Labeled neurons were also identified in the thin ventral-most S(0) Layers. Although most of the cells within the thin interlaminar regions in the LGN of both species were labeled following injections of HRP, many unlabeled neurons were identified within the large cell-rich interlaminar region (IL) between the internal parvocellular and internal magnocellular layers in the LGN of the owl monkey, suggesting that IL may be a specialized region containing a large number of intrinsic neurons. Finally, measurement of the cell diameters of neurons within the densely labeled areas in relay layers revealed that labeled and unlabeled neurons could not be distinguished on the basis of cell body size alone and that some of the smallest cells of the LGN project to striate cortex. These findings indicate that nearly all of the neurons of the main relay layers of the LGN in these two primates are relay cells and that the organization of the LGN in primates may differ significantly from that of other mammals with respect to the percentage of interneurons.


The central projections of the glossopharyngeal and vagus ganglia in the mallard, Anas platyrhynchos L. The central projections of the glossopharyngeal and vagus nerves in the mallard have been studied with the Fink-Heimer I method and are compared to those of the trigeminal and facial nerves. The N. vagus projects ipsilaterally and contralaterally upon the central nuclei of the solitary complex, except the most rostral part of it, upon the n. sulcalis dorsalis, the parasolitary nuclei and the n. commissuralis. The glossopharyngeal nerve contributes to the rostral pole of the n. centralis anterior and to the n. ventrolateralis anterior of the solitary complex, but it has also terminal fields in a cellgroup sIX of the principal sensory trigeminal nucleus, in a small cellgroup sIXd on the dorsum of the descending trigeminal tract, in the n. interpolaris of this tract and in nuclei of the cuneate complex. There is hardly any overlap of the respective terminal fields. The convergence of projections from N VII and N IX can be connected with the presence of tastebuds in upper and lower bill and in the soft palate. The converging projections from N V and N IX in "trigeminal" nuclei may reflect the functional coherence of the mechanoreceptors in bill and tongue. It is suggested that these nuclei play a role in the feeding behavior.


Projections to the visual cortex in the golden hamster. Retrograde transport of horseradish peroxidase (HRP) was used to determine the origins of afferent connexions to the visual cortex (areas 17, 18a and 18b) in the hamster. The distribution of neurons projecting to the visual cortex from other cortical areas, from the thalamus and from the brainstem was studied using a computer technique for three-dimensional reconstruction. There is a topographically organized projection from the dorsal lateral geniculate nucleus to area 17, but probably to no other of the areas studied. The lateral posterior nucleus of the thalamus (LP) projects to area 18a and weakly to area 17. The lateral nucleus (L) projects to area 18b and also, probably, weakly to area 17. The cortical projections from LP and L are also organized topographically but relatively grossly compared with the geniculo-cortical pathway. There are reciprocal association projections between area 17 and areas 18a and 18b. Areas 18a projects weakly to 18b. The main commissural connexions of the posterior neocortex are between the area 17/18a boundary zones in the two hemispheres, with little between the bodies of area 17. Labelled neurons were found bilaterally in the locus coeruleus, more ipsilaterally than contralaterally, after multiple injections into the visual cortex: single, small injections sometimes resulted in the labelling of a single cell body in the locus coeruleus.


An autoradiographic study of the rubroolivary tract in the rhesus monkey. Autoradiographic tracing methods were employed to study the course and distribution of the rubroolivary tract following unilateral injections of tritiated leucine into the rostral red nucleus of seven rhesus monkeys. A topographic organization of projections to the ipsilateral principal nucleus of the inferior olivary complex was demonstrated. Lateral and medial portions of the rostral red nucleus projected to medial parts of the dorsal and ventral laminae of the principal inferior olive respectively; neurons in intermediate lateralities emitted fibers which terminated in lateral parts of the principal olive. Injections involving the oral end of the rostral red nucleus elicited label overlying the medial accessory olive in addition to the principal nucleus. Projections to the medial accessory olive may have arisen from the rostral end of the red nucleus and/or the immediately adjacent tegmentum. There were no projections to the dorsal accessory olive. Fibers of rubral origin also were distributed ipsilaterally to several reticular nuclei including the pedunculopontine, pontis oralis, caudalis, and gigantocellularis.


Ca2+-stimulated phosphorylation of muscle glycogen synthase by phosphorylase b kinase. Phosphorylase b kinase from rabbit muscle phosphorylates glycogen synthase purified from the same tissue. The reaction is markedly stimulated by Ca2+ and results in a decrease in the synthase %I activity. Phosphorylase b kinase action leads to the incorporation of phosphate (0.6 to 0.8 mol/mol of subunit) preferentially into a single cyanogen bromide fragment of synthase (fragment III). Cyclic AMP-independent synthase kinase also shows a specificity for the site(s) contained in fragment III whereas the cyclic AMP-dependent protein kinase exerts a preference for the site(s) located in a distinct cyanogen bromide fragment (fragment II). A Ca2+-stimulated endogenous kinase also results in the phosphorylation of fragment III and can be attributed to the presence of phosphorylase b kinase. The finding of a Ca2+-stimulated phosphorylation of glycogen synthase has important implications for the regulation of glycogen metabolism and particularly those processes thought to be controlled by cytoplasmic Ca2+ concentration.


Lymphatic vessels of healthy and inflamed gingiva. Lymphatic vessels were traced in healthy and early inflamed gingivae of various nonhuman primates. Lymphatic capillaries and primary collecting vessels originated in the connective tissue papillae and stroma near or adjacent to the attachment epithelium. They emptied into thin-walled irregular-shaped collecting vessels that drained into the periodontal ligament. In early inflammation, all the lymphatic vessels of luminal caliber were distended within and/or surrounding the inflammatory infiltrate.


Effect of chewable fluoride tablets on dental caries in schoolchildren: results after six years of use. Two groups of children, initially in the first  and second grades, chewed, rinsed with, and swallowed an acidulated phosphate-fluoride tablet containing 1 mg of fluoride either once or twice a day in school. A control group of children followed the procedure once a day using a placebo tablet. An evaluation after six years of treatments showed that both the once-a-day and the twice-a-day procedures with fluoride tablets effectively reduced the incidence of dental caries.


Reduced allergenicity of high molecular weight ragweed polymers. Polymerized ragweed antigens of different molecular weight ranges were studied to determine if the degree of allergenicity was dependent on molecular size of the polymers. In this study, allergenicity is defined as the ability to elict IgE-mediated skin reactivity. Ragweed antigen treated with glutaraldehyde was fractionated into one preparation with a molecular weight range of 200,000 to 20,000,000 and another with molecular weights less than 200,000. Except for the difference in molecular sizes of the two molecular sizes of the two preparations, all ragweed polymers had been treated in an identical fashion. The low molecular weight fraction had cutaneous endpoint reactivity greater than 10(5) to 10(8) times that of the high molecular weight materials. In these and other studies, immunogenicity defined as the ability to induce an IgG antibody response was retained by the high molecular weight material. The results are consitent with the hypothesis that allergenicity decreases as the molecular weight of the polymerized allergen increases.


Human and macaque mastication: a quantitative study. Significant differences exist between human and Macaca fascicularis patterns of mandibular movement during mastication. Macaque patterns display less asymmetry, more uniformity, and limited lateral excursions when compared to humans for mastication of the same food. Different anatomical structures between the two species offer explanations of the different patterns that were observed. Researchers should use caution when using macaques as models for human mastication.


In vivo analysis of bone strain about the sagittal suture in macaca mulatta during masticatory movements. In vivo strain gauge analysis demonstrated that tensile bone strain is transmitted along the cranial vault to the parasagittal region during isotonic temporalis contraction. This strain is sufficient to cause measurable separation of the sagittal suture, and thus could influence growth at the sutural margins.


