Immunological study of anthranilate synthetase. An immunological study of anthranilate synthetase (ASase) has been initiated using quantitative precipitation, enzyme neutralization, and immunodiffusion methods. Cross-reactivity of anthranilate synthetase-anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (ASase-PRTase) from Escherichia coli, Klebsiella aerogenes, and Salmonella typhimurium and ASase from Serratia marcescens and Pseudomonas putida was detected with antibodies to ?E. coli trypsin-treated ASase. Cross-reactivity of antigens was also obtained with S. marcescens anti-ASase. Indices of dissimilarity verified the overall structural similarity of ASase-PRTase from E. coli, K. aerogenes, and S. typhimurium and the divergence from S. marcescens ASase. Further divergence of these enzymes from ASase in B. subtilis and P. putida was apparent. Precipitation of ASase components I and II (ASase CoI and ASase CoII) was obtained using anti-ASase or antiserum fractionated to contain component-specific antibodies. Anti-ASase inhibited enzyme activity to binding to determinants on both subunits. Anti-ASase CoI inhibited the ammonia-dependent reaction and interfered with amide transfer from glutaminyl-ASase CoII. Anti-ASase CoII inhibited the glutamine reaction by blocking amide transfer. Enzyme neutralization experiments indicate more conservation of determinants at the active site region of ASase CoII compared to ASase CoI in the enterobacteria. A particulate form of ASase-PRTase in E. coli, K. aerogenes, and S. typhimurium could be distinguished by quantitative precipitation and immunodiffusion.


Effects of reducing and oxidizing agents on the action of bleomycin. The effects of reducing agents, such as 2-mercaptoethanol, dithiothreitol, L-ascorbic acid, or sodium borohydride, and oxidizing agents, such as hydrogen peroxide or dehydroascorbic acid, on the in vitro action of bleomycin were investigated.  After the incubation of DNA with a low concentration of bleomycin and a reducing or oxidizing agent, single strand breaks were mainly caused in the DNA molecules.  The degradation of DNA was largely prevented by the removal of oxygen, or by the addition of divalent cations or of S-(2-aminoethyl)isothiuronium bromide hydrobromide, a radical scavenger, to the incubation mixture.  Preincubation of bleomycin with these reducing or oxidizing agents reduced the DNA-degrading activity of the antibiotic.  However, this reduction in activity was observed even in the absence of oxygen, or in preincubation mixture supplemented with radical scavenger.


Characterizations of acylagmatine amidohydrolase and carboxypeptidase from Fusarium anguioides. Previously an enzyme, named acylagmatine amidohydrolase, hydrolyzing bleomycin B2 to bleomycinic acid and agmatine was found in the mycelia of Fusarium anguioides Sherbakoff.  In this work the enzyme was purified further, but not completely.  The crude enzyme preparation hydrolyzed various acylagmatines and also peptidyl arginine, but the latter activity could be separated from acylagmatine amidohydrolase activity by gel filtration on Sephadex G-100.  The enzyme was inhibited by PCMB and its molecular weight was estimated as 65,000 by gel filtration.  It showed substrate specificity with respect to the alkyl-chain length of the amine moiety.  The other hydrolase fraction with activity toward Bz-Gly-Arg was found to be of a sort of carboxypeptidase, which preferentially hydrolyzed peptides with arginine or lysine at the carboxyl terminus, including bradykinin, but liberated neutral amino acids as well from the terminus when the penultimate residue of the substrates was phenylalanine.  With Bz-Gly-Arg as substrate Fusarium carboxypeptidase was sensitive to chelating agents but not to diisopropyfluorophosphate, and its molecular weight was estimated to be 145,000.


Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase. Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.


Transmission of stability (telestability) in deoxyribonucleic acid. Physical and enzymatic studies on the duplex block polymer d(C15A15) - d(T15G15). The properties of the duplex block polymer d(C15A15) - d(T15G15) were examined by thermal denaturation and nuclease susceptibility studies in the absence and presence of drugs (actinomycin and netropsin) which bind specifically to only one end of the block polymer. The nucleotide composition of one region of this synthetic double-helical DNA affected the properties of a contiguous but remote region. Furthermore, the binding of actinomycin influenced the properties of both the binding and nonbinding regions. These findings suggest a mechanism for gene regulation at a distance.


Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase. One- and two-diminsional tryptic and chymotryptic peptide maps of 125-I-labeled alpha and alphabeta avian myeloblastosis virus DNA polymerase demonstrate that the alpha polypeptide of the one and two subunit enzymes are structurally similar, if not identical. Furthermore, the beta subunit contains the same major 125I-labeled peptides as alpha, plus several additional peptides. These relationships and the fact that aging of purified alphabeta avian myeloblastosis virus DNA polymerase increases the proportion of alpha DNA polymerase that can be isolated from the alphabeta enzyme by phosphocellulose chromatography, suggests that alpha is derived from beta by proteolytic cleavage.


Further studies on telestability in DNA. The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20). The synthesis and characterization of the duplex block polymers d(C20A10) - d(T10G20) and d(C20A15) - d(T15G20) are described. Thermal denaturation studies on these DNAs in the absence and presence of actinomycin, which binds only to the GC portions of these molecules, have confirmed and extended our previous observation that the properties of one region of a DNA can be influenced (telestabilized) by a remote region. In addition, the large scale synthesis of d(C15A15) - d(T15G15) is described.


Vitamin K and the biosynthesis of prothrombin. V. Gamma-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin. Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.


Analysis of insect chemosterilants.

Analysis of insect chemosterilants. Background information concerning the concept of insect chemosterilization and analytical methods for determining trace levels of 19 P- and/or S- containing chemicals of current interest are presented. GC retention times of the compounds on 6 different columns and their p-values in 11 solvent systems are tabulated. The utility of the flame photometric detector for determining subnanogram levels of the sterilants in biological substrates is illustrated.


Mutual modification of glucose-stimulated serum insulin responses in female rhesus monkeys by ethinyl estradiol and nortestosterone derivatives. Changes in iv glucose tolerance (IVGTT) and serum insulin responses to glucose infusion have been measured in intact female rhesus monkeys treated per os with norethindrone or medroxyprogesterone acetate (500 mug/day) both alone and in combination with mestranol or ethinyl estradiol (10 mug/day) orally for 3 weeks. When administered as the sole contraceptive steroid, neither norethindrone, medroxyprogesterone acetate, mestranol, or ethinyl estradiol produced consistent changes in fasting serum insulin or glucose concentration, mean intravenous serum glucose disappearance rates (K) or mean integrated serum insulin response to glucose (sigmal40). By contrast, concurrent administration of norethindrone with mestranol or ethinyl estradiol resulted in a significant increase in the fasting serum insulin concentration and the mean sigmal40. An increase in the mean K was also observed after norethindrone + mestranol. These results show that synthetic estrogens have the ability to potentiate the metabolic effects of norethindrone. However, the improvement in glucose tolerance produced in rhesus monkeys by concurrent mestranol + norethindrone treatment was marginal because of wide variation in glucose assimilation rates uncer control conditions. Thus, the IVGTT in the rhesus monkey appears to have limited use as a model for studying glucose homeostasis in man.


Cooperation between human thymus-derived and bone marrow-derived lymphocytes in the antibody response to ragweed antigen E in vitro. Human T lymphocytes from patients with ragweed hay fever, when exposed to ragweed antigen E (AgE) in vitro, produced an activity that, in the presence of antigen, induced B cells from AgE-sensitive donors to synthesize and secrete IgE and IgG antibodies to AgE. Anti-AgE specificity was assessed both in vitro and in vivo. B lymphocytes from ragweed-sensitive individuals exposed in vitro to AgE alone failed to transform or to secrete antibody to AgE. The T cells activity had no effect on B cells of individuals not sensitive to AgE. The results of this study suggest that the human reaginic antibody response requires T and B cell cooperation. The experimental approach used may be a useful model for the investigation of the antibody responses of allergic individuals.


Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes. Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A (alha) and B (beta) chains of fibrinogen by various enzymes. Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was couple to desaminotyrosine and radiolabeled with 125I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slowere cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.


Autoradiographic studies of the projections of the midbrain reticular formation: descending projections of nucleus cuneiformis. The descending projections of nucleus cuneiformis in the cat were traced by autoradiography in the transverse and sagittal planes following stereotaxically placed injections of 3H-leucine. Many descending axons are organized into distinct fiber systems, of which the largest and most well-defined crosses directly in the midbrain and descends through the ventromedial tegmentum of the brain stem. This fiber system first terminates profusely in n. reticularis tegmenti pontis and then proceeds through the rhombencephalic tegmentum emitting transversely oriented branches to n. reticularis pontis caudalis and gigantocellularis, the raphe magnus and the facial nucleus...


Translaminar growth of axons in the kitten dorsal lateral geniculate nucleus following removal of one eye. The distribution of retinal afferents to the dorsal lateral geniculate nucleus of the cat following the early removal of one eye has been studied using autoradiographic as well as degeneration techniques. The degeneration techniques have confirmed earlier observations that axons from the remaining, normal eye grow across laminar borders into the deafferented lamina A. Autoradiographic techniques, however, have shown further examples of translaminar growth between lamina A nadlamina A1 as well as between lamina A1 and lamina C. Although the possibility exists that such growth also occurs between the other C laminae (C1, C2 and C3) such growth is harder to demonstrate since no well defined interlaminar regions exist in this part of the nucleus. In the monocular segment of the nucleus degeneration techniques have shown some translaminar invasion of the deafferented lamina A. Autoradiographic techniques have demonstrated similar growth as well as an abnormal invasion of the most lateral parts of the monocular segment. This latter growth originates in the optic tract as it curves around the nucleus on the way to the superior colliculus.


