Mononuclear phagocyte proliferation, maturation and function. The mononuclear phagocytic system is a continuum of cells beginning with the bone marrow monoblast and promonocyte, through the monocyte to the larger tissue macrophages and multinucleate giant cells. This system of cells is widely distributed throughout the body in the blood and bone marrow; the pleural, peritoneal, and alveolar spaces; the lymph nodes, spleen, liver, and other parenchymal organs. The activity and composition of the cell varies with the level of maturation, changes in cellular environment, and with various cellular activities. The monocyte-macrophage group of cells plays an active role in defense reactions against certain microorganisms, and in the removal of dying cells and cell debris. They are an integral part of both the inductive phase of the immune response, and of cell-mediated immune reactions. In addition, they probably play a role in the defence against spontaneously arising tumours, in the control of granulopoiesis, and possibly in erythropoiesis.


beta2-Microglobulins.

beta2-Microglobulins. A low-molecular-weight protein, beta2-mu, isolated from urine and other biological fluids was shown to have an amino acid sequence related to constant regions of the immunoglobulin heavy chain--to CH3 comain in particular. Several homologues of this protein were also isolated and partially sequenced. The protein was shown to be localized in the membranes of nucleated cells and found to be associated with HL-A antigens. Its biological and/or immunological function is still unknown; however, it binds to complement and is cytophilic to guinea pig macrophages as well as to other cells. Antibodies to beta1-mu can act as mitogens and can blcok stimulation in MLCs but not recognition by "killer" lymphocytes.


The radiation biology of the thyroid.

The radiation biology of the thyroid. The structure and function of the thyroid gland are described. A detailed analysis of population kinetics in the gland suggests a method of measuring cell survival after irradiation that has many features in common with methods used in other mammalian cell systems. This method is used to obtain survival curves for thyroid cells afer irradiation. The effects on survival of splitting the radiation dose into two or multiple fractions, radiation type, and radioprotective agents are also examined. In the light of these data the tolerance of thyroid tissue to radiation exposure under various conditions is discussed. The dosimetry and biological effects of 125I and 131I are described in detail, and compared with X-rays. Radioiodine treatment of thyrotoxicosis is presented in relation to the known biological effects of the isotopes on the gland. Carcinogenic action of ionizing radiations in the thyroid are reviewed with particular reference to the clinical consequences of observations in this field.


DNA replication patterns of human chromosomes from fibroblasts and amniotic fluid cells revealed by a Giemsa staining technique. A technique is described for visualizing late-replicating regions by a Hoechst 33258-Geimsa-staining procedure combining the techniques of Latt (1973) and of Perry and Wolff (1974). The advantages are two-fold: distinct bands are obtained and many possible mistakes and interpretation difficulties with autoradiography are avoided. The time sequence of late-replication patterns (excepting C-group chromosomes) has been established in human fibroblasts of adults, and these results have been compared with three of four different cell types from amniotic fluid. No significant differences in late-replication patterns and time sequence of the different cells could be discovered. As expected, the replication patterns are in good concordance with the patterns of G- and Q-bands. Some exceptions are described.


Airway obstruction due to spontaneous retropharyngeal hemorrhage. A patient is described with polycythemia vera who was taking anticoagulants and developed a spontaneous retropharyngeal hemorrhage after a violent sneezing episode. This progressed to tracheal compression with stridor after he had taken some aspirin for relief of the neck pain. An emergency tracheostomy was life saving.


A high resolution study of the DNA replication patterns of chinese hamster chromosomes using sister chromatid differential staining technique. Chinese hamster cells were grown for 1+ and 2+ cell cycles in the presence of BrdU and then treated by the sister chromatid differential staining technique (SCD). Those regions of a chromosome which had replicated twice in the presence of BrdU were pale staining and by selecting appropriate metaphase cells an accurate reconstruction of the DNA synthetic patterns was possible. A direct correlation between the staining intensity of the G bands and the order in which they replicate was found. Dark staining G bands were always the last region of a chromosome to replicate while G negative bands were first. It is concluded that each G band may be a cluster of replicons capable of initiating DNA synthesis simultaneously.


Visualization of nucleolar organizer regions im mammalian chromosomes using silver staining. A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.