Glycosyltransferases in plant and animal tissues effects of fixation and lead on enzyme activity. As the initial step toward the cytochemical localization of glycosyl-transferases in situ, biochemical determinations of these enzyme activities from onion root tips and L1210 cells were performed before and after fixation as well as in the presence of lead ions. Glycosyltransferase activity from roots fixed in buffered formaldehyde or glutaraldehyde before homogenization decreased as the concentration of the fixative or fixation time was increased. Formaldehyde fixation was less inhibitory than glutaraldehyde; 35% of the glycosyltransferase activity was retained after 30 min fixation in 2% formaldehyde while 25% of the enzyme activity remained after a similar fixation in glutaraldehyde. Substantially higher levels of L1210 cell glycosyltransferase activity were retained after a 30 min 2% formaldehyde fixation (60% sialyltransferase; 82% galactosyltransferase), but inhibition by glutaraldehyde was similar to that observed for onion root galactosyltransferase. Glycosyltransferase from formaldehyde-fixed roots was inhbited 35% by lead nitrate, but sialytransferase from formaldehyde-fixed L1210 cells was unaffected by lead ions. These findings are encouraging for further studies aimed at the development of cytochemical technique to localize glycosyltransferase in plant and animal tissues.


Immunofluorescent staining of histaminase (diamine oxidase) in human placenta. An immunofluorescent procedure for the localization of histaminase in human tissue sections has been developed by using a specific antiserum against human placental histaminase. For localization of this enzyme in placental sections, fixation in equal volumes mixture of absolute ethanol and acetone provided the optimum visualization of this enzyme in both frozen sections and paraffin-embedded sections. The immunofluorescent staining of this enzyme in placenta is found to be localized in areas within the maternal decidua, both within the cytoplasm of the decidual cells and in tissue space between the cells. The chorionic villi are completely void of the immunofluorescent stain. Variations in patterns of histaminase localization have been found between term and premature placentas, with the former showing a predominantly intercellular localization and the latter a predominantly intracellular localization. The intercellular localization of this enzyme in the decidua may represent a nonspecific diffusion of the enzyme associated with delivery of the placenta or may reflex a specific functional role of the enzyme in the intercellular space during pregnancy.


An attempt at immunotherapy of murine virus erythroleukemia. It was demonstrated that the nonvirulent strain of Listeria monocytogenes isolated from vegetable matter prolonged significantly the survival time of mice inoculated with murine virus erythroleukemia. The most significant survival time was recorded when live listeriae were administered on the same day but into another site than the leukemia virus was.


A new B cell differentiation antigen (BDA) on normal and leukemic human B lymphocytes that is distinct from known DR (Ia-like) antigens. A newly defined human B cell differentiation antigen, designated as BDA, has been defined and partially characterized. BDA is expressed on normal human B cells and lymphocytic leukemia cells at all stages of known differentiation (pre-B cells to plasma cells). It is distinct from DR (Ia-like) determinants and other known B cell surface constituents.


C3-mediated cytoadherence. II. Dependence of cell attachment on similar topographic distribution of the receptors (for C3) and the ligands (C3/C3b). Particles carrying C3 in a random distribution (Etan-C3) bound to C3 receptor (Raji+) cells independent of temperature and irrespective of whether the Raji cells were fixed with glutardialdehyde. In contrast, the reactivity of EAC43b, having grouped C3b in clusters, was dependent on temperature. The interaction with Raji cells was inhibited if the latter were treated with a fixative. The reaction of both Etan-C3 and EAC43b was a function of the concentration of C3 and C3b, respectively. The conclusion is drawn that for the interaction between C3/C3b-carrying particles and C receptor cells the receptors and the ligands have to be grouped in a similar typographic distribution, irrespective if this arrangement is provided already from the beginning or if it is obtained only by lateral movement of one or both reaction partners.


The structural heterogeneity among degraded gamma heavy chain disease proteins. The amino terminal sequence of seven newly studied and three previously reported gamma Heavy Chain disease proteins show that they begin at several sites ranging from position 215--234 in the hinge region. The presence of more than one species of molecule in several of the sera and the susceptibility of this region to proteolytic cleavage, taken together with the finding of a blocked Heavy Chain disease protein in the serum of one and the cells of another patient support the concept that these molecules are the result of proteolytic cleavage of typical Heavy Chain disease proteins.


Hemolysis-in-gel (HIG) test for antibodies to influenza A, measles and mumps using liquid nitrogen freezed erythrocytes coupled with the respective viral antigen. A drawback with the hemolysis-in-gel test is the constant need for fresh erythrocytes which must be treated with virus before incorporation in the gel. This problem was overcome by freezing small droplets containing erythrocytes to which antigen had been attached. The droplets were stored at -70 degrees C or in liquid nitrogen. This modification was applied in detecting antibodies to influenza, measles and mumps viruses and the results were shown to equal those obtained with fresh erythrocytes.


A simple method for quantifying IgM of low molecular weight. A new and simple method for quantitation of IgM of low molecular weight in sera by radial immunodiffusion in 7% agar gel is described. The test is easy to perform and permits exact determination of low molecular weight IgM concentration down to very small quantities (1 mg%). Results obtained with the method described agree with previously used assay procedures and offer the advantage of convenience and rapidity.


Rocket immunoelectrophoresis in the presence of denaturing agents. Modifications of the Laurel rocket technique for assaying antigen antibody reactions are described. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents (e.g., SDS and urea) and subjected to electroimmunoassay without loss of sensitivity or specificity. Methods are also presented for obtaining rockets using gel slices from polyacrylamide gel electrophoresis either in the presence of urea or SDS. Results obtained using keratins, the insoluble proteins of epidermis, hair and nail are summarized.


Xenogenic response of human peripheral blood lymphocytes against murine cells in vitro. Human peripheral blood lymphocytes (HPBL) were sensitized to mitomycin-treated murine spleen cells and mitomycin-treated mastocytoma P-815. A DNA proliferative response occurred to both cell types. The sensitized HPBL lysed chromium-51-labelled mastocytoma P-815 cells in a cell-mediated cytotoxicity assay (CMC). Evidence is presented that this human-murine xenogeneic system involves T cell response with specificity in CMC mainly toward H-2 antigens. DBA/2 splenocytes were superior to mastocytoma cells as sensitizers in the xenogeneic DNA proliferative assay.


Artefactual variations in the B and T subpopulations of rabbit blood lymphocytes depending on method of isolation, and blocking of C3 receptors due to in vitro activation of complement. Marked differences were found in the proportions of lymphocyte subpopulations in rabbit peripheral blood depending on the techniques used for the purification of lymphocytes. Rosette-forming reactions were used to find the numbers of T lymphocytes, Ig-bearing cells and cells with receptors for C3 or IgG-Fc. Some of the methods used for lymphocyte separation altered the relative numbers of T and B lymphocytes, through a disproportionate loss of T cells. Other changes were due to in vitro activation of complement detectable by the presence of C3 on the lymphocyte cell-membrane and causing partial blocking of C3 receptors. Highest yields of lymphocytes were obtained from defibrinated blood treated with carbonyl iron to remove phagocytes and methyl cellulose to sediment erythrocytes. This procedure was accompanied by in vitro activation of complement, with the consequences mentioned. Complement activation was inhibited by taking the blood into either EDTA or citrate. As EDTA was cytotoxic for rabbit T lymphocytes, citrate was considered best although the resulting lymphocyte suspensions were contaminated with up to 25% granulocytes and monocytes owing to the inhibition of carbonyl iron uptake by the prior exposure to citrate.


Polyglutaraldehyde: a new reagent for coupling proteins to microspheres and for labeling cell-surface receptors. Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hypdrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrically as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations.


A method for the preparation of protein-protein conjugates of predetermined composition. A novel procedure for the synthesis of well-defined protein-protein conjugates is described using ovalbumin (OA) and IgG as test proteins. This procedure involves the highly selective and rapid reaction of alkyl halide and sulfhydryl groups, which have been grafted, respectively, onto the proteins to be conjugated. Accordingly, iodoacetylated IgG, (ICH2CO)nIgG, was prepared by the reaction of the epsilon-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA), the degree of iodoacetylation (n) being proportional to the concentration of NHIA. OA was reacted with S-acetylmercaptosuccinic anhydride (SAMSA) under conditions yielding, on the average, a monosubstituted derivative. Following removal of the protective S-acetyl group, the resulting -SH derivative of OA was reacted with (ICH2CO)nIgG. The OAx-IgG conjugates so produced were characterized by gel filtration, specific radioactivity (using tritiated OA) and immunodiffusion. It was found that the average number of OA molecules coupled per IgG molecule could be controlled by varying the degree of iodoacetylation of IgG.