The pulvinar nucleus of Galago senegalensis. The present study was undertaken to analyze the connections of the pulvinar nucleus in a prosimian. The experiments, which rely on the Fink-Heimer ('67) method for staining degenerating axons and their terminals, fall into two parts: first, the tracing of ascending tectal projections to the caudal thalamus and second, the tracing of projections from this thalamic target to the cortex. Large lesions of the superior colliculus resulted in dense degeneration in the caudal half of the inferior subdivision of the pulvinar complex. This pathway could be identified when the lesion was restricted to the superficial layers of the superior colliculus, signifying that it is a visual pathway. In general, the projections of the deep and superficial layers of the superior colliculus were distinct and in this respect Galago resembles Tupaia. The inferior pulvinar nucleus in turn projects to area MT, a conspicuous subdivision of the temporal cortex. The superior division of the pulvinar, in contrast to the inferior division, is not a major target of ascending projections from the superior colliculus and projects to the areas of the occipital and temporal lobe intercalated between areas MT and 17. When these results are compared with similar studies in nonprimates, notably studies of Tupaia, a striking difference in organization emerges. In Tupaia, and in distantly related mammals such as the squirrel, the target of the tecto-pulvinar system includes area 18 adjacent to area 17. This feature is important since the two parallel projection systems seem to be related to each other in terms of the way in which the zero vertical meridian is spatially represented. However, in Galago the subdivision of the pulvinar receiving projections arising from the superior colliculus does not project to area 18. Area 18 is indeed the target of pulvinar projections, but these projections arise from that portion of the pulvinar which is not a recipient of ascending tectal projections. It is not easy to see how this primate organization, if indeed the Galago is representative of primates, evolved from the organization reflected in Tupais.


A cytoarchitectonic and Golgi study of the red nucleus in the rat. The cytology of rubral neurons was investigated using both paraffin and 1 mu thick Epon sections. The neurons were divided into four size categories which form a continuum with regard to cellular characteristics. Giant neurons (greater than 40 mu) and large neurons (26-40 mu) predominate in the caudal one-third of the nucleus. Large neurons extend up the ventral, medial and lateral borders of the nucleus into its middle one-third. The caudal one-third of the nucleus together with this extension of large cells is designated as the magnocellular part of the nucleus. The more rostral part of the nucleus contains predominantly small (less than 20 mu) and medium size (20-25 mu) neurons, and this constitutes the parvocellular part of the nucleus. The characteristics of each cell type are described. Camera lucida drawings of the rubral neurons were made in three different planes of section, and the extent of the magnocellular and parvocellular portions of the nucleus was determined. Dorsomedial and ventrolateral subgroups of the magnocellular part of the nucleus are found 300-400 mu from the caudal pole. A lateral horn of small and medium size neurons with the addition of a few large neurons extends from the lateral part of the nucleus 500-700 mu from the caudal pole. Golgi preparations of the red nucleus were examined in three planes of section. Giant and large neurons display short spines on the soma and also, along the entire length of the dendrites. Inaddition, more elongate spinous processes are seen on these dendrites and are frequently aggregated into tufts at loci on the dendrites or at their terminations. The dendrites of these neurons radiate in all directions from the soma but remain within the confines of the nucleus. Medium size neurons demonstrate radially arranged dendrites. Sparsely positioned spines are seen along the length of the dendrites but are absent on the soma. A number of such neurons demonstrate tufts of elongate spines on their dendrites. The dendrites of small neurons branch infrequently and remain within the confines of the nucleus. A few dendritic but no somatic spines are seen in association with these neurons.


Golgi studies in the substantia gelatinosa neurons in the spinal trigeminal nucleus. This Golgi study identifies three neuronal cell types in the substantia gelatinosa (SG) layer of the spinal trigeminal nucleus. The SG neurons are distinguished from each other based on: (1) dendritic branching pattern, (2) denritic spine distribution, (3) geometric shape of the denritic tree, (4) laminar distribution of the dendrites, (5) axonal branching pattern and (6) laminar distribution of the axonal arbor. The islet cell is found in small clusters and its dendrites and axonal arbor are confined within the SG layer. Its dendrites span the full width of the SG layer and extend up to 500 mum in the long axis of the layer. Dendritic spines are generally sparse with small clusters of spines found on the higher order dendritic branches. The islet cell axon extends for at least 1 mm in the long axis of the layer. Each of its collaterals divide every 50-100 mum with one branch doubling back in the direction of the cell body and the other branch continuing on in the direction of its parent. In this manner each islet cell generates a profuse axonal plexus in the SG layer. The stalked cell is found individually within the SG layer. Its cell body is usually found in the inner half of the SG layer and its sinuous dendrites cross the SG layer and enter the marginal layer. The stalked cell dendrites emit numerous fine stalk-like branches and dentritic spines. Its axon emits branches in the SG and marginal layers. The spiny cell is found singly between groups of islet cells. Its extensive dendritic tree spans up to 500 mum rostrocaudally and mediolaterally crossing into both the marginal and magnocellular layers. Spiny cells have evenly distributed dendritic spines along their dendrites in the SG layer. The spiny cell axon sends branches into all three layers of nucleus caudalis. Numerous branches enter the outer 300 mum of the magnocellular layer where they undergo further branching with some branches returning in recurrent fashion toward the SG layer. The three neuronal cell types of the SG layer satisfy all of the morphological criteria for Golgi type II interneurons. Their highly branched axons generate many collaterals within the confines of their dendritic trees and do not project out of nucleus caudalis. The SG neurons are considered to be inhibitory interneurons interposed between V nerve primary afferent axons which arborize in the SG layer and second order neurons of nucleus caudalis.


Demonstration of a pallido-nigral projection innervating dopaminergic neurons. Afferents to the substantia nigra from the neostriatum and globus pallidus were studied in the rat by means of the autoradiographic tracing technique. 3H-leucine was injected stereotaxically into either the globus pallidus or neostriatum. Twenty-four hours later the axoplasmic transport of labelled proteins to the substantia nigra was studied by light and electron microscopic autoradiography. In animals used for electron microscopy, degeneration of dopaminergic cells in the substantia nigra was induced by intraventricular injection of 6-hydroxydopamine 72 hours before sacrifice. After neostriatal injections, light microscopic analysis revealed heavy labelling of the globus pallidus and entopeduncular nucleus, but only background labelling of the subthalamic nucleus. There was preferential labelling of the zone reticulata of the substantia nigra, with significantly less labelling of the zone compacta. After pallidal injections, light microscopic analysis showed very light labelling of those parts of the caudate-putamen in the vicinity of the injection site. There was intense labelling of the subthalamic nucleus and heavy labelling of the entopeduncular nucleus. The zona compacta of the substantia nigra was also heavily labelled. There was considerably less labelling of the zona reticulata. The electron microscopic analyses showed that after neostriatal injections, autoradiographic grains in the substantia nigra were located preferentially over boutons which terminated on normal dendritic processes. After pallidal injections, however, grains were preferentially located over boutons synapsing with degenerating dendritic processes. The degeneration produced in these dopaminergic processes by 6-hydroxydopamine was invariably of the dark type. Except for the different association with degenerating vs. non-degenerating dendrites, the subcellular distribution of autoradiographic grains in the substantia nigra was the same after injection into either the globus pallidue or caudate-putamen. Approximately 80 percent of the grains were over axons or boutons which invariably made symmetrical synaptic contacts. These observations demonstrate the existence of a pallido-nigral projection which terminates preferentially on dopaminergic cells in the pars compacta of the substantia nigra. They also confirm previous studies indicating that the strionigral projection terminates mainly in the pars reticulata. These terminations appear to be principally to non-dopaminergic cells.


Acute anaphylaxis associated with serum complement depletion. A 45-year-old woman with rheumatoid arthritis developed anaphylaxis 6 min after receiving a subcutaneous injection of lidocaine, followed by an intra-articular injection of lidocaine mixed with methprednisolone acetate. One hour after the onset of anaphylaxis, serum complement component levels were markedly depressed and remained so far 18 hr. Circulating immune complexes and antibodies to lidocaine could not be demonstrated. Neither lidocaine nor methylprednisolone acetate activated the patient's complement system in vitro. Subsequently, total hemolytic activity (CH50) levels were variable, complement component protein concentrations of C1q, C1s, C4, C2, C3, C5, C6, C9, and Factor B were normal, but hemolytic activity of C4 and C2 was diminished. Serum C1 inhibitor concentrations were normal or slightly depressed. The patient has never had any symptoms suggestive of angiodema. It is postulated that the endogenous complement abnormality present in this patient may have contributed to the anaphylactic reaction.


Media in teaching college level nutrition. Is it effective and efficient? Several techniques have been used, studied, and tested to teach nutrition at Syracuse University. One self-paced course in nutrition and food science tutors students completely through audio tapes integrated with films, slides, video tapes, discussion groups, laboratory manual, and computer-assisted instruction. Evaluation is by computerized tests given after each module at the student's discretion. Compressed-speech tapes are used to increase learning efficiency. Dietetic, nutrition, nursing, and pre-medical students are taught nutrition via these methods for selected modules, but they mainly learn by lectures supplemented by pertinent films, slides, transparencies, television commercials, telectures, videotapes, and simulations. Multi-media "happenings" are presented which gain students' attention and change attitudes while imparting nutritional information which is well retained.