Analysis of the replication pattern of Chinese hamster chromosomes using 5-bromodeoxyuridine suppression of 33258 Hoechst fluorescence. Chinese hamster fibroblasts were synchronized and given 5-bromodeoxyuridine for DNA synthesis except during one hour of the S phase when thymidine was present in the medium. In the next mitosis, chromosomes stained with 33258 Hoechst were banded in appearance when photographed by fluorescence microscopy. The bright regions corresponded to the chromosome segments replicated during the thymidine exposure in the S phase. The segments replicated together during any one hour produced three distinct patterns which were characteristic of early, middle, and late S phase. Most of the fluorescent regions corresponded in size and position with G-bands of these chromosomes. There was no correlation between the staining behavior of a band in G-band procedure and its time-or-replication, i.e., both light and dark G-bands were replicated during early, middle, and late S phase. However, it appears that all of the DNA within a single band is replicated together within one third of the S phase.


Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining. The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.


Harmonic composition and topographic distribution of responses to sine wave modulated light (SML), their reproducibility and their interhemispheric relationship. 1. Responses to sine wave modulated light (SML) were recorded bipolarly from the occipital, parietal and temporal scalp areas on the right and the left sides of 9 normal individuals. Their harmonic composition was determined by means of Fourier analysis. 2. The wave form of the responses was rather complex in relation to the stimulus and differed according to stimulation frequency and localization. 3. The components of the responses were principally the 1st and 2nd harmonics. 4. The amplitudes of the harmonics were usually largest over the occipital and smallest over the parietal scalp areas. 5. Considerable intra-individual amplitude variabilities of both harmonic component amplitudes existed in all areas of all individuals. 6. The interhemispheric amplitude correlations of 1st and 2nd harmonic components, simultaneously recorded in homologous occipital, parietal and temporal areas, were measured by means of Kendall's rank correlation test. 7. The interhemispheric synchrony of the harmonic components was measured by determining the phase differences between each of the response components in homologous right and left scalp areas. 2. In subjects with a high amplitude correlation a strong synchrony existed and vice versa. 9. It is suggested that the interhemispheric relationships between harmonic components of the responses to SML might be of importance for clinical application. This opinion is supported by the fact that through SML evoked responses a considerable data reduction is obtained in comparison with the responses to light flashes.


Cyclic EEG changes in subacute spongiform and anoxic encephalopathy. Polygraphic recordings of EEG, EMG, EKG and respiration were made on three patients with histologically verified subacute spongiform encephalopathy and one patient with anoxic encephalopathy both before and after intravenous diazepam. The records showed cyclical activity occurring about every half minute in which the EEG changes were correlated with myoclonus and cardio-respiratory changes. It is suggested that these cycles represent spontaneous cycles of arousal to which the 1/sec sharp waves are related wnd which result from the same mechanisms that produce other cyclical autonomic phenomena.


[Maturation of interhippocampal responses. Report and localization of inhibitory and excitatory synapses in the horn of Ammon of the young rabbit]. Extracellular recording from hippocampal areas of new-born rabbits up to 3 months of age was carried out to examine the responses evoked by stimulation of the contralateral alveus of CA1. In CA1, the extracellular potential fields show a positive wave whose amplitude is maximal at the somata of the pyramidal cells at any age. This positive wave has the same latency as an inhibitory postsynaptic potential induced in the same conditions and presumably represents inhibition at this level. In stratum oriens and at the top of stratum pyramidale, this positive wave is accompanied by ripples (at a frequency of about 200 c/sec) that are produced by basket cells. These ripples exhibit a very small amplitude up to 10 days of age. This positivity decreases in the depth towards the apical dendrite, reverses at the beginning of stratum radiatum and becomes rapidly negative. A second positive wave with ripples could be recorded from the layer of deep pyramidal cell somata belonging to the underlying CA3-CA4, only after 7 days. It is concluded that, in both the new-born and the adult rabbit, inhibitory synaptic action from contralateral stimulation is present on or close to the pyramidal cell somata, whereas excitatory action is located in the apical dendrites near the main branching.