Immunodiffusion using tissue sections.

Immunodiffusion using tissue sections. A simple radial immunodiffusion technique is described for determining relative antigen content of frozen tissue sections. Its use is illustrated with frozen sections of chick intestine containing the vitamin D-induced calcium-binding protein (CaBP). Diffusion of the intestinal CaBP from the sections placed on agar containing specific anti-CaBP anti-serum produced a precipitin ring, whose diameter was proportional to the CaBP content of the intestine. The technique allowed the determination of the relative antigen content in sections of tissue adjacent to others in which immunocytochemical localization or other histochemical studies were to be performed.


Control of intestinal flora in animals and humans: implications for toxicology and health. The importance of the intestinal flora in disease management is discussed, and apparent relationships noted in experimental and clinical reports are reviewed. It is suggested that investigation of the influence of the intestinal flora upon body organs and systems would productively lay the groundwork for abetter understanding of certain synergistic mechanisms.


Genetic analysis of vitellogenesis in Drosophila melanogaster: the identification of a temperature-sensitive mutation affecting one of the yolk proteins. A number of female sterile mutations on the first and third chromosomes of Drosophila melanogaster have been screened for defects in the yolk proteins using polyacrylamide gel electrophoresis. Two new mutants were identified. 6m45 accumulates all three yolk proteins (YP1, YP2 and YP3) in the haemolymph but they are all absent from the ovaries suggesting it is a yolk-protein-uptake mutant. In contrast, 1163 is a temperature-sensitive mutation with a large reduction in the quantity of YP1 in the haemolymph and ovaries at 29 degrees C. Both mutants are autonomous in ovary transplant experiments.


Renal effects of repeated administration of calcium disodium ethylenediaminetetraacetate during excessive exposure to lead in rats. The renal effects of repeated administration of EDTA were studied in rats given 0.5% lead as lead acetate in drinking water for 24 weeks. Repeated treatment of lead toxic rats with EDTA during continuous exposure to lead reduced blood lead levels and brain and bone lead but not to a statistically significant level (p greater than 0.05). Liver lead concentration in the EDTA treated, lead exposed rats changed only slightly upward, but kidney lead levels decreased greatly when compared to lead exposed, non-EDTA treated rats, (group IV vs. group III), (p less than 0.05). Light and electron microscopy of kidneys of lead treated rats showed evidence of an early interstitial nephropathy characterized by peritubular fibrosis and variation in size of tubular cells. Nuclei of renal tubular cells of lead exposed rats had inclusion bodies, but these were not present in lead exposed, EDTA treated rats. The present study suggests that repeated treatment of rats with EDTA during continuous exposure to lead reduces renal lead concentration but does not significantly influence the progression of lead induced nephropathy.


Interaction of hydroxychlorobiphenyls--polychlorinated biphenyl metabolites--with the human erythrocyte membrane. Effects of hydroxychlorobiphenyls (polychlorinated biphenyl metabolites) and chlorobiphenyls on membranes have been studied with the human erythrocyte membrane as a model. Many of the hydroxychlorobiphenyls are very effective hemolytic agents, whereas the parent chlorobiphenyls are generally quite ineffective at inducing hemolysis. The hemolytic potency of the hydroxychlorobiphenyls varies with the degree of chlorination and, more importantly, with the position of the chloro- and hydroxy- substituents. At lower concentrations, the hydroxychlorobiphenyls protect the erythrocyte against hypotonic hemolysis, while they induce hemolysis at higher concentrations. In the range of concentrations of each hydroxychlorobiphenyl required for maximum protection, the erythrocytes exist in altered morphological forms as opposed to normal discocytes. The chlorobiphenyls at lower concentrations also protect the erythrocytes from hypotonic hemolysis, but they do not induce hemolysis at higher concentrations. These studies suggest that products of the metabolism of chlorobiphenyls may be more biologically active than the parent compounds themselves. Effects on membranes may thus play a role in the mammalian toxicity of the hydroxychloro- and chlorobiphenyls.


Metabolic behaviour and cleavage capacity in the amphibian egg. During the winter season the full grown Bufo arenarum oocyte shows the metabolic behaviour characteristic of differentiated tissues of the same species. Due to seasonal variations, during the amplexus period, it acquires the metabolic behaviour of the segmenting egg. Short-time-induced ovulations (5-6 h) determine germinal vesicle breakdown immediately before the expulsion of the oocyte, without modifying the ovarian metabolism of the same. The incidence of the operative type of metabolism upon their capacity to cleave after insemination and needle pricking, has been studied in coelomic oocytes, which have attained nuclear maturation and have not experienced oviducal secretion effects. The results obtained indicate that the segmenting capacity of the egg is attained only when, through biochemical modifications, the oocyte acquires the metabolic behaviour characterizing embryonic cells. It is postulated that the metabolic changes observed in the oocyte constitue a fundamental aspect of cytoplasmic maturation.


Uptake of the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate by HeLa cells. The uptake of 12-0-tetradecanoyl-phorbal-13-acetate (TPA) by HeLa cells, cells of a line responsive to the action of this tumor promoting agent, was studied. Our results indicate that most of the uptake is due to a physical nonsaturable process which is not grossly affected by inhibitors of energy metabolism or macromolecular synthesis. Uptake occurs mainly in membranous fractions of the cell, is largely reversible, and is temperature dependent. These results suggest that 3H-TPA uptake is largely due to partitioning of this lipophilic substance into the lipid phase of the cell membranes.


On a regulatory gene controlling the expression of the murine lambda1 light chain. We describe here two alleles, an allele of the lambda1 locus present in the SJL strain (rlambda1lo) and an allele of the lambda1 locus present in the BALB/c strain (rlambda1 +), of a regulatory gene locus which specifically influences the expression of the mouse lambda1 light chain structural gene. The rlambda1 regulatory gene is not linked to either the major histocompatibility complex or to the heavy-chain allogroup but appears to be linked to the lambda1 structural gene locus. In the homozygous state, the present of the rlambda1lo allele results in a 50-fold reduction in the number of lambda1 antigen-sensitive, bone-marrow derived lymphocytes (ASCs) compared to the presence of the rlambda1 + allele. However, those few lambda1ASCs present in rlambda1lo homozygotes can be induced normally to produce lambda1 light chains indistinguishable from those found in rlambda1 + homozygotes. The reduction in lambda1ASC's due to the rlambda1lo allele results both in a reduction in the amount of lambda1 Ig in the serum and also in a large variation in the magnitude of the lambda1 antibody response to alpha(1,3) dextran by individual animals. This variation permits the estimate that, on the average, 50 B cells of anti-alpha(1,3) specificity must be present per animal to permit a measurable response. Surprisingly, the expression of a gene locus regulating lambda1 light chain expression (rlambda1 locus) shows a clear gene dosage effect with rlambda1lo/rlambda1 + heterozygotes having 1/2 the number of lambda1ASCs and 1/2 the amount of serum lambda1 Ig as rlambda1 +/rlambda1 + homozygotes. This fact permits an analysis of the relationship between germ-line v-genes and their individual expression in serum Ig. The rlambda1 locus controls specifically a DNA-level event which occurs in stem cells as they become committed to lambda1 light chain expression. We postulate that the rlambda1 locus represents one of the DNA level recognition sites involved in the translocation event which places the vlambda1 and clambda1 structural genes in a transcriptional unit.