Senile degenerative brain lesions and dementia. In a study of senile degenerative lesions-including Alzheimer's neurofibrillary changes, senile plaques and amyloid angiopathy-the hippocampal area of the brain was examined by thioflavine T fluorescence microscopy in 146 consecutive autopsy patients over the age of 49. The incidence and quantity of neurofibrillary changes and senile plaques rose with age, and an approximate positive correlation in quantity was noted among the three kinds of degenerative change. The quantity of neurofibrillary lesions and senile plaques was significantly different between the demented and non-demented patients, but not between the severely and less severly demented patients. The cause of dementia was studied retrospectively, based on the extent of morphologic changes in the brain, thus classifying dementia into three types: degenerative, vascular, and mixed. Clinically, the mixed type resembled the vascular type with regard to major neurologic signs, and there was some similarity to the degenerative type with regard to mental features.


Studies with Brugia pahangi 10. An attempt to demonstrate the sharing of antigenic determinants between the worm and its hosts. Infective stage Brugia pahangi that were reared in Aedes aegypti survived equally well in cats that had previously been immunized against mosquito tissue and in a normal cat. The survival of third, fourth, juvenile, adult and microfilarial stages of B. pahangi that were recovered from cats was similar in jirds that had been immunized against cat antigens and in normal jirds. Host antigenic determinants were not detected on the surface of larvae in substantial amounts using fluorescent antibody techniques. It is unlikely that B. pahangi evades the immune response of its vertebrate hosts by masquerading as "self" behind host antigens.


Simultaneous differential staining of nucleic acids, proteins, conjugated proteins and polar lipids by a cationic carbocyanine dye. The multiple interactions of the cationic carbocyanine dye, 1-ethyl-naptho-[1,2d]thiazolin-2-ylidene)-2-methylpropenyl]naptho[1,2d]thiazolium bromide, 'Stains-described. Many of these substances could be distinguished from one another on the basis of color in conjunction with chemical and enzymatic digestions. Further studies with this dye have shown that under certain conditions polar lipids as well may be distinguished from these substances. Stains-all has a highly sensitive metachromatic reaction for the presence of polar lipids. It is possible to detect the lipids in large part because they are green and contrast with a red- or pink-stained protein background and with the blue-purple of nuclei or cartilage. Where other green substances occur as in sialoglycoproteins of mucous or membranes, the lipids can be distinguished because they are extracted by chloroform-methanol (2:1) or pyridine.


Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits. The biophysical properties of the adr subtype of HBSAg were determined and found to be identical to those of the adw and ayw phenotypes. These properties were used to purify the 22 nm spherical form of HBSAg/adr from the serum of a chronic antigen carrier by using zonal centrifuge techniques. The purified antigen was injected into 15 rabbits which were bled weekly to follow the development of antibodies to HBSAg by hemagglutination, and to the subtype determinants of HBSAg by counterelectrophoresis and agar gel diffusion (AGD) assays. From these data and two pilot studies to produce anti-w using an adw and an ayw antigen the relative immunogenicities of the various HBSAg determinants could be ranked as a greater than r greater than d greater than y greater than w. A selected pool of the sera from the rabbits immunized with HBSAg/adr was prepared as an interim reference reagent; when diluted 1:4 in saline and tested by AGD, it contains anti-a and anti-r without demonstrable anti-d or anti-normal human serum activity.


Long-lasting in vitro immune response to a distinct antigenic determinant of a bacterial protein. Cyclic changes of antibody titer and affinity. Long-lasting (60 days or more) antibody responses in vitro by rabbit lymph node fragments to a distinct determinant of Escherichia coli beta-D-galactosidase were obtained by supplementing culture medium with fetal calf and horse serum. Antibodies released in the supernatant were removed every 3rd to 5th day together with the spent medium, without pooling to minimize intermixing of molecules synthesized far apart in time. Antibody titer, association constant, and heterogeneity index were measured in medium samples collected throughout the response in order to draw profiles of their changes under conditions whereby a limited number of clones synthesize antibodies in a closed system without connection to antigen depots, central lymphoid organs, and circulating cell and antibody pools. It was found that antibody affinity changes cyclically and that such cycles may be repeated. Cycles are composed of an ascendant limb with a gradual increase in affinity and a parallel diminution of heterogeneity. A descendant limb follows with the opposite modifications. High affinity antibodies predominate at the peak of the cycles, whereas low affinity molecules take over at the end of the cycles until the next ascendant limb begins; these persist after the last cycle has waned.


Radioimmunoassay of human serum antibody specific for adenovirus type 5-purified fiber. A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with 125I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at minus 20 degrees C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per millimeter was sufficient to inhibit the 125I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.


In vitro studies on information transfer in cells from allotype-suppressed rabbits. Data are presented which show that cells from allotypically suppressed rabbits resist in vitro "rescue" attempts in which informational ribonucleic acid (i-RNA) coding for the suppressed allotypic marker is used. It is shown that peritoneal exudate cells from such thoroughly suppressed animals contain cells that yield i-RNA coding for the suppressed marker, and that central lymphatic tissue possesses cells that produce Ig of the suppressed type.


Demonstration of a tumor-associated surface antigen in Marek's disease. Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.


Blocking of spleen cell activity against target mammary tumor cells by viral antigens. Spleen cells from BALB/c females exposed to or neonatally infected with mammary tumor virus (MTV) are cytotoxic to MTV-induced mammary tumor cells in microcytotoxocity assay. This activity can be partially or completely blocked by pretreatment of spleen cells with MTV purified from milk. Murine leukemia virus (MuLV) has no effect. T cell responses of virgin and multiparous BALB/cfC3H females are effectively blocked. Non-T cell responses of multiparous BALB/cfC3H females or of virgin BALB/c females are blocked by some but not all of the MTV antigen preparations. MuLV, but not MTV, can block activity of spleen cells from MuLV-sensitized donors against target MuLV-producing tumor cells.


Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro. It was established previously that committed precursors of T cells, which reside in bone marrow and spleen and lack T cell surface differentiation antigens, can be induced by thymopoietin and certain other agents to differentiate rapidly in vitro into T cells bearing typical surface antigens, including Thy-1 and TL (Komuro-Boyse assay). To relate this differentiative step observed in vitro to physiologic events in vivo, a system was devised to trace the migration of precursor cells to the thymus, and their maturation to T cells. Lethally irradiated mice of a TL- strain received spleen cells from TL+ hybrids i.v., and the TL+ population of the thymus was enumerated 13 to 20 days later. Donor TL+ cells first became detectable at 13 days and increased thereafter. Preliminary tests showed that cells capable of migrating to the thymus have a similar density to the cells that are inducible in the Komuro-Boyse assay, this being lower than that of mature of T cells. The thymus-repopulating properties of the donor spleen population were not affected by: 1) pre-treatment in vitro with thymus extract or thymopoietin, which initiates differentiation of T cells precursors, nor b) pre-treatment with anti Thy-1 serum plus complement, which eliminates differentiated T cells. But pre-treatment a) and b) applied in sequence markedly reduced the capacity of spleen cells to repopulate the thymus. These results can be interpreted as follows: induction of Thy-1-TL- precursor cells (pro-thymocyte) in vitro yields Thy-1+TL+ cells (early thymocytes) which have not yet lost their property of repopulating the thymus; therefore, thymus-repopulation was not depleted by treatment a) alone, which induced Thy-1 +TL+ cells, nor by treatment b) alone, which did not affect thymus-repopulation by Thy-1-TL- cells, although treatments a) plus b) did eliminate the newly induced Thy-1+TL+ cells and thus impaired repopulation of the thymus. We conclude that the cell which responds to thymopoietin in the Komuro-Boyse assay by expressing the T cell surface phenotype is the same cell (pro-thymocyte) that normally migrates in vivo from hemopoietic tissues to the thymus and is there induced by thymopoietin to express the phenotype of an early T cell.


Antigenicity of carcinogen and viral induced sarcomas in inbred and random bred guinea pigs. A tumor specific transplantation antigen (TSTA) has been detected in a methylcholanthrene (MCA) induced guinea pig tumor. It was possible to induce resistance to rechallenge with the tumor by immunization with irradiated cells in CFA. In contrast, the same technique failed to detect TSTA in two viral (Kirsten strain mouse sarcoma virus, Ki-MSV) induced guinea pig tumors; these results are similar to observations made with mouse Ki-MSV-induced tumors. Transplantation studies with these tumors in both inbred and random-bred guinea pigs showed a complexity of growth and rejection patterns. The B alloantigen, a major serologically defined antigen of the guinea pig histocompatibility complex, seemed to play a central role in acting as a guniea pig transplantation antigen. In all cases studied, the absence of B antigens in the recipient led to tumor rejection and anti-B antibody protection.