A temporal component of the auditory evoked response. We studied the 75-225 msec portion of the auditory evoked response (AER) in 32 normal adults at vertex (Cz) and temporal (T3 and T4) placements referred to a balanced, noncephalic reference electrode using a monaural 1 msec click stimulus delivered every 4.7 sec at 60 dB above threshold. The tape-recorded EEG was filtered at 1-25 c/sec, and 128 individual responses were summed, sampling every 0.5 msec for 250 msec post-stimulation. The Cz AERs showed the classic vertex response, a negative peak, N1, at 100 msec, followed by a positive peak, P2, at 160-200 msec. The T3 and T4 AERs were similar to the Cz AERs from 0 to 80 msec and from 200 to 250 msec. They differed significantly from the Cz AERs from 80 to 200 msec. The difference is best explained by the hypothesis that the Cz AERs consisted of N1P2, while the T3 and T4 AERs consisted of N1P2 plus an additional superimposed component, which we called the T complex, comprising a positive peak, Ta, at 105-110 msec, and a negative peak, Tb, at 150-160 msec. By computer, the corresponding Cz and T3 or T4 AERs were normalized to equalize their amplitudes, and the former was subtracted from the latter, thus isolating the T complex. The Ta peak was found to occur 1.5 +/- 1.6 msec earlier at the electrode contralateral to stimulation, and 2.2 +/- 4.0 msec earlier at the T4 (right) electrode. Both differences were statistically significant. The T complex amplitude was greater at the electrode contralateral to stimulation and at the T4 electrode. These findings appear to resolve current controversies concerning the form of the temporal AER. While N1P2 is apparently a product of widespread areas of cortex, we conclude that the T complex is probably a product of secondary auditory cortex.


Role of biogenic amines in the effects of marijuana on EEG patterns in cats. The results of pretreatment with drug having selective action on brain biogenic amine levels on the subsequent effects of acute marijuana treatment were assessed for behavior and EEG measures. Behavioral changes and alterations in the spontaneous EEG indicated that pretreatment with drugs affecting brain levels of 5-HT was most effective in altering the dual properties of acute marijuana treatment. The present findings support the interpretation that the availability of serotonin interacts with the effects of acute marijuana treatment but do not eliminate the possible involvement of other neurotransmitters in the action of marijuana.


Dynamics of nap sleep during a 40 hour period. Following 1 baseline night, the sleep of 8 adult males in equally space 1 h naps during a 40 h period was examined. Ten additional subjects were sleep-deprived for 40 h with 1 h periods of exercise given in place of naps. One recovery night followed the 40 h period for both groups. Total sleep time and the amount of stage REM during the naps were negatively related to the circadian-temperature cycle. Stage REM frequently appeared within 10 min of stage 1 onset and the normal sequence of stages REM and 4 were altered, demonstrating that the organization of sleep within a nap is quite different from that in monophasic nocturnal sleep. Auto-correlation and cross-correlation analyses showed that the relation of sleep stages from hour to hour in normal continuous baseline sleep was altered in nap-to-nap comparison. The timing of REM onset may be controlled by a sleep-dependent ultradian clock; the clock may stop upon awakening and resume at the next sleep onset. Naps had recuperative value in terms of maintaining the normal amounts of sleep stages on the recovery night; recovery sleep for the exercise group showed typical sleep-loss effects.


Regulation of unit activity in nucleus reticularis thalami by the mesencephalic reticular formation and the frontal granular cortex. Recruiting responses and related synchronous activities appear to be mediated by thalamic inhibition originating in nucleus reticularis thalami, a structure jointly regulated by an ascending projection from mesencephalic reticular formation and a descending influence from the frontal cortex. Extracellular unit activity was recorded in the anterior nucleus reticularis thalami (RVA) during recruiting responses, augmenting responses, stimulation of the mesencephalic reticular formation (MRF), and cryogenic blockade of the inferior thalamic peduncle (ITP). During recruiting responses, RVA units responded to medial thalamic (MT) stimulation with prolonged high frequency bursts. Analysis of the post-stimulus time histograms of these responses showed tham to have the same latency, duration, incrementing character, and envelope shape as the phasic thalamic inhibitory postsynaptic potentials (IPSPs) which appear to mediate recruiting responses. Brief stimulation of the MRF, which abolishes recruiting responses and thalamic IPSPs, prevented the response of RVA units to MT stimuli, and inhibited the spontaneous discharge of these units for 20 sec or more. We propose that the desynchronizing effect of MRF activation results from the abolition of thalamic inhibition originating in RVA. Cryogenic blockade of the ITP, which abolishes recruiting responses in the thalamus and cortex, also prevented R units from responding to MT stimuli. This result suggests that the MT activates R units via a thalamo-frontocortico-R pathway and explains the long latency of R bursts and thalamic IPSPs following MT stimulation. R cells that fired prolonged bursts during recruiting responses did not respond during augmenting responses. This result suggests that separate thalamic inhibitory mechanisms are involved in these two types of synchronization.