The histocompatibility restrictions on macrophage T-helper cell interaction determine the histocompatibility restrictions on T-helper cell B-cell interaction. To study the histocompatibility restriction between macrophages and helper T cells, carrier primed guinea pig T cells were positively selected in vitro with antigenpulsed macrophages for 7 days and the selected T cells were then mixed with hapten-primed B cells and stimulated with antigen in a modified Mishell-Dutton system. Helper T cells could only be selected with syngeneic, but not allogeneic, antigen-pulsed macrophages and would then collaborate only with syngeneic, but not allogeneic, hapten-primed spleen cells. When F1 T cells were selected with antigen-pulsed parental macrophages they would only collaborate with B cells of the same parental strain as the macrophages used in the selection culture. These results are strongly in support of the view that the primed T cell is activated by carrier determinants of the nominal antigen in association with Ia antigens on macrophages and the helper T cell, in turn, activates B cells which bear the same Ia antigens and determinants of the nominal antigen bound to immunoglobulin receptors on their surface. In addition, in experiments with antigens the response to which is controlled by I-linked genes, we demonstrated that primed (responder X nonresponder)F1 T cells would only collaborate with B cells of the responder parent. The defect appeared to be at the level of the B cell in that the addition to the cultures of antigen-presenting cells of the responder type did not restore the ability of F1 T cells to collaborate with non-responder B cells.


Antigen-specific suppressor T-cell activity in genetically restricted immune spleen cells. Virgin spleen cells develop comparable primary antibody responses in vitro to syngeneic or allogeneic macrophages (Mphi) bearing the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), whereas immune spleen cells primed with syngeneic or allogeneic GAT-Mphi develop secondary responses preferentially when stimulated with GAT-Mphi syngeneic to the GAT-Mphi used for priming in vivo. These restrictions are mediated by products of the I-A subregion of the H-2 complex and are operative at the level of the GAT-Mphi-immune helper T-cell interactions. To investigate why these immune spleen cells fail to develop a significant antibody response to GAT-Mphi other than those used for in vivo immunization and determine the mechanism by which the restriction is maintained, spleen cells from virgin and syngeneic or allogeneic GAT-Mphi-primed mice were co-cultured in the presence of GAT-Mphi of various haplotypes. Antibody responses to GAT developed only in the presence of GAT-Mphi syngeneic to the Mphi used for in vivo priming; responses in cultures with GAT-Mphi allogeneic to the priming Mphi, whether these Mphi were syngeneic or allogeneic with respect to the responding spleen cells, were suppressed. The suppression was mediated by GAT-specific radiosensitive T cells. Thus, development of GAT-specific suppressor T cells appears to be a natural consequence of the immune response to GAT in responder as well as nonresponder mice. The implications of stimulation of genetically restricted immune helper T cells, and antigen-specific, but unrestricted, suppressor T cells after immunization with GAT-Mphi in vivo are discussed in the context of regulatory mechanisms in antibody responses.


Suppressor T-cell activity in responder X nonresponder (C57BL/10 X DBA/1)F1 spleen cells responsive to L-glutamic acid60-L-alanine30-L-tyrosine10. The ability of spleen cells from (responder X nonresponder)F(1) mice immunized with various GAT-Mphi, GAT-MBSA, and soluble GAT to develop IgG GAT-specific PFC responses in vitro after stimulation with responder and nonresponder parental and F(1) GAT-Mphi, was investigated. F(1) spleen cells from mice immunized with F(1) GAT-Mphi or GAT-MBSA developed secondary responses to responder and nonresponder parental and F(1) GAT- Mphi, but not to unrelated third party GAT-Mphi. Spleen cells from F(1) mice immunized with either parental GAT-Mphi developed secondary responses to F(1) GAT-Mphi and only the parental GAT-Mphi used for immunization in vivo. Soluble GAT-primed F(1) spleen cells responded to F(1) and responder parental, but not nonresponder parental, GAT-Mphi. Simultaneous immunization in vivo with the various GAT-Mphi or GAT-MBSA plus soluble GAT modulated the response pattern of these F(1) spleen cells such that they developed secondary responses only to F(1) and parental responder GAT-Mphi regardless of the response pattern observed after immunization with the various GAT-Mphi or GAT-MBSA alone. These observations demonstrate the critical importance of the physical state of the GAT used for immunization in determining the subsequent response pattern of immune F(1) spleen cells to the parental and F(1) GAT-Mphi. Further, suppressor T cells, capable of inhibiting primary responses to GAT by virgin F(1) spleen cells stimulated by nonresponder parental GAT-Mphi, were demonstrated in spleens of F(1) mice immunized with soluble GAT, but not those primed with F(1) GAT-Mphi. Because responder parental mice develop both helper and suppressor T cells after immunization with GAT-Mphi, and soluble GAT preferentially stimulates suppressor T cells whereas GAT-Mphi stimulate helper T cells in nonresponder parental mice, these observations suggest that distinct subsets of T cells exist in F(1) mice which behave phenotypically as responder and nonresponder parental T cells after immunization with soluble GAT and GAT- Mphi.


Malaria immunization in Rhesus monkeys. A vaccine effective against both the sexual and asexual stages of Plasmodium knowlesi. Rhesus monkeys were immunized with a preparation of Plasmodium knowlesi parasites containing principally microgametes with lesser numbers of macrogametes and asexual trophozoites. The antigen mixture was emulsified in Freund's complete adjuvant (FCA) and administered intramuscularly. After one or two inoculations of from 10(5) to 10(7) microgametes in FCA, monkeys showed high levels of circulating anti-gamete antibodies as demonstrated by various in vitro microgamete immobilization or transmission blocking tests. After challenge with P. knowlesi, immunized monkeys developed low level asexual parasitemias and were not infectious to feeding mosquitoes as measured by growth of the parasite on the mosquito gut. Control monkeys developed rapidly rising, usually fatal infections and were highly infectious to mosquitoes. Anti-gamete antibodies appear to neutralize the sexual parasites and prevent mosquito infection within the gut of the recently fed mosquito vector. Suppression of asexual parasitemia in immunized monkeys may be due to the presence of asexual trophozoites in the antigen mixture or to antigens common to both sexual and asexual stages of the parasite. A vaccine effective as a single injection capable of interrupting malaria transmission from man to man whereas reducing the severity of the disease in infected individuals offers a new approach to the control of one of the major diseases affecting man.


The involvement of suppressor T cells in Ir gene regulation of secondary antibody responses of primed (responder X nonresponder)F1 mice to macrophage-bound L-glutamic acid60-L-alanine30-L-tyrosine. (Responder [R] X nonresponder [NR])F1 mice give indistinguishable primary in vitro plaque-forming cell (PFC) responses to either R or NR parental macrophages (Mphi) pulsed with the Ir-gene controlled antigen L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). However, such (R X NR)F1 mice, if primed to GAT, retained in vitro responsiveness to GAT-R-Mphi, but no longer responded to GAT-NR-Mphi. This suggested (a) a possible Mphi-related locus for Ir gene activity in this model, and (b) the occurrence of active suppression after priming with GAT leading to a selective loss of the usual primary responsiveness of (R X NR)F1 mice to GAT-NR-Mphi. This latter interpretation was tested in the current study. [Responder C57BL/6 (H-2b) X nonresponder DBA/1 (H-2q)]F1 mice were primed with 100 microgram GAT in pertussis adjuvant. 4-8 wk later, spleen cells from such mice were tested alone or mixed with normal unprimed F1 spleen cells for PFC responses to GAT-R-Mphi and GAT-NR-Mphi. The primed cells failed to respond to GAT-NR-Mphi, and moreover, actively suppressed the normal response of unprimed F1 cells to GAT-NR-Mphi. If the primed spleen cell donor had been treated with 5 mg/kg cyclophosphamide 3 days before priming or with 5-10 microliter/day of an antiserum to the I-Jb subregion [B10.A(5R) anti B10.A(3R)] during the first 4 days postpriming (both procedures known to inhibit suppressor T-cell activity), cells from such mice responded in secondary culture to both GAT-R-Mphi and also GAT-NR-MPhi. In addition, such spleen cells no longer were capable of suppressing normal F1 cells in response to GAT-NR-Mphi. Similar data were obtained using [CBA (H-2k) X DBA/1 (H-2q)]F1. Further, it was shown that (a) primary responsiveness to GAT-NR-Mphi was not an artifact of in vitro Mphi pulsing, because in vivo GAT-pulsed Mphi showed the same activity and (b) the secondary restriction for Mphi-antigen presentation was controlled by H-2 linked genes. These data suggest an important role for suppressor T cells in H-2 restricted secondary PFC responses, and also provide additional support for the hypothesis that Ir-gene controlled differences in Mphi antigen presentation are related to both suppressor cell generation and overall responsiveness in the GAT model.