Identification of the cell population involved in viral-specific cell-mediated cytotoxicity in man: evidence for T cell specificity. The nature of the cell population involved in lymphocyte mediated cytotoxicity to baby-hamster-kidney (BHK-21) target cells persistently infected with rubella virus was investigated by a 51Cr-release microassay. After depletion of the T cell population with an antiserum to human0thymus-lymphoid tissue antigen (HTLA), the purified B cell population showed a decrease in E-rosette formation (9.0 +/- 2.2% compared to 69.6 +/- 9.1% before treatment) and an insignificant degree of cytotoxic activity against rubella-infected target cells (specific immune release of 51Cr was 0.9 +/- 2.6% compared to 24.2 +/- 3.8 before treatment). A purified T cell population, prepared by depletion of B cells with an anti-human immunoglobulin serum and complement, was found to show no alteration in E-rosette formation (85.2 +/- 6.2%) or cytotoxicity (30.3 +/- 4.4% SIR) but showed decreased EA- and EAC-rosette formation (2.7 +/- 1.5% and 10.5 +/- 3.2%, respectively, compared to 19.4 +/- 2.9% and 28.0 +/- 4.1% before treatment). A monocyte-depleted population prepared by removal of the plastic adherent mononuclear cells showed no significant alteration of rosette formation or cytotoxicity. These experiments suggest that the predominant lymphoid population responsible for direct cell-mediated cytotoxicity to virus infected target cells in the 51Cr-release microassay appears to be effected by a thymus-dependent lymphocyte population.


Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. I. Resolution and characterization. Addition of high molecular weight dextran to culture medium prevents the initiation of T lymphocyte-mediated killing by holding the cytolytic T lymphocytes (CTL) and target cells in suspension and preventing intercellular contact. Suspension in 10% dextran was used to interrupt the ongoing formation of adhesions between CTL and target cells already in contact in a centrifuged pellet. The results demonstrate that 1) firm adhesions form between CTL and target cells within 1 min at 37 degrees C; 2) once formed, these adhesions are stable at low temperature and are resistant to mechanical shearing forces; 3) these adhesions can be disrupted by EDTA; 4) immediately after the adhesions form, separation of the CTL from the target cells prevents lysis of the latter; 5) after incubation of targets adhering to CTL for an additional 6 min at 37 degrees C, removal of the CTL no longer prevents target cell lysis. Thus, target cells become "programmed" for subsequent lysis within a few minutes after contact with CTL, after which lysis occurs during the next several hours without further participation of the effector cell. At 15 degrees C, adhesions form 1/17 as fast as at 37 degrees C. Programming of target cells for lysis occurs 1/76 as fast at 15 degrees C as at 37 degrees C. Thus, the programming for lysis step is about 4-fold more temperature dependent than the adhesion step. In addition to being detected by subsequent target cell lysis in 10% dextran, the adhering cell clusters can be counted with low power microscopy. This permitted verification that EDTA separates the clusters after programming for lysis is complete. Moreover, the great majority of the clusters seen at 37 degrees C are antigen-specific. Knowledge of the cluster size distribution and the subsequent level of lysis permits the deduction that not less than 6% of the sensitized peritoneal cell populations used were CTL.


Induction of T lymphocyte responses to a small molecular weight antigen. I. Failure to induce tolerance in azobenzenearsonate (ABA)-specific T cells in guinea pigs with an ABA conjugate of A copolymer of D-glutamic acid and D-lysine (D-GL). 2,4-Dinitrophenyl (DNP) coupled to the copolymer D-glutamic acid and D-lysine (D-GL) induces B cell tolerance but not T cell tolerance. This implies either a lack of DNP determinant recognition by T cells or a substantial difference in tolerance mechanisms for the two cell types. In the present study D-GL was conjugated with the well-defined determinant azobenzenearsonate (ABA) coupled to single amino acids shown here and previously by others to trigger effectively T lymphocytes. The experiments presented here demonstrate that these ABA conjugates of D-GL, although capable of diminishing anti-ABA antibody production, completely fail to render ABA-specific T lymphocytes tolerant thus drawing us to conclude that there are significant operational differences in the mechanisms of tolerance induction in T and B lymphocytes, respectively.


Concanavalin A-induced histamine release from normal rat mast cells. Concanavalin A- (con A) induced release of histamine from normal rat mast cells was studied. In the presence of phosphatidylserine (PS) con A induced a concentration and temperature-dependent, noncytotoxic histamine release at con A concentrations ranging from 0.1 to 100 mug/ml. The optimal con A concentration, 100 mug/ml, caused a 27.3% (+/- 2.7 S.E.M.) net histamine release. Release began approximately 30 sec after addition of con A and was complete within 45 min. In the absence of PS, no net con A-induced release occurred. The effect of PS was concentration dependent from 1 to 100 mgg/ml. PS alone, however, did not cause histamine release. Binding studies indicated that mast cells bound up to 16 X 10(6) con A molecules per cell without histamine release. Upon removal of unbound con A and the addition of PS, normal histamine release occurred. Alpha-Methyl-D-mannose (50 mM) prevented both con A binding and histamine release and if added after Con A, caused a rapid cessation of histamine release and a reversal of con A binding. This study indicates several important advantages of the con A-induced histamine release system. Binding of con A to mast cells can be dissociated from histamine release by omitting PS from the medium. Release can then be induced by the addition of PS. Alpha-Methyl-D-mannose can be used to terminate rapidly the ongoing release reaction at any phase of the interaction. This system is potentially very useful for investigation of metabolic events during histamine release.


Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. I. Differences in reactivity with Poly (U) and Poly-(A) Poly (U). In a previous study, all 40 sera from patients with scleroderma, 20 of 40 sera from SLE patients, but none of 40 sera from normal controls, were found to have antibodies to ssRNA. All scleroderma sera were also found to react with HSA-coupled uridine and UMP and their reaction with HSA-coupled uridine and UMP and their reaction with ssRNA could be inhibited by uracil, uridine, and UMP. To characterize further these uracil-specific anti-RNA antibodies found in scleroderma and compare them with the anti-RNA antibodies found in SLE, we tested their reactivity with Poly (U) and with Poly (A)-Poly (U) and all but one failed to react with Poly (A)-Poly (U). This same serum was the only one in which the reaction with Poly (U) could not be inhibited with uracil. Reactivity of SLE sera was strikingly different from that found in scleroderma sera. Seventeen of 34 SLE sera studied reacted with ssRNA but only four of these reacted with Poly (U). Conversely, two SLE sera that reacted with Poly (U) did not react with ssRNA. Fifteen reacted with Poly (A)-Poly (U) and only two of these failed to react with ssRNA. Five SLE sera which were reactive with ssRNA did not precipitate with Poly (A)-Poly (U). All SLE sera which reacted with Poly (U) could be inhibited with uracil, although less effectively than in scleroderma. Reactivity with Poly (A)-Poly )U) was not inhibited with uracil nor with adenosine. These findings confirm that antibodies to RNA that are found in scleroderma are directed to uracil and thus specific to ssRNA, whereas RNA antibodies found in SLE sera are heterogeneous and directed to either the base, to the site of union of the base and sugar moiety to the ribose backbone, or to the helical structure of double stranded RNA. These differences and the respective antigenic specificities of these anti-RNA antibodies found in scleroderma and SLE may be theoretically important.


Normal tissue alloantigens and genetic control of susceptibility to tumors: microcytotoxicity studies on resistant C3Hf and susceptible (A X C3Hf) F1 mice inoculated with transplacentally induced C3Hf lung tumor. The immune response of mice to a transplacentally induced lung tumor was investigated with the microcytotoxicity (MC) assay. The tumor, originally induced in C3Hf mice, does not grow readily when transplanted to normal syngeneic C3Hf recipients. It grows readily, however, in (A C3Hf)F1 hybrids and in strain C3H mice, which express in their normal lung tissue a component which constitutes a strong lung tumor-associated transplantation antigen (TATA) in C3Hf mice. Both lung tumor-immunized C3Hf and tumor-bearing (A X C3Hf)F1 and C3H mice possessed lymphoid cells reactive against cultured lung tumor cells in the MC assay. Reactivity was also observed against cells cultured from normal lungs of (A X C3Hf)F1 and C3H mice, but not against cells similarly cultured from C3Hf of C57BL/6 mice. Anti-tumor MC was inhibited by serum-blocking factors present in some but not all tumor-bearing and tumor-immunized mice. The MC assay and detection by it of serum-blocking factors does not distinguish the effective anti-C3Hf lung tumor immune response of immunized C3Hf mice from the ineffective immune response of tumor-bearing (A X C3Hf)F1 and C3H mice. Furthermore, in lung tumor-bearing mice cells reactive in the MC assay may be directed against a normal tissue antigen rather than a tumor-associated antigen.


The immune response to a synthetic amino acid terpolymer in man: relationship to HL-A type. The immune response to the synthetic amino acid terpolymer (L-glutamic acid-55 L-lysine-33 L-tyrosine15)n (GLT) was studied in normal human volunteers. Delayed skin test reactivity to this antigen was seen in 34 of 61 subjects immunized with 150 mug of GLT. No antibody to GLT was detected in these responding individuals. There was a close correlation between the in vivo skin reactivity of volunteers to GLT and the ability of their lymphocytes to produce migration inhibitory factor (MIF) in response to GLT in vitro. However, a similar correlation was not seen when the in vitro proliferative response of lymphocytes to GLT, as measured by [methyl-3H] thymidine ([3H] T dR) incorporation, was assayed. HL-A typing of volunteers was studied to determine if responsiveness to GLT was correlated to HL-A type. No statistical association was seen after correction was made for the number of individual HL-A antigens.


Immunologic activity of myelin basic protein in strain 2 and strain 13 guinea pigs. The resistance of Strain 2 guinea pigs to experimental allergic encephalomyelitis (EAE) induced by inoculation with whole CNS tissue in complete Freund's adjuvant (CFA) has been confirmed. The resistance is even more pronounced when myelin basic protein (BP) is used in attempts to induce EAE. Strain 2 guinea pigs are also resistant to an immunization schedule (multiple injections with BP in IFA followed by a single injection of BP in CFA) known to induce significant levels of antibody in susceptible strains. The poor response of Strain 2 guinea pigs to BP is not the result of lack of specific B cells--antibody equivalent to that produced by Strain 13 animals is obtained when the inoculum contains 0.5 mg BP and 2.5 mycobacteria.