Neuronal activities in human epileptic foci and surrounding areas. 1. Extracellular microelectrode recording was carried out in human epileptogenic cortex and in the area surrounding the focus in three cases and in the hippocampus of two cases. 2. It was impossible to identify primary "epileptic" or "pacemaker" neurons. Emphasis is placed on the interactions among the neuronal aggregates at the focus for generating the epileptic discharges in the interictal stage. The possibility of plasticity in each neuronal element in the epileptogenicity is considered. Surround inhibition was not observed. The slow wave burst was accompanied by a long repetitive rhythymic burst of unitary discharge. It was shown that the slow wave burst was a modified feature of epileptic activity. 3. Various types of activity of hippocampal neurons are described, which are, however, considered as normal discharges of those neurons.


Interaction between the activity of an epileptic focus and discrete skilled movements in rats. Sixteen male hooded rats were trained to reach into a narrow feeding tube for small food pellets. The paw movements were photoelectrically detected. An epileptic focus established by local application of 1% picrotoxin on the exposed motor cortex increased the frequency of reaching with the ipsilateral paw and impaired reaching with the contralateral paw. Interictal discharge rate of all ipsilateral foci was increased by reaching in the same way as the slow activity (less than 0.5/sec) of contralateral foci. On the other hand, fast activity (greater than 0.5/sec) of contralateral foci was decreased by reaching. Computer analysis of interictal discharge indicence during 512 msec before and after reaching onset showed that the brief facilitation of discharge (50 msec) during the actual movement was often preceded and followed by more prolonged inhibition (200 msec). The inhibition was better expressed in the contralateral hemisphere. The results are interpreted as due to changes of cortical excitability associated with reaching and to interference of the epileptic focus with the cortical elaboration of the skilled movement.


The sleep of the baboon, Papio papio, under natural conditions and in the laboratory. The sleep pattern of sixteen baboons (Papio papio) was studied under two very different conditions: (1) in a laboratory at Marseilles, the monkey being immobilized in a restraining chair in a soundproof cubicle; (2) in an African reserve, the monkey being housed in a large cage placed in its natural environment. Some very marked differences emerged. Sleep in the laboratory was longer (by 24 min) and richer in stage 3 and paradoxical sleep. In Africa, however, the sleep showed much more stage 1, was more fragmented and stages 2 and 3 and paradoxical sleep episodes were of shorter duration. Records made in Africa indicate that sleep is independent of slight environmental changes (day length, brightness of the moon, variations in temperature, calls of predators). But the comparison of the two series of results reveals the reorganization which occurs when the monkey is exposed to such different conditions. This adaptation to the environment affects, unequally, the various slow sleep stages and paradoxical sleep. In fact, the major modifications occur in stages 1 and 3 of slow sleep and in paradoxical sleep, while stage 2 appears to constitute the stable, unmodifiable nucleus of sleep.


Sleep-waking cycle in rabbits after cerebral ischemia. In rabbits experimental cerebral ischemia of 4-6 min was followed by degradation of the electroencephalographic sleep-waking cycle, as determined from 3 h afternoon records: I. Hyposomnia i.e., reduction of slow wave and paradoxical sleep lasting for about 2 days, was seen, with gradual normalization in case of survival. II. In the first postischemic days abundant 14-17 c/sec spindles appeared in the motor cortex against a low voltage desynchronized background, making the EEG of waking qualitatively different from control records. The results are discussed with reference to polygraphic studies in comatose patients, EEG phenomenology of drowsiness, and cerebral monoamines.


An instrument for stable single cell recording from pulsating human cerebral cortex. An instrument has been developed for positioning microelectrodes near neurons in the exposed pulsating cerebral cortex of man or animals. The electrode moves with the cortex and maintains a fixed relationship with the desired neuron. Stable extracellular single cell recordings have been obtained for 17.5 min from an exposed human brain that pulsated 1.5 mm at the surface.


Urinary metabolites from gamma- and beta-BHC in the mouse: chlorophenol conjugates. Metabolites produced from gamma- and beta-BHC in the mouse urine were purified and characterized. Most metabolites from both isomers were not extractable by non-polar organic solvents and were conjugates such as sulfates and glucuronides. After hydrolysis with an appropriate enzyme, the conjugates gave chlorophenols, among which 2,4,6-trichlorophenol existed most abundantly (total, about 25% in the urine metabolites). 2,4-Dichlorophenol also constituted a significant portion.