Pre-B and B cells in rabbits. Ontogeny and allelic exclusion of kappa light chain genes. Pre-B cells in developing rabbits were identified by immunofluorescence as cells containing small amounts of cytoplasmic IgM (cIgM) but lacking surface immunoglobulin (sIg). During ontogeny the first pre-B cells appeared in fetal liver at 23 days gestation, 2 days before the appearance of sIgM+ B lymphocytes. Pre-B cells were relatively frequent in fetal and adult bone marrow, but were not found in other tissues except rarely in fetal spleen. Allelic exclusion is apparently established at this early stage of development, because individual pre-B cells and B lymphocytes from heterozygous rabbits expressed only one of the alternative alleles in amounts sufficient for detection. Development of isotype diversity among rabbit B lymphocytes followed the general pattern seen in mouse and man. sIgM+ cells were detected before birth. Expression of sIgG was detected in neonatal rabbits on cells which were also sIgM+ but in older animals most sIgG+ cells lacked sIgM. Cells bearing sIgA were not found until 5-6 days of age, and had no other isotype on their surface.


The natural abundance of lambda2-light chains in inbred mice. The amino acid sequence of the constant (C) domain of the light chain of the mouse myeloma protein M315 has not been identified so far in any other myeloma protein. In this study, serological analysis with antiserum to the C-domain of this light chain (L315) showed that approximately equal to 1% of Igs in normal mouse serum have L chains of the L315 type (called lambda2). Corroborative evidence was obtained by analysis of the carboxyterminal amino acid removed from normal light chains by carboxypeptidase A. A survey of 35 inbred mouse strains showed that all had lambda2; the serum level of Igs with lambda2-chains ranged from approximately equal to 140 microgram/ml in AL/N mice to approximately equal to 25 microgram/ml in SJL, BSVS, and eight other strains. In accord with the anti-Dnp activity of M315, sera from mice immunized with Dnp-KLH had three- to fivefold more lambda2 than sera from control mice immunized with KLH. It was also possible to measure serum immunoglobulin molecules bearing the lambda2 variable region of M315 (VL315). In BALB/c sera, the concentration of VL315 was about sixfold lower than that measured for lambda2. Thus, lambda2-chains are divided into at least two subsets: those whose V domain is indistinguishable from VL315 and those whose VL differs from VL315. A 10-fold increase in VL315 was obtained by immunizing BALB/c mice with Dnp-KLH. The relationship of the VL domains of normal immunoglobulin lambda2-chains to the embryonic Vlambda gene recently sequenced by Tonegawa et al., is discussed.


The role of H-2 linked genes in helper T-cell function. IV. Importance of T-cell genotype and host environment in I-region and Ir gene expression. We have studied the properties of helper T cells specific for sheep erythrocytes (SRBC), keyhole limpet hemocyanin (KLH), or poly-L-(Tyr,Glu)-poly-DL-Ala-poly-L-Lys [(T,G)-A--L]. These T cells differentiated and were primed in vivo in irradiation chimeras constructed of various combinations of F1 and parental bone marrow donors and irradiated recipients. Primed T cells were then tested for helper activity in the in vitro response of B cells and macrophages (Mphi) of parental or F1 origin to the hapten trinitrophenol coupled to the priming antigen. When testing either SRBC or KLH-specific T cells of parental H-2 type which had differentiated in F1 hosts, we found that they cooperated equally well with B cells and Mphi of either parental H-2 type. On the other hand, when testing F1 T cells which had differentiated in parental hosts, we found that they cooperated well only with B cells and Mphi which had the K-IA region type of the parental host. In similar experiments we found that (T,G)-A--L-specific T cells of low responder H-2 type which had differentiated in (high responder X low responder) F1 hosts induced high responses in high responder B cells and Mphi (T,G)-A--L-specific F1 T cells which differentiated in high responder but not those which differentiated in low responder hosts induced high responses in high responder B cells and Mphi. Low responder B cells and Mphi yielded low responses in all cases regardless of the source of (T,G)-A--L-specific T cells with what they were tested. Our results support the conclusion that I-region and Ir genes function via their expression in B cells and Mphi and in the host environment during helper T-cell differentiation, but not, at least under the conditions of these experiments, via their expression in the helper T cell itself. These findings place constraints upon models which attempt to explain the apparent dual recognition of antigen and I-region gene products by helper T cells.


Activation of mouse lymphocytes by anti-immunoglobulin. II. A thymus-independent response by a mature subset of B lymphocytes. Mouse spleen cells can be stimulated to proliferate in vitro by purified anti-mu or anti-gamma,kappa antibodies. These responses can be obtained in cell populations bearing  membrane immunoglobulin (Ig), purified by the fluorescence activated cell sorter (FACS), but they are not observed in FACS-purified Ig- cell populations. Furthermore, treatment of spleen cell populations with anti-Thy 1.2 and complement does not impair the response, nor does addition of nylon wool-purified T lymphocytes enhance it. These results indicate that B lymphocytes respond to anti-Ig and that their response does not require T cells. On the other hand, cells from athymic nude (nu/nu) mice respond slightly less well to anti-mu than do cells from heterozygous littermate (nu/+) controls; nu/nu cells are almost unresponsive to anti-gamm,kappa and addition of nylon wool-purified T cells from nu/+ controls does not restore the response. This suggests that T lymphocytes or the thymus may control the appearance of cells responsive to anti-gamma,kappa. Responsiveness of normal mice to anti-mu does not appear until 4 wk of age and does not reach maximum levels until 8 wk of age. Acquisition of full responsiveness to anti-gamma,kappa is even more delayed. This, together with the failure of mice with the CBA/N B-cell defect to respond to anti-Ig, suggests that cells stimulated to proliferate by anti-Ig are a mature subset of B cells. Depletion of adherent cells by Sephadex G-10 treatment or by treatment with carbonyl iron and exposure to a magnetic field does not diminish anti-mu or anti-gamma,kappa responses, suggesting that the responsiveness does not require the presence of macrophages. Thus, activation of B-cell proliferation by anti-Ig appears to be a T-cell independent, macrophage-independent process in which membrane Ig plays a direct role in signal generation.


The surface properties of Neisseria gonorrhoeae: determinants of susceptibility to antibody complement killing. Monovalent rabbit antisera were prepared to highly purified gonococcal lipopolysaccharide (LPS), to pili and to two major purified outer envelope proteins. All these antisera were free from significant specific IgM antibody and were standardized to 4 microgram specific IgG antibody per test, permitting accurate comparisons between the different gonococcal surface antigens as triggers of the complement-dependent bactericidal reaction. LPS was the most effective antigen at inducing a bactericidal response to homologous and heterologous gonococci, followed by the two individual outer envelope proteins. Pili were relatively ineffective. Strain P9 gonococci grown in vivo or which possessed a 'capsule' in vitro were more resistant to serum killing than the non-capsulated parent strain. One highly susceptible strain, F62, which was killed by complement in the absence of any LPS antibody, was able to directly activate complement by the alternative pathway.


The surface properties of Neisseria gonorrhoeae: topographical distribution of the outer membrane protein antigens. Gonococci were labelled with 125I using the lactoperoxidase system. The amount of label incorporated was similar with all strains including those which appeared capsulated. Electrophoresis on sodium dodecyl sulphate-polyacrylamide gels revealed that the major proteins labelled were those found in outer membrane preparations. Comparison of variants of one strain showed that the major outer membrane protein (protein I) was always present and heavily labelled. The second major protein (protein II) was present in variable amounts but labelling was proportional to the amount present. A third protein (III) was only present in outer membranes from a freshly isolated variant but was present in whole cells of each strain. Protein III was not labelled in whole cells but was labelled in outer membrane preparations suggesting that many membranes have their inner surface exposed. The labelling of a strain adapted to growth in guinea-pig chambers failed to reveal any new major surface proteins. The results demonstrate the variation in surface topography possible with variants of one strain of gonococcus but show that one major protein antigen is always expressed on the surface.