Receptors for antigen on lymphoid cells. II. The nature of the molecule responsible for plaque-forming cell adherence. The nature of specific adherence of rat anti-TNP PFC to TNP-GRBC has been investigated with PLL-fixed antigen monolayers as cellular immunoadsorbents and as plaque indicators. The immunoglobulin nature of the molecule responsible for PFC adherence is suggested by the fact that pretreatment of the PFC with rabbit anti-rat Ig antisera, but not anti-histocompatibility antisera, inhibits adherence. Removal of the adherence capacity of early PFC with the proteolytic enzymes papain and pronase, or by "capping" with anti-Ig is followed by slow regeneration of the ability to adhere, suggesting that adherence is due to membrane rather than secreted immunoglobulin, the latter being detectable within minutes after enzyme treatment. Several time-related events relating to PFC adherence were observed. 1) Both direct and indirect PFC are capable of specific adherence; the ability to adhere, however, tends to decline with time, especially after secondary immunization. 2) Although early PFC adherence is unaffected by trypsin treatment, later populations become increasingly sensitive. 3) Pretreatment of PFC at various times after primary immunization with antisera specific for rat mu-chain indicates that IgM and possibly early IgG-secreting PFC have mu heavy chains on their surface. These data suggest that the PFC membrane is progressively changing during the maturation of the antibody response.


Macrophage-lymphocyte interaction: antigen-independent binding of guinea pig lymph node lymphocytes by macrophages. The antigen independent binding of guinea pig lymph node lymphocytes by glass-adherent macrophages was investigated. Binding was found to be mediated by a trypsin digestible, divalent cation-dependent, temperature-sensitive macrophage receptor mechanism that was not competitively inhibited by excess immunoglobulin. Data are presented to indicate that in the absence of antigen, macrophages were capable of binding both thymus-derived and bone marrow-derived lymphocytes without apparent selectivity, and further, that the binding of neither cell was mediated by surface membrane-associated immunoglobulin.


Induction of mouse homocytotropic antibodies to Timothy pollen antigens. The IgG1 and IgE homocytotropic antibody responses of LAF and C3H mice to timothy pollen antigens are defined. Both mouse strains responded to low doses of crude timothy pollen extract (WST) or a major antigen of timothy pollen coupled to a purified fraction of Ascaris suum (Antigen B-Ascaris). Titers in LAF mice were greater than those in C3H mice. Regardless of the immunogen, antigen B was the major determinant recognized by the homocytotropic antibodies; PCA titers with WST or antigen B for challenge were equivalent and PCA activity could be inhibited by antigen D, a dialyzable fraction of timothy pollen possessing the antigen B determinant in monovalent form. The possible usefulness of antigen D for in vivo and in vitro studies of specific immune suppression of cellular activity is discussed.


Regulation of lymphocyte trapping: the negative trap. Suppression of 51Cr-labeled lymphocyte homing to the spleen occurs after administration of insoluble protein antigen and as a late sequela to the graft-vs-host response (GVHr). This decrease in splenic localization of labeled cells, termed "negative trapping," has elements of immunologic specificity in that tolerance induction or presensitization abrogates the negative trap. Increasing the dose of antigen accelerates the appearance of the negative trap, which is, however, evanescent, lasting from 24 to 48 hr. Synergistic and antergistic regulation of the GVHr-induced lymphocyte trap is produced by populations of 850 R-irradiated thymocytes and by F1 cortisone-resistant thymocytes. The time at which these subpopulations cause suppression or amplification of the lymphocyte trap correlates with the activity of the GVHr-producing inoculum. These findings suggest that regulation of lymphocyte traffic may provide a mechanism for control of immune responses in vivo.


Lysis of tumor cells by antibody and complement. III. Lack of correlation between antigen movement and cell lysis. The increase in susceptibility to killing by rabbit antibody and guinea pig complement of guinea pig hepatoma cells (line-10), after treatment with certain metabolic inhibitors, did not correlate with the mobility of antigen on the cell surface as measured by indirect immunofluorescence.


A surface membrane determinant shared by subpopulations of thymocytes and B lymphocytes. Utilizing a quantitative fluorescence assay with the fluorescence-activated cell sorter (FACS), we have demonstrated that a rabbit antiserum obtained by immunization with cells of a mouse IgM-producing plasma cell tumor (MOPC104E) is reactive with at least two surface determinants, designated Th-B and ML2, on subpopulations of normal murine lymphocytes. The ML2 determinant is restricted to B lymphocytes. The Th-B determinant is shared by splenic B lymphocytes and a large subpopulation of thymocytes, the latter of which express a 3-fold higher density of Th-B on their surface than do the B lymphocytes. Neither Th-B nor ML2 were found on peripheral T cells or on brain, liver, or kidney cells. The available evidence suggesting that Th-B may be a stem cell determinant that is lost upon maturation is discussed.


Intravascular effects of IgE antibody upon basophils,, neutrophils, platelets and blood coagulation in the rabbit. Rabbits synthesizing only IgE antibody to BSA were challenged intravenously with 50 mg BSA and the associated blood cell and coagulation alterations were examined. Basophils decreased by 90% within 1 min of challenge. This was followed by a 70% decrease in neutrophils and a 50% decrease in platelets by 15 min. These changes were found to be highly significant when compared to control unimmunized rabbits similarly challenged. By 60 min, the neutrophils and platelets in the experimental rabbits had returned to 50 and 80% of their prechallenge levels respectively, but no basophils were demonstrable by toluidine blue staining. Lymphocyte counts in the experimental rabbits did not differ from controls at 0, 1, 15, or 60 min after challenge. Blood coagulation alterations also occurred after BSA challenge in the rabbits synthesizing IgE. A significant shortening of the whole blood clotting time in plastic tubes at 25 degrees C occurred in blood samples taken at 30 sec after antigen challenge. Similarly, in vitro addition of BSA to blood samples taken prior to challenge significantly shortened the whole blood clotting time. In addition, significant prolongation of whole blood clotting times was observed in blood obtained 60 min after challenge, but only among the rabbits positive for systemic anaphylaxis. This in vivo study corroborated the known in vitro effects of IgE upon the rabbit blood basophil and platelet. In addition, the results indicate that the intravascular interaction of antigen with specific IgE antibody induced neutrophil and blood coagulation alterations.


IgE-induced blood coagulation alterations in the rabbit: consumption of coagulation factors XII, XI, and IX in vivo. Intravenous administration of BSA into 3-month-old rabbits producing detectable anti-BSA antibody only of the IgE class of immunoglobulin induced a variety of intravascular blood coagulation alterations observed in the plasma 15 min after antigen challenge included: a) the intravascular consumption of intrinsic blood coagulation factors XII, XI, and IX and possibly the reduction in clottable fibrinogen; b) a significant prolongation of the activated partial thromboplastin time but not the prothrombin time; and c) the production of an inhibitor affecting the last stage of blood coagulation. The observed blood coagulation alterations were not caused by the manipulative procedures utilized, the presence of anti-BSA, IgG or IgM antibody, histamine-induced alterations in the vascular endothelium or the development of hypotensive shock. It is proposed that specific IgE antibody can induce directly or indirectly the activation of intrinsic blood coagulation in vivo.


Immunochemical analysis of a Smith-like antigen isolated from two human strains of Staphylococcus aureus. A surface antigen consisting of aminoglucuronic acid and N-acetyl-L-alanine was isolated from the culture filtrates of two human strains of Staphylococcus aureus. Double diffusion analysis in agar suggested that the antigen is immunologically similar to the alanyl-aminoglucuronic acid capsule of the Smith strain of S. aureus. Quantitative precipitin inhibition studies indicated that N-acetyl-L-alanine is the immunodominant determinant of the acidic antigen. In addition, conjugates consisting of N-acetyl-L-alanine coupled to bovine serum albumin gave a significant precipitin reaction with anti-staphylococcal serum which is rich in alanyl-aminoglucuronic acid polymer antibodies. Antibodies with N-acetyl-L-alanine specificity were isolated from N-acetyl-L-alanine-Sepharose immunoabsorbent columns. Double diffusion analysis in agar indicated that the eluted antibodies were serologically reactive and belonged to the IgG class of immunoglobulins.


Cytoplasmic immunofluorescence of bone marrow plasma cells producing immunoglobulins of the four IgG subclasses. Immunofluorescence studies were performed on bone marrow samples from 21 individuals with normal or slightly elevated serum IgG levels. On the average, 40.7 percent of the IgG-positive plasma cells contained molecules of the subclass IgG; 30.6 percent had intracellular IgG2; 21.7 percent, IgG3, and 7.0 percent, IgG4. These figures were compared to the relative serum IgG subclass concentrations. The principal finding was the discrepancy between the high relative number of IgG-3 producing bone marrow plasma cells and the low relative IgG3-producing bone marrow plasma cells and the low relative IgG3 serum concentration (5.5 percent of the total IgG). It was explained by the short biologic half-life of  molecules of this subclass.


Suppressor cell control of unresponsiveness to experimental allergic encephalomyelitis. Lymph node cells (LNC) from Lewis rats rendered unresponsive to experimental allergic encephalomyelitis (EAE) by pretreatment with myelin basic protein markedly suppressed clinical (but not histologic) EAE in normal recipients later challenged with an encephalitogenic emulsion. Unresponsiveness was immunologically specific, and required viable LNC; serum transfer was ineffective. These findings suggest that suppressor cells exert control over this autoimmune disease.


Class, amounts and affinities of anti-dinitrophenyl antibodies in chickens. I. Production of 7S and 17S antibodies of equal affinity by intravenous injection of antigen. Repeated intravenous injections of maximally coupled dinitrophenylated bovine gamma-globulin elicited both 7S and 17S anti-dinitrophenyl antibodies in chickens. Only antibodies of low affinity were produced regardless of the priming dose, the interval between injections, and the number of injections. The 7S and 17S antibodies isolated from invididual animals had identical affinities and heterogeneity indices. The concentrations of antibodies formed were uniformly low despite many injections over a prolonged period. These studies indicate that stimulation by antigen alone may not be sufficient for the induction of predominant 7S antibody formation and for the synthesis of high affinity antibody.


Class, amounts, and affinities of anti-dinitrophenyl antibodies in chickens. II. Production of a restricted population of high affinity 7S antibodies by injection of antigen emulsified in adjuvant. The response of chickens given a single intramuscular injection of maximally coupled dinitrophenylated-gamma-bovine beta-globulin in either Freund's complete (FCA) or incomplete (FIA) adjuvants was characterized by an initial synthesis of 7S and 17S antibodies followed by the exclusive and persistent production of 7S antibodies. The 17S antibodies were not detected either 3 to 4 weeks after a single injection or after an intravenous boost 16 months later. Injections of low doses of antigen in FCA induced the synthesis of 7S antibodies of high affinity at least by 4 months. Analyses of the Sips plots generated from equilibrium dialysis data indicated that a shift in the distribution of 7S antibody affinities occurred because of the production of a restricted population of high affinity antibodies. The changes in the binding properties of antibody during the immune response from chickens given antigen in FIA were less apparent, although qualitatively similar, to those found in birds given antigen in FCA. Three possibilities were presented to explain the effect of adjuvant on the class and affinity of the antibody: a) the requirement of a second signal for B cell differentiation, b) the presence of subpopulation of B cells, and c) somatic mutation events.


Macrophages activated as suspension cultures with lymphocyte mediators devoid of antigen become cytotoxic for tumor cells. Normal peritoneal exudate cells (PEC) were activated as suspension cultures either in mediator-rich supernatants from o-chlorobenzoyl-bovine gamma-globulin (OCB-BGG) stimulated lymphocytes or in antigen-free Sephadex fractions from these supernants. After 24 hr incubation thration. The adherent cell fractions of PEC, recovered by trypsinization from monolayers and activated by this technique, were as cytotoxic as unfractionated PEC. Lymphocyte supernatants and antigen-free fractions of the supernatants induced comparable macrophage-mediated tumor cytotoxicity. Treatment of activated macrophages with trypsin did not alter their cytotoxic capacity.


Further studies on the biologic properties of guinea pig IgG1 antibodies. II. In vivo activation of C3 by anti-glomerular basement membrane antibodies. Normal rats were injected with guinea pig anti-rat glomerular basement membrane antibodies of the IgG1 or IgG2 class or with their F (ab') 2 fragments, in order to study which antibody site triggers the alternate complement pathway in vivo. Both IgG classes were able to induce a heavy proteinuria and led to C3 deposition in the glomeruli in a pattern similar to their own distribution along the glomerular basement membrane, as shown by the immunofluorescence technique. The Fab(ab')2 fragment of IgG2 did not produce C3 binding or proteinuria. The F(ab')2 fragment of IgG1 was difficult to obtain devoid of Fc determinants. A F(ab')2 fragment of IgG1 still bearing Fc determinants led to C3 binding and proteinuria, whereas the true F(ab')2 fragment of IgG1 had none of these effects in two out of three animals.


Immunologic effects of morphine administration in rabbits. Long-term effects of morphine administration or immunologic test responses were studied in female rabbits. Implantation of morphine-containing pellets was found to be more effective than injection of morphine sulfate solutions in promoting increased serum binding of 140-morphine. A large part of the increased morphine binding by sera associated with administration of morphine was found in serum fractions containing gamma-globulin and was absent in gamma-globulin-free fractions. These sera showed some degree of specificity for the morphine configuration in tests with other narcotics. They also gave positive immunologic test reactions in passive hemagglutination and radial immunodiffusion tests involving serum albumins conjugated with morphine derivatives. Other evidence for immunologic responsiveness against morphine by morphine-pretreated rabbits was shown by cutaneous hypersensitivity reactions against morphine-carrier conjugates and by a diminution of the serum morphine-binding response in rabbits given an immunosuppressive dose of cyclophosphamide. Failure of naloxone, a morphine antagonist, to alter the serum morphine-binding response suggested that serum levels of the morphine-binding globulin studied here were not direclty related to morphine withdrawal.


Existence of VHIII subgroup in rabbit antibody heavy chains. Sequence analysis of CNBr fragments from the heavy chains of a rabbit anti-p-azobenzoate antibody of restricted heterogeneity has revealed a VHIII-like heavy chain sequence which was present in 6 to 10% of the heavy chain preparation.


Preparation of specific anti-thymocyte and anti-bursacyte sera in rabbits. A method of producing anti-thymocyte and anti-bursacyte sera in rabbits is described. Chickens which served as donors of cells were thoroughly perfused with saline to remove plasma proteins and circulating blood elements. For immunization, thymocytes were obtained from neonatally bursectomized birds, and bursacytes from chickens thymectomized at hatching. The purification of rabbit anti-lymphocyte sera included absorptions with leucocyte-free suspension of chicken erythrocytes, chicken liver cell membranes, thymocytes from bursectomized-irradiated and bursacytes from thymectomized-irradiated chickens, and chicken IgM and IgG immunoadsorbents. Cytotoxicity and fluorescent-antibody assays revealed that anti-thymocyte and anti-bursacyte sera thus produced clearly distinguished the surface antigenic determinants of thymocytes from those of bursacytes.


Non-specific interference of certain components of tissue culture media with the radioimmunoassay of rat alpha-foetoprotein. Interferences of 'Williams' tissue culture medium, used for cultivating rat hepatocytes, upon rat alpha-foetoprotein (AFP) radioimmunoassay have been investigated; they are not due to foetal calf serum proteins which are added as growth factor and can be abolished by dialysis which appears to be necessary for the distinction between AFP non-producer and low-producer cell lines. Of the three major groups of non-mineral components examined, amino acid solution played a major role; when individual amino acids were examined using the double antibody technique, arginine was found to interfere predominantly; its dose-response curve was parallel to that of rat AFP, which confirmed that an immunological identity between two substances cannot be established on the basis of parallelism as the only criterion.


Isolation of hepatitis B surface antigen (HBsAg) by affinity chromatography on antibody-coated immunoadsorbents. Hepatitis tb surface antigen (HBsAg) was isolated from human serum by two steps of affinity chromatography on antibody-coated gels. HBsAg-positive serum was passed through a column packed with guinea pig anti-HBsAg antibodies covalently bound to CNBr-activated beaded agarose gel. The majority of non-specifically bound proteins was removed by washing the gel with increased concentrations (0.5 M) of NaCl in Tris buffer. Elution of the specifically bound HBsAg was carried out with 3 M NaSCN. Residual normal human serum proteins present in the eluate were removed by passing the partially purified HBsAg through an immunoadsorbent coated with rabbit antibodies directed against human serum proteins. After this treatment normal human serum proteins could no longer be demonstrated by passive hemagglutination in the isolated HBsAg. Cross-reactions between HBsAg and normal human serum proteins could not be demonstrated. Both antibody-coated immunoadsorbents could be used over ten times without significant loss of their binding capacity.


The use of indirect immunofluorescence to evaluate the gel filtration method of fractionating human immunoglobulins. The indirect immunofluorescent technique has been shown to be a more sensitive method than either immunoelectrophoresis or gel filtration for testing the efficacy of the gel filtration method in fractionating human immunoglobulins. It has been confirmed that IgM fractions of some sera from patients with multiple sclerosis, contain antibody which reacts with measles virus-infected tissue culture cells.


Determination of antibody avidity at the cellular level by the plaque inhibition technique: effect of valence of the inhibitor. Inhibition of plaque formation by multivalent and univalent ligands was compared as an assay of avidity of antibody produced by PFC. Multivalent ligands are much more effective as inhibitors and their use tends to impart an appearance of lack of heterogeneity and high avidity to the PFC populations being studied. It is thus probably generally advisable to employ univalent ligands in such studies.


Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods. Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.


Bovine keratohyalin: anatomical, histochemical, ultrastructural, immunologic, and biochemical studies. Salt extraction studies showed that keratohyalin (KH) could be solubilized and extracted from fresh bovine hoof epidermis. The solubility of KH varied in relation to the molarity of the salt solution used for extraction. Using this information, the extracted KH was aggregated in vitro by dialyzing the high salt extract against distilled water. Histochemical, ultrastructural, and immunologic studies of the resultant particles or macroaggregates showed that the latter had the same properties and immunogenicity as the KH granule in situ and produced antibodies against it. Fractionation of the macroaggregates by polyacrylamide gel electrophoresis demonstrated that the macroaggregates were compsed of sets of 20 polymers whose subunits or monomers had a molecular weight of 16,900. Amino acid analyses showed that the macroaggregates and the various fractionated polymers were similar and that the protein ahd 116 amino acid residues. Serine, arginine, glycine, glutamic acid, and histidine constituted 78% of all residues, and serine alone represented 27%. The molecular weight by amino acid analyses was 16,150 after correction for the 8% ribonucleic acid which appears to be complexed to the protein.