Degeneration and regeneration of autonomic nerve endings in the anterior part of rhesus monkey ciliary muscle. The autonomic nerve plexus of the ciliary muscle was examined with the electron microscope in normal rhesus monkeys of different ages. In the anterior region of the muscle, at the boundary with the poorly innervated scleral spur and trabecular meshwork, 3.8-7.1% of the axons exhibit either degenerative or regenerative features. The cytoplasm of degenerating axons contains lamellated, dense and multivesicular bodies, vesicles, whorls of filaments, and membranous debris. The plasma membrane is often discontinuous and, on occasion, axonal debris and degenerative organelles are freely dispersed in the connective tissue spaces of the muscle. Degenerating axons contain a granular reaction product when stained for acid phosphatase activity. Regenerating axons are characterized by tightly packed mitochondria, glycogen particles, and aggregates of synaptic vesicles; they synapse with muscle cells and are negative to the acid phosphatase reaction. A quantitative analysis showed that in the anterior region of the ciliary muscle degenerating and regenerating axons increase in number with age, although the total number of axonal profiles remains constant. In the age groups examined, degenerating axons occurred with the same frequency as regenerating axons, thus, the age-dependent increase in axonal degeneration is accompanied by a parallel increase in axonal regeneration. We conclude that autonomic nerve endings in the anterior part of the ciliary muscle undergo a continuous process of renewal that is more prominent in old age.


The role of calcium ions in the synthesis and transport of noradrenaline carrier vesicles in guinea-pig sympathetic neurons in vitro. Guinea-pig inferior mesenteric ganglia (IMG)/hypogastric nerve preparations were incubated in tissue culture media containing ethylene glycol-bis-(beta-aminoethyl ether)N,N'tetra-acetic acid (EGTA) or colchicine and examined either by fluorescence microscopy or by transmission electron microscopy. Two millimolar EGTA inhibited the accumulation of noradrenaline (NA) fluophore proximal to a crush, but did not produce an increase in the fluorescent intensity of, or the number of dense-cored vesicles (DCVs) within the neuronal perikarya. Calcium ions, but not magnesium ions, were able to block this effect of EGTA. Preparations incubated in the presence of colchicine (2.5 microgram/ml) showed a reduction in the amount of fluorescent material accumulating proximal to a crush, but an increase in both the fluorescent intensity and the number of DCVs within the neuronal perikarya. The suggestion that calcium ions are required for the synthesis of some part of the NA-containing vesicle rather than for their loading onto the axoplasmic transport mechanism is discussed.


Hemangioma calcificans. Case report of an intraparenchymatous calcified vascular hematoma with epileptogenic potential. A middle-aged woman, with a previous history of medically suppressed absence attacks, presented with mild changes in mental status and a skull film demonstrating several areas of mottled, granular, intracranial calcifications. These lesions, although readily visible on computerized tomography, appeared avascular during the course of cerebral angiography. At the time of surgery the masses, which were densely calcified and generally circular, demonstrated numerous areas of superficial, white, verrucous excrescences. Microscopic, pathological evaluation confirmed the diagnosis of hemangioma calcificans. The literature describing this rare entity is briefly reviewed.


Collection of blood from the pituitary stalk and portal veins in monkeys, and from the pituitary sinusoidal system of monkey and man. A transorbital, transsphenoidal microsurgical approach to the pituitary stalk and gland was used to collect blood from the hypothalamo-hypophyseal portal system in monkeys. Specimens may be obtained from the entire pituitary stalk, individual long portal veins, or the pituitary sinusoidal bed, with little risk of mortality. Continuous stalk blood sampling was carried out for periods of up to 10 hours. Pituitary sinusoidal-system blood was also collected during transsphenoidal surgery in man. The uses of data concerning hypothalamic-hypophyseal regulation obtained by these methods are illustrated.


Evidence for the promotion of bone mineralization by 1alpha,25-dihydroxycholecalciferol in the rat unrelated to the correction of deficiencies in serum calcium and phosphorus. Concurrent administration of 1alpha,25-dihydroxycholecalciferol [1alpha,25-(OH)2-CC] to intact and thyroparathyroidectomized rats treated with ethane-1-hydroxy-1,1-diphosphonate (EHDP) prevented or reversed the EHDP-induced inhibition of bone mineralization as measured by changes in epiphyseal plate width and ash content of bone. An analog, 1alpha-droxycholecalciferol, was also effective. Recovery of bone after EHDP treatment was also significantly improved by administration of 1alpha,25-(OH)2-CC as evidenced by enhanced uptake of 45Ca by epiphyseal plates and decreased plate widths. Cholecalciferol (CC), ergocalciferol, dihydrotachysterol2, 5,6-trans-CC, 25-OH-CC, 5,6-Trans-25-OH-CC, and 1alpha24R,25-(OH)3-CC also blocked EHDP-induced epiphyseal plate widening, but required high, pharmacological dose levels. 24R,25- (OH)2-CC was inactive at doses up to 10 microgram/day. Since EHDP-treated rats are not deficient in calcium or phosphate, these data suggest that 1alpha,25-dihydroxycholecalciferol promoted bone mineralization independently of effects upon the intestinal absorption of calcium and phosphate.


The relationship between the dose of dentogingival plaque and the in vitro lymphoproliferative response in subjects with periodontal disease. The in vitro lymphoblastic response to autologous dentogingival plaque (DGP) extract at nine concentrations was measured in 28 individuals (Periodontal Index [Russell 1956] 0.8-7.4). Significantly elevated responses were obtained in 25 of the patients but the levels of the peak responses were unrelated to the severity of the diseases. There was a significant (n = 28, r = -.734) negative linear relationship between the PI of the individuals studied and the logarithmic concentration of DGP producing the peak lymphoblastic response. Lower concentrations of DGP extract would appear to be required to achieve peak responses in patients with severe periodontal disease.


Electron microscopy of cells displaced into the dentinal tubules due to dry cavity preparation. Using dogs' teeth, cells displaced into the dentinal tubules due to dry cavity preparation were observed using electron microscopy. In order to obtain adequate fixation a new local-perfusion method was employed. Most of the cells which moved into the dentinal tubules were odontoblast nuclei and partly neutrophilic leukocytes and erythrocytes. Not only odontoblast nuclei but also cytoplasmic organelles, such as mitochondria, rough endoplasmic reticulum, free ribosomes, and lysosomes were displaced into the tubules. The weavings of dentinal fibers in the tubules were observed in some areas.


Colitis cystica superficialis: a report of a case in a Barbary ape (Macaca sylvana). A case of colitis cystica superficialis is described in a Barbary ape. Early mucosal herniations through the muscularis mucosae were identified, suggesting a possible relationship between this condition and colitis cystica profunda.


The bone inducing capacity of syngeneic thyroid tissue in guinea-pig muscle. Syngeneic thyroid extract injected into the rectus femoris muscle of guinea-pigs resulted in the appearance within the muscle of osteoblasts and the formation of bone. Injection of irritant failed to do so. It is thought that thyroid extracts exert their effect by inducing osteoblast transporation in muscle fibroblasts.


The pathology of experimental Ebola virus infection in monkeys. Six rhesus and two vervet monkeys were infected intraperitoneally with Ebola virus. They developed an acute haemorrhagic fever with skin rash 4 days later and died 6--12 days after infection. Histopathological lesions of acute necrosis were present in the liver, spleen, lymph nodes, lungs and testes. The presence of fibrin thrombi in several organs was suggestive of the occurrence of disseminated intravascular coagulation during the infection.


Partial characterisation of an elastase-like enzyme secreted by human and monkey alveolar macrophages. An elastinolytic activity on soluble and insoluble elastin has been demonstrated in the culture medium of human and monkey alveolar macrophages. This activity is potentiated by latex particles in the medium. An enzyme resembling some other elastases was partially purified and characterised. It differs from other elastases in its affinity for elastin and a synthetic substrate, its susceptibility to various kinds of inhibitors and by its metal ion requirement.