Desmosomes, filaments, and keratohyaline granules: their role in the stabilization and keratinization of the epidermis. Components of desmosomes, filaments, and keratohyaline granules were studied by electron microscope and biochemical methods to clarify their role in the stabilization and keratinization of the epidermis. Isolated desmosomes are composed of 76% protein, 17% carbohydrate, and 10% lipid. The bulk of protein consists of a "spectrin"-like fibrous protein, presumably present in the plaque, and of glycoproteins in the desmosomal interspace. The main component of filaments, prekeratin, is a low-sulfur alpha-protein composed of a pair of three-chain subunits with non-alpha-helical segments separated by 200 A-long alpha-helical regions. The major component of isolated keratohyaline granules, the amorphous particulate material, is formed by a high-sulfur protein with a single-type of polypeptide chain. Polypeptide chains comparable to those found in prekeratin and keratohyaline granules were recovered from extracts of horny cells. Within the living part of the epidermis, filaments hypothetically form a cytoskeletal system which is anchored to desmosomes by a filamentous plaque protein. Glycoproteins are involved in the formation of strong junctions between the cells which enable the living part of the epidermis to respond as a whole to mechanical stress. The stratum corneum is stabilized by a similar system in a consolidated state which is less extensible. Horny cells are enveloped by a thickened membrane and the interfilamentous spaces are filled with various proteins including the sulfur-rich amorphous protein found in keratohyaline granules.


Complement activation in bullous skin diseases. Pemphigus, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, and herpes gestationis are members of the chronic vesiculobullous skin diseases of man. The complement system, including both the classical and alternative pathways, may be important in the pathogenesis of these diseases. In pemphigus, early complement components (C1, C4, and C2) appear to be activated in addition to later components (C3 and C5), suggestive of classical pathway activation. Participation of properdin in addition to early complement components suggests local activation of both complement pathways in bullous pemphigoid and cicatricial pemphigoid. Herpes gestationis and dermatitis herpetiformis may be bullous skin diseases entirely mediated by the alternate or properdin pathway. The specific immunopathologic findings in these diseases are discussed.


Ordered structure of the epidermis.

Ordered structure of the epidermis. Recent reports on the ordered structure of the epidermis are reviewed. The regular relationship between Langerhans cells and cell columns in the mouse is not typical of the epidermis of other animals. The pattern of mitotic activity previously described in mouse epidermis has also been demonstrated in the hamster, which suggests control of keratinocyte activity rather than the exclusion of keratinocytes from the central region beneath cell columns. These findings are discussed in relation to mechanisms which may be responsible for the formation of cell columns.


Basement membranes: structural and biosynthetic considerations. Basement membranes are extracellular matrices synthesized by a variety of cells including the basal cells of the epidermis; the respiratory, gastrointestinal, and glandular epithelium; the capillary endothelium; the epithelial cells of the glomerulus, the renal tubule, and the lens capsule; and the endothelium of Descemet's membrane. Basement membranes in the mature animal are free of lipids, DNA, and proteoglycans and are composed of dissimilar protein subunits. One of these is a procollagen-like molecule associated with a noncollagenous matrix glycoprotein. The proportion of the latter component varies among basement membranes. These various subunits are stabilized by hydrogen bonds, disulfide bonds, and aldehyde-derived cross-links which are so extensive that they render the basement membranes highly insoluble. Immunochemical studies indicate three distinct antigenic components which correspond to the collagenous moiety, its nonhelical extension, and the matrix glycoprotein. The collagen component of basement membranes, free of the nonhelical extension, is composed of three identical alpha-chains. It is highly rich in hydroxylysine, 3- and 4-hydroxyproline and contains 4 to 8 residues of half-cystine. It contains 38 residues of glucosyl-galactosyl-hydroxylysine per chain and minimal amounts of mannose, glucosamine, and fucose. Newly synthesized basement membrane collagen is secreted in the extracellular space as the precursor molecule "procollagen." This molecule does not undergo conversion to collagen but interacts with the matrix glycoprotein to give rise to the appropriate structure.


Ultrastructural localization of in vivo-bound complement in bullous pemphigoid. Antihuman complement component C3 labeled with horseradish peroxidase was used to reveal the ultrastructural localization of complement in two cases of bullous pemphigoid. The complement deposits were shown to be exclusively located in the space between the plasma membrane of the basal cells and the basal lamina. This corresponds exactly to the ultrastructural localization of immunoglobulins in bullous pemphigoid.


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins of newbonr rat skin. II. Keratohyalin and stratum corneum proteins. Keratohyalin extracts from newborn rat epidermis were prepared by potassium phosphate and citric acid-detergent extraction procedures. These preparations were compared by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and amino acid analysis. The major band of the potassium phosphate extract has a molecular weight of 48,000. The major bands of the citric acid-detergent preparation have molecular weights of 64,000, 61,500, 57,000 and 54,000. Electrophoresis of S-carboxylmethylated (SCM)-fibrous protein results in two major bands of approximately 57,000 and 64,000. SDS gels of the two preparations of keratohyalin and the SCM-fibrous protein were compared with gels of the insoluble proteins of granular and eluted cornified cells. All of the major bands in the preparations of keratohyalin can be seen in gels of the granular preparation. The two SCM-fibrous protein bands correspond with two prominent bands in gels of the cornified cell preparation. Two bands of the citric acid-extracted keratohyalin sample also have the same mobility. The major band of the potassium phosphate-extracted preparation of keratohyalin corresponds with a third prominent band of the cornified cell preparation. These results suggest that biochemical components of the preparations of keratohyalin are present in both the granular and the cornified layers of newborn rat epidermis.


Ultrastructural and immunohistochemical studies in five cases of Argentine hemorrhagic fever. Ultrastructural and immunohistochemical studies on tissues from five patients with Argentine hemorrhagic fever revealed previously undetected lesions caused by the viral infection. Two types of particle were seen in the cells of all organs examined. The particles had some characteristics similar to those described for arenaviruses. However, the virus-like particles were intracellular, had a single membrane, and apparently originated by a process of budding into the endoplasmic reticulum cisternae. Intranuclear bodies and three types of cytopolasmic change were observed in conjunction with the virus-like particles; Antigenic determinants of Junin virus were demonstrated in cells of all organs examined. Immunohistochemical experiments also indicated alterations in the cellular mechanisms of protein synthesis. Until now the pathogenesis of human diseases produced by arenaviruses has not been established. The results of this study suggest that in Argentine hemorrhagic fever the virus is responsible for a direct pathogenic action.


Sepharose-bound erythropoientin. Studies with anti-erythropoietin. Anti-erythropoietin antibodies (anti-ESF) were determined by the capacity of the immunoglobulins to neutralize the biological activity of erythropoietin (ESF). Affinity chromatographic methods were used to initiate the purification of anti-ESF by use of ESF covalently linked to Sepharose. It was observed that anti-ESF bound the ESF-Sepharose. In order to remove the nonspecific immunoglobulins, the anti-ESF preparation was subjected to further chromatography on affinity colums where either serum or urinary proteins from a polycythemic patient were covalently linked to Sepharose. It was demonstrated the affinity chromatography could be used as a method isolate anti-ESF.


5alpha-dihydrotestosterone stimulation of human prostate in organ culture. Explants of human benign hypertrophied prostate were cultured for 8 days. The effect of 5alpha-dihydrotestosterone on the morphology, DNA and glucose utilization of 14 cases was investigated. In four cases proliferation was clearly more extensive in the dihydrotestosterone-treated explants than in the controls. In six explants, there were no morphological differences between those cultured with or without dihydrotestosterone and in half of these, proliferation was present. Four explants were either necrotic or did not contain epithelial cells. The mean DNA content of the treated explants was higher than that of the controls (P smaller than 0.01). There was no significant difference in the rate of glucose utilization by the explants treated with dihydrotestosterone, and by the controls.


Changes in the endometrium of mice after the induction of implantation by actinomycin D. Actinomycin D can induce a small number of implantations in pregnant mice undergoing progestin-induced delayed implantation following ovariectomy. However, the response of the uterus to the blastocyst is considerably retarded compared with the response observed when implantation is precipitated by oestradiol. With the electron microscope the attachment reaction between the trophoblast and uterine epithelium is evident about 48 h after administration of the drug. However, the differentiation of the luminal surface of the epithelial cells in areas of uterus distant from a blastocyst (2nd stage of closure), which normally accompanies implantation, and can be induced by oestradiol in progesterone-treated animals, is not seen. Thus actinomycin D, although allowing implantation to proceed, does not completely mimic the actions oestradiol on the progesterone-treated uterus.


Experimental study of the formation of the heart tube in the chick embryo. A study was made of the development of the heart tube beginning from Hamburger & Hamilton (1951) stage 8+ up to stage 12. We used labelling with particles of iron oxide followed with time-lapse cinemicrophotography, staining with methylene blue, serial section and cutting the embryo in two halves. Our results led to the conclusion that the tubular heart is formed by the addition of precardiac material into its posterior end, but in addition it is necessary to consider the fusion of the myocardium in a cephalic direction, starting with the fusion of both heart primordia at the rostral end. By this fusion the most anterior part of the heart up to stage 12 is formed.


Microfilaments in the external surface layer of the early amphibian embryo. A comparison was made by transmission electron microscopy of the microfilaments in the surface layers of the early embryos of Triturus alpestris and Xenopus laevis at stages of development up to neurulation. Actin-like filaments which bound heavy meromyosin (HMM) were found in cell extracts of all stages, but were comparatively rare in the newly fertilized egg. Ten nm microfilaments were present throughout development in Xenopus, and from the mid-neurula stage in Triturus. Both kinds of microfilament were located in the circumferences of superficial ectoderm cells at the level of the apical junctions, the 10 nm microfilaments in association with desmosomes which began to develop before gastrulation in Xenopus. The accumulations of microfilaments in the apical constrictions, which form in ectoderm cells of Triturus early gastrulae when dissociated in a calcium-free medium, suggest that they are contractile elements. In the absence of such accumulations in the cell apices, the reverse curling exhibited by Xenopus ectodermal explants is attributed rather to a separation of the cells' lateral borders. Cytochalasin B (5 mug/ml) caused ectodermal explants from the early gastrulae of both species to disaggregate. With the rupture of the apical junctions there was a disorganization of the associated microfilamentous layer.


Morphogenesis in Micrasterias. I. Tip growth. Observations on lobe growth in the lateral wings of the developing primary cell wall in the desmid Micrasterias rotata are reported and discussed. Patterns of incorporation of methyl-[3H]-methionine and C-1[3H] glucose into the primary wall as revealed in autoradiograms indicate that formation of the new wall is concentrated at the tips of lobes. Patterns follow the predictions of Robertson's model for tip growth in fungal hyphae; thus they link growth in M. rotata lobes with mechanisms of cell elongation found in other cells. Damage done to selected regions of the cell surface with a laser microbeam demonstrates that only certain regions are required for continued growth and morphogenesis while much of the surface plays only a passive role. In growth stages at which lobes are already well defined (stage 4 and later) continued growth of each lobe requires the participation of an area no more than 4-5 mum in diameter, here termed a singularity, at its tip. At early stages (prior to stage 3) singularities per se cannot be demonstrated. At these stages the capacity to intitiate lobes and hence to form singularities is not fixed at specific points, but is distributed over an area of the surface no less than 10 mum in diameter. Singularities, by their persistence and repeated duplication, are directly responsible for the spatial form of the two lateral wings.


The mechanism of plasticization of the abdominal cuticle in Rhodnius. 1. The mechanism of plasticization of the abdominal cuticle in Rhodnius larvae has been investigated, using the properties of loops of cuticle under varying test conditions as a model for the behaviour of the cuticle in vivo. 2. It is supposed that plasticization is effected by a change in the intracuticular environment. A number of model mechanisms for plasticization may be proposed, which suppose that the epidermis is capable of regulating (a) pH, (b) ionic strength,(c) Ca and/or Mg, (d) urea, within the cuticle. 3. Analyses of cuticle ash show that models(b) and (c) are not responsible for plasticization in vivo. The levels of inorganic ions within the unplasticized cuticle are not sufficiently high to allow plasticization upon their removal. 4. No evidence for model (d) has been found; urea does not occur in the cuticle in detectable quantities. 5. Exact measurements of the intracuticular pH have not been achieved but straining experiments strongly suggest that a change in pH occurs within the cuticle on plasticization. This pH change is probably large enough to account for the increased extensibility shown by plasticized cuticle.


Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. I. Demonstration of similar or identical idiotypes on IgG molecules and T-cell receptors with specificity for the same alloantigens. Antigen-binding receptors on T lymphocytes and IgG antibodies with the same antigen-binding specificity as the T-cell receptors display shared or identical idiotypes. This was shown using a system where adult F1 hybrid rats between two inbred strains were inoculated with T lymphocytes from one parental strain. Such F1 hybrid rats produce antibodies directed against idiotypic determinants present on IgG alloantibodies, produced in the T donor genotype strain and with specificity for the alloantigens of the other parental strain. The idiotypic nature of the F1 antialloantibody serum against the parental alloantibodies was demonstrated both by indirect hemagglutination tests or by gel diffusion using alloantisera with different specificity as targets. Furthermore, the F1 anti-T-lymphocyte sera could be shown to contain antibodies against idiotypic parental T lymphocytes as well. This was shown by the capacity of the antisera, in the presence of complement, to wipe out the relevant parental T-cell reactivity against the other parental strain (as measured in MLC or GVH) whilst leaving the T-lymphocyte reactivity against a third, unrelated allogeneic strain intact. These findings demonstrate that F1 hybrid rats inoculated with parental T lymphocytes make anti-idiotypic antibodies directed against both the T cell receptors and IgG alloantibodies of that parental strain with specificity for alloantigens of the other parental strain. In order to prove identity between the anti-idiotypic antibodies against the B and T-cell antigen-binding molecules the following experiments were carried out; highly purified IgG from relevant alloantibody-containing serum in immunosorbent from could be shown to selectively remove both anti-idiotypic activities from the F1 antiserum. Further more, parental normal T lymphocytes could be shown capable of removing from the anti-idiotypic antisera all those antibodies that would cause agglutination of the relevant alloantibody-coated erythrocytes in the indirect agglutination assay. We would thus conclude that T and B lymphocytes reactive against a given antigenic determinant use receptors with antigen-binding areas coded for by the same variable gene subset(s).


Suppression of in vitro antibody response by a serum factor (SAA) in experimentally induced amyloidosis. Serum from CBA/J mice made amyloidotic by chronic casein injections has been shown to suppress in vitro antibody response to SRBC. Similar suppression was also found with normal mouse serum but to a much lesser degree. This suppressive activity of both amyloidotic serum and normal serum was removed by absorption of the sera with antiserum to protein AA, the major constituent of casein-induced (secondary) amyloid fibrils. This antiserum to the amyloid fibril protein AA (mol wt 8,400 daltons) detects an immunologically cross-reacting serum alpha globulin (SAA) (mol wt approx. 100,000). It is postulated that the serum factor (SAA) is a regulator of antibody response and may be present in elevated amounts as the result of chronic antigenic stimulation.


Cross-resistant relationships among the aminoglucoside antibiotics in Mycobacterium tuberculosis. Phenotypes of isolates of Mycobacterium tuberculosis H37RV showing resistance to the aminoglucoside antibiotics streptomycin, viomycin, kanamycin, capreomycin, tuberactinomycin N, lividomycin and paromomycin could be grouped into the following types: (I) resistant only to different levels of streptomycins; (2) resistant only to a low level of kanamycin; (3) triply resistant, to low levels of viomycin, tuberactinomycin N and capreomycin; (4) triply resistant, to a low level of kanamycin and high levels of lividomycin and paromomycin; (5) quadruply resistant, to a low level of capreomycin and high levels of kanamycin, lividomycin and paromomycin; (6) hextuply resistant, to high levels of viomycin, tuberactinomycin N, capreomycin, kanamycin, lividomycin, and paromomycin. Three modificatied types of the latter were also observed. Appearance rates of the six types were estimated as 10(-6) to 10(-9), 10(-6), 10(-6) to 10(-7), 10(-8), 10(-8), and 10(-8) to 10(-9), respectively, in a total viable population of the parent strain. Mutations to all phenotypes were considered to be produced by single mutations. According to cross-resistance relationships, aminoglucoside antibiotics were classified into three groups: (I) streptomycin; (II) viomycin, tuberactinomycin N and capreomycin; (III) kanamycin, lividomycin and paromomycin. No cross-resistance relationship between streptomycin and other antibiotics was observed. Resistances to viomycin, tuberactinomycin N and capreomycin occurred by single mutation to type 3. Resistances to kanamycin, lividomycin and paromomycin occurred by single mutations to types 4 and 5. Low resistance to capreomycin was produced by mutation to type 5. Therefore capreomycin was considered to be an intermediate between the second and third groups. These two groups had a close relationship, as resistance to all six agents in these groups could be produced by a single mutation to type 6 (and its modified types).


Glycerol teichoic acid as an antigenic determinant in a Gram-negative bacterium Butyrivibrio fibrisolvens. An antigenic determinant isolated from a strain of the Gram-negative bacterium Butyrivibrio fibrisolvens reacted with specific antisera to the polyglycerophosphate backbone of membrane teichoic acids of lactobacilli. It gave a reaction of identity with membrane glycerol lipoteichoic acid and glycerol teichoic acid preparations from lactobacilli, and with phenol extracts of other Gram-positive bacteria. The antigen-antibody reactions was strongly inhibited by glycerol-phosphoryl-glycerol-phosphoryl-glycerol and the chemical composition was consistent with glycerol teichoic acid. It was concluded that this Gram-negative bacterium contained a glycerol teichoic acid whose polyglycerophospate backbone was acting as antigenic determinant. Extracts of 33 out of 52 other strains of butyrivibrios examined gave similar reactions.


Localized intracellular polyphosphate formation by Desulfovibrio gigas. The dissimilatory sulphate-reducing bacterium Desulfovibrio gigas, frequently sub-cultured, often contained spherical granules which stained metachromatically with some basic dyes. The granules were examined in situ by transmission electron microscopy of whole organisms and thin sections. The granules were isolated from broken bacteria as a water-insoluble, non-crystalline, white material containing magnesium, phosphorus and organic carbon, but devoid of sulphur and nitrogen. The molar ratio of phosphorus to magnesium (1 to 17) was close to the proportions in magnesium tripolyphosphate. Infrared absorption spectra for the white material and magnesium tripolyphosphate were similar.