Long-term effects of myochrysine on the synovial membrane and aurosomes. Mysochrysine injected into the rabbit knee joint produced regressive and destructive changes in the synovial membrane, ultimately leading to fibrosis. Aurosomes, containing characteristic electron-dense deposits indicating the presence of gold, formed in the synovial intimal cells and subsynovial macrophages. The number of aurosomes decreased with the passage of time but some were found even 2 yr after the injection of Myochrysine. Electron-probe X-ray analysis showed that the aurosomes contain gold, sulphur and phosphorus. A comparison was made between the atomic ratios of these elements in 3-day and 18-mth-old aurosomes but no significant difference was detected.


Immunoglobulin in Reed-Sternberg and Hodgkin cells. In eight cases of Hodgkin's disease of various types, Ig was found within approximately one-third of the RS and HD cells. The Ig within each RS and HD cell consisted of gamma heavy chains and both k and lambda light chains. The most probable origin of the Ig would appear to be uptake of IgG-containing immune complexes. The presence of both types of light chain in individual RS and HD cells is inconsistent with an origin of these types of cell from B lymphocytes.


Oral iron absorption in infantile protein-energy malnutrition. The ability of infants with protein-energy malnutrition to absorb iron was assessed using the serum iron response to a dose of ferrous sulfate providing 3 mg elemental iron per kg body weight. Responses were grouped as flat (delta serum Fe less than 30 microgram/dl), intermediate (30 to 100 microgram/dl), and normal (greater the 100 microgram/dl). Of 25 consecutively admitted children studied, seven had a flat, five an intermediate, and 13 a normal curve (mean delta serum Fe: 10 microgram/dl, 66 microgram/dl, and 175 microgram/dl, respectively). There were no differences among the three groups in hematocrit, fasting serum iron or transferrin saturation, severity of malnutrition, or evidence of other malabsorption sufficient to explain these differences. Although hematocrits, fasting serum iron, and transferrin saturations did not change appreciably during nutritional rehabilitation, all children with initially abnormal responses subsequently had normal tests.


Ventricular involvement in experimental Hemophilus influenzae meningitis. We determined the frequency of ventricular involvement during Hemophilus influenzae type b meningitis in ten bacteremic infant rhesus monkeys. Meningitis was defined as cerebrospinal fluid obtained from the lumbar subarachnoid space or cisterna magna containing bacteria and ten or more leukocytes per mm3. HIB were cultured from 22 of 22 ventricular CSF samples. In 17 of 18 comparisons of bacterial density in ventricular and cisternal CSF, the values were within one log10: similarly, 5 of 8 quantitative ventricular-lumbar comparisons were within one log10. This concordance was present at bacterial densities of 2 x 10(1) to 1 x 10(9) CFU/ml. When discordance was present, the ventricular CSF contained more bacteria. In 15 of 20 comparisons of leukocyte density in ventricular and cisternal CSF, the ventricular pleocytosis was lower (mean ventricular/cisternal ratio 0.08). We conclude that infection of the lateral cerebral ventricle is a uniform feature of HIB meningitis in infant monkeys, but the cellular inflammatory component is less.


Synthesis and biological activity of cocaine analogs I: N-alkylated norcocaine derivatives. N-Allylnorcocaine, N-dimethylallylnorcocaine, and N-cyclopropylmethylnorcocaine were prepared and examined for cocaine-like activity. The compounds were prepared by alkylation of norcocaine, which was obtained by demethylation of cocaine with 2,2,2-trichloroethyl chloroformate followed by zinc--acetic acid reduction. The compounds were evaluated by comparison with cocaine in causing disruption of milk intake in rats, behavioral modification in squirrel monkeys, and inhibition of 3H-serotonin uptake by rat synaptosomes. The compounds showed cocaine-like activity less potent than cocaine in the latter two tests and were inactive in the milk intake test.


Calcium fluxes in single muscle fibres measured with a glass scintillator probe. 1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p-mole . cm-2 . sec-1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca-loaded fibre into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular 'cleft space'. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA-injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6--22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19--48 p-mole . cm-2. External D600 (5 x 10(-4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6--11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p-mole . cm-2. The upper value was 145 p-mole . cm-2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA.


The physiological effects of monocular deprivation and their reversal in the monkey's visual cortex. 1. 1127 single units were recorded during oblique penetrations in area 17 of one normal, three monocularly deprived and four reverse sutured monkeys. 2. In all animals most cells outside layer IV c were orientation-selective, and preferred orientation usually shifted from cell to cell in a regular progressive sequence. 3. The presence in layer IV c of non-oriented, monocularly driven units, organized in alternating right-eye and left-eye 'stripes' (LeVay, Hubel & Wiesel, 1975) was confirmed. 4. Early monocular deprivation (2--5 1/2 weeks) caused a strong shift of ocular dominance towards the non-deprived eye. However, even outside layer IV c, neural background and some isolated cells could still be driven from the deprived eye in regularly spaced, narrow columnar regions. In layer IV c the non-deprived eye's stripes were almost three times wider, on average, than the deprived. 5. Later monocular deprivation (11--16 months) had no detectable influence on layer IV c but seemed to cause a small shift in ocular dominance outside IV c. Deprivation for 6 1/4 months in an adult had no such effect. 6. After early reverse suturing (at 5 1/2 weeks) the originally deprived eye gained dominance over cells outside layer IV c just as complete as that originally exercised by the eye that was first non-deprived. 7. The later reverse suturing was delayed, the less effective was recapture by the originally deprived eye. Reversal at 8 weeks led to roughly equal numbers of cells being dominated by each eye; fewer cells became dominated by the newly open eye after reverse suturing at 9 weeks and most of them were non-oriented; reversal at 38 1/2 weeks had no effect. 8. Binocular cells, though rare in reverse sutured animals, always had very similar preferred orientations in the two eyes. The columnar sequences of preferred orientation were not interrupted at the borders of ocular dominance columns. 9. Even within layer IV c there was evidence for re-expansion of physiologically determined ocular dominance stripes. After early reverse suture, stripes for the two eyes became roughly equal in width. Possible mechanisms for these changes are discussed.


Chloride dependence of active sodium transport in frog skin: the role of intercellular spaces. 1. In agreement with previous observations the replacement of Cl by a nonpenetrating anion in the solution bathing either the outside or both sides of the frog skin causes a fall in the short-circuit current. 2. When Cl is replaced by a non-penetrating anion in the solution bathing the outside of the frog skin the Isc is still a correct measure of the net Na transport. 3. Under the same conditions both active and shunt paths seem to be affected since there is a decrease in Isc, Na influx, amiloride-dependent conductance, and initial Na uptake across the external barrier, together with a decrease in Cl-backfluxes and amiloride-independent conductance. There is also a decrease in water permeability and a reduction in size of the intercellular spaces. 4. The removal of Cl does not appear to affect the entry step of Na but may have an effect on the shunt path. This in turn may change the active Na transport.


Relaxation of mammalian single cardiac cells after pretreatment with the detergent Brij-58. 1. The influence of load and activation on relaxation of heart muscle has been studied. 2. Cardiac cells devoid of functioning sarcolemma were isolated from rat and cat ventricular myocardium. Pretreatment with the detergent Brij-58 destroyed residual sarcoplasmic reticulum function. In order to analyse the mechanical properties of relaxation in these cells, a new miniature transducer was designed which could measure force by feedback sensing (resolution of 1 microgram). Contraction was induced by ionophoretically released calcium ions. Activation, sequestration of calcium and loading conditions could be controlled independently. 3. The time course of relaxation was shown to be governed by the amount of calcium released, and unlike intact preparations from rat or cat heart (but like those from frog), to be independent of load and of alterations in load. 4. We conclude that relaxation of the cardiac contractile system is determined basically by an activation-dependent mechanism, which is masked by load dependence in intact muscle preparations with a well developed calcium sequestering membraneous system.


Movement-sensitive and direction and orientation-selective cutaneous receptive fields in the hand area of the post-central gyrus in monkeys. 1. In the hand area of the post-central gyrus of three alert Macaca speciosa monkeys neurones related to cutaneous receptors but not activated by simple touch on the receptive field were recorded using the transdural micro-electrode recording technique. Thirty-six cells were found to have complex cutaneous receptive field properties. These neurones were subdivided into the following three groups. 2. Nine neurones were not activated by punctate stimuli on the receptive fields but responded well to movement along the skin. The activity of these neurones was not affected by the direction of movement; nor was it sensitive to different textures of the moving surface. 3. Eighteen neurones responded to cutaneous movement along the skin surface in a particular direction giving no response to stimulation in the opposite direction and intermediate responses to intermediate directions. Similar responses were evoked from different subparts of the receptive field. 4. Nine neurones responded well to an edge placed on the skin in an optimal orientation or moved along the skin in a direction perpendicular to the edge. A maximal response was produced by stimuli of the same optimal orientation in different parts of the receptive field. The significance of the stimuli to the monkey had only a minor influence on the magnitude of the responses of these neurones and no influence on the receptive field properties. 5. The occurrence of the complex cutaneous cells increased from anterior to posterior within the post-central gyrus and most of them were found in Brodmann's area 2. Thus we postulate that the complex receptive field properties arise as a consequence of cortical processing in a network in which postsynaptic one-way lateral inhibition generates the directional properties of the neurones. 6. The complex cutaneous neurones constituted only 6% of the neurones studied in the hand area of the post-central gyrus. Thus the prevalence of neurones with elongated and direction-selective receptive fields is low in the primary somatosensory cortex in comparison with the visual cortex. These neurones may, however, serve the sterognostic capcity of the hand by contributing information about stimulus motion, orientation and direction of movement on the skin.


Receptive field integration and submodality convergence in the hand area of the post-central gyrus of the alert monkey. 1. An exploration of the occurrence of different functional cell types was made in the three cytoarchitectural subdivisions (areas 3, 1 and 2) of the hand area of the post-central gyrus of the monkey. The functional properties of 632 cells were studied using the transdural micro-electrode recording method. 2. Over half of the neurones studied (57%) belonged to the class of simple skin neurones that were related either to rapidly adapting (272 neurones) or slowly adapting (seventeen neurones) cutaneous receptors or to both (seventy-one neurones). The simple skin neurones were particularly common in the anterior part of S I where they constituted 60% of the cells. More complicated cutaneous neurones made up 10% of the total sample. They were more common in the posterior part of the gyrus. 3. Altogether ninety-two neurones (15%) were related to subcutaneous or deeper receptors. Another seventy-one neurones (11%) exhibited convergence of skin input and input from deep receptors. A smaller group of forty-seven undamaged neurones (7%) were unrelated to stimuli of the types described above. 4. In tangential electrode penetrations made along the anterior and posterior banks of the gyrus, functional columns were found to be 500 micrometers wide on the average; this width is comparable with that of ocular dominance columns and visual orientation hypercolumns. 5. Correlation of the functional types of cells with cytoarchitecture showed that the complexity of the functional properties of the neurones increased posteriorly. The receptive field size also increased toward posterior. The changes that take place in the functional properties of cells when moving across different cytoarchitectural areas suggests intracortical information processing which leads to handling of larger body regions and more complex combinations of information in the cellular elements of the posterior part of the post-central gyrus.


An electrophysiological study of the regulation of ciliary beating frequency in Paramecium. 1. The role of the surface membrane in the control of ciliary beat frequency in Paramecium was examined by intracellular electrophysiological techniques and pressure injection of Ca2+ and EGTA. Experiments were done on wild type P. caudatum and on both the wild type and a pawn mutant of P. tetraurelia. 2. The increased frequency of beating that accompanies reversal of power stroke orientation in response to depolarization in the wild type fails to occur during depolarization in the mutant pawn, which also fails to exhibit ciliary reversal upon depolarization. 3. Injection of moderate amounts of EGTA blocked the frequency increase without interfering with reversal of the beat in response to depolarization of the wild type. Larger injection of EGTA also prevented reversed beating. 4. The beat frequency in the normal (forward-swimming) direction increased during hyperpolarization in pawn. The hyperpolarizing frequency-voltage relations were quantitatively similar to those of the wild type. 5. Injection of EGTA to a final concentration of 10 mM into wild type cells neither modified the resting frequency nor blocked the frequency increase which normally accompanies hyperpolarization. 6. Transient ciliary reversal in both pawn and wild type produced by injection of Ca2+  could be terminated by the passage of inward current. The power stroke returned to the normal forward-swimming direction and the ciliary beating frequency increased. Upon termination of the inward current the cilia of Ca2+-injected cells again beat in reverse for many seconds. 7. The results support previous reports that increased frequency of beating and ciliary reversal seen in response to depolarization both require the entry of Ca2+ through the surface membrane. On the other hand, the results indicate that frequency increase with hyperpolarization is independent of an altered rate of Ca2+ entry. 8. Increased frequency during hyperpolarization appears to be related more closely to electrotonic membrane current than to membrane potential. It is proposed that inward current might activate high frequency beating by altering the ionic environment of the axoneme within the restricted volume of the cilium by electrophoretic means.


Some problems of projections and actions of cortico- and rubro-spinal fibres. A review of a number of known, partly known and to be established properties of the cortico- and rubro-spinal tract systems in relation to: (1) multiple projections of individual neurones, or functional subgroups of neurones in the motor cortex and in the red nucleus, (2) identification of spinal target cells of these neurones, and (3) the mechanisms of interactions between the two systems.


Properties of the pyramidal tract neuron system within the precentral wrist and hand area of primate motor cortex. 1. To obtain basic anatomical data that will be useful in interpreting the results of studies of primate pyramidal tract neurons (PTNs), extracellular, single-unit recording techniques were used to determine a number of the properties of the PTN population within the electrically defined, precentral wrist zone of the monkey's motor cortex. 2. Recordings were obtained from a total of 1,375 antidromically identified PT and corticospinal tract (CST) cells. A mathematical model was then used to correct the statistics of the sample for variations in the probability of unit detection, which arise from variations in neuronal size and extracellular field dimensions. 3. Both the experimentally observed and theoretically corrected results suggest that the PT projection from this cortical zone is derived principally from slowly conducting, and presumably small to medium-sized cells (an estimated 85% of the resident PTN population). 4. Both the fast and slow cell subpopulations were found to be concentrated within cortical layer V, where they tend to congregate in small, mixed clusters of 2 to 5 neurons. Estimates of the total packing density of PTNs within layer V of this cortical zone suggest that they account for only 10-20% of the neurons within this major efferent layer. 5. 70% of the slow and 82% of the fast PT neurons within this cortical area were found to send their axons into the contralateral, lateral corticospinal tract. Thus, in futur functional studies of PTNs in this cortical area, it can be assumed that three of every four neurons will in fact influence segmental cells of one category or another directly. 6. Extensive data are also presented on the incidence of axon collateral branching from PT and CST cells to the red nucleus, the medial medullary reticular formation and the cuneate nucleus. 7. Some general implications of these findings for the design of future functional studies of anatomically identified motor cortex cell systems are then discussed.


An intracortical microstimulation study of output organization in precentral cortex of awake primates. We investigated the output organization of the forelimb control area in primate precentral cortex by using low-current (less than 30 microamperemeter) intracortical microstimulation (ICMS). Movement about a joint was selected as the index of response. Penetrations perpendicular to the cortical surface and deep into the rostral bank of the central sulcus were made in two awake unanesthetized monkeys (Macaca arctoides). Cortical areas were designated by the joint about which movements occurred. 1. ICMS loci which produced movements about finger joints were found to tightly cluster in a central zone, and were surrounded by loci controlling movement about the wrist. This wrist zone was in turn approximately encircled by an elbow zone, which itself was enclosed by a shoulder zone. 2. Appreciable overlap between these zones controlling movements about contiguous joints was observed. 3. The observations indicate a nested-ring organization of the forelimb output zones of precentral cortex, such that a cortical zone controlling movement of a more distal joint is partly encircled by the zone controlling a more proximal joint.