Recent status of organochlorine pesticide residues in foods in Japan. In Japan, organochlorine compounds, especially BHC, were used in large quantities. Consequently, their accumulation in the human body or in live-stock through feed crops, and the contamination of mother's and cow's milk caused serious public concern. This prompted not only initial stricter regulation of their use but the eventual prohibition of BHC and DDT use in 1971. Fortunately, the contamination of mother's and cow's milk is decreasing by these steps. The Japanese daily BHC intake was 0.57, 0.63 and 1.34 mg./kg. body weight on the average, which is higher than both in England and the United States. But the daily intake of DDT and other organochlorine compounds is the same as or less than in these countries. Two researches in 1971 to 1972 indicate that beta-BHC contained in mother's milk was 0.096 and 0.115 mg./kg. on the average.


Direct stimulation of the sinus node in man. A possible explanation for an unusual finding during premature atrial stimulation. An atypical behavior of the postextrasystolic pauses was observed in a 19-yr-old patient studied by His bundle electrography and programmed premature atrial stimulation. In the normal case there is a prolongation of the postextrasystolic pauses compared to the spontaneous cycle length allowing calculation of sinoatrial conduction time (SACT). In this case there were constant postextrasystolic pauses during the whole range of prematurity which were identical to the spontaneous cycle length. It is suggested that the catheter tip was accidentally positioned at the sinus node itself. The lengths of the spontaneous cycles, of the return cycles, and of the postreturn cycles showed no significant differences. Thus, one may assume that sinus node automaticity was not influenced in this case.


Regulation of blood cell diferentiation. The hemopoietic (blood forming) system contains pluripotent stem cells able to give rise to a variety of differentiated progeny, including erythrocytes, granulocytes, megakaryocytes, monocytes, macrophages, and possible other cell types. Although a good deal is known about cell lineage relationships in the hemopoietic system, only limited information is available about the mechanisms regulating the proliferation and differentiation of the stem cells and their progeny. An approach to this latter problem has been provided by the develoment of new techniques for the cultivation of hemopoietic cells in short-term cultures. In such cultures, the proliferation and differentiation of hemopoietic cells can be studied under controlled conditions. Two areas of investigation show particular promise: elucidation of the role of the cell surface membrane in regulation; and the possible development, through a detailed investigation of the properties of leukoviruses, of new methods for the genetic analysis of hemopoietic cells.


Effect of estrogen plasma binding on sexual differentiation of the rat fetus. alpha-Fetoprotein, the estradiol-binding plasma protein (EBP), binds estradiol but not R 2858 (11beta-methoxy-17-ethynyl-estradiol) specifically. R 2858 interferes more markedly than estradiol with the sexual differentiation of the male rat fetus following treatment of the mother during the final stages of gestation. Moreover, its tissular uptake is higher. These facts suggest that alpha-fetoprotein protects the fetus from the high circulating hormone concentrations present in the pregnant mother. The hormone, once transferred to the fetus, is retained in its vascular bed by EBP.


Fast and slow axoplasmic flow in sciatic nerve of diabetic rats. Accumulation of acetylcholinesterase (AChE) and choline acetylase (ChAc) activities proximal to a tie placed on the sciatic nerve was measured in control, untreated diabetic, and insulin-treated diabetic rats. In the diabetic animals AChE accumulation was reduced by about 20% and ChAc accumulation by about 40%. Insulin treatment eliminated the impairment. It remains an open question whether these reversible functional changes in rat have any counterpart in the diabetic neuropathy of man.


[Investigations on the influence of Rh immunoglobulin prophylaxis on the immune response to postpartum rubella vaccination (author's transl)]. A preliminary investigation showed that patients with rubella HHT antibody titres of 1:8 or greater did not show a significant rise in the antibody titre following rubella vaccination. The rubella antibody titre was determined in 651 obstetric patients. Of these, 43 (6.6%) had no significant antibodies to rubella (HHT less than 1:8) and were included in the present investigation. Patients in Group A received 0.5 ml. of the rubella vaccine Meruvax on the fifth postpartum day. Patients of Group B(Rh negative and Rh positive) received 250 mug anti-D in a 16% gammaglobulin solution intra-muscularly 48 hours postpartum and the rubella vaccination 3 days later. Three weeks following the rubella vaccination the mean geometric rubella antibody titre had risen +/- 1 Standard deviation to 19.6 +/- 7.7 in Group A (17 patients) and to 18.0 +/- 6.3 in Group B (12 patients). Six weeks following the rubella vaccinations Group A (19 patients) showed titres of 61.7 +/- 2.9 and Group B (14 patients) showed titres of 70.0 +/- 2.6. There was no statistically significant difference (greater than 0.5). The conversion rate in both groups was 100%. Patients can therefore be vaccinated against rubella in the postpartum period even though they will receive a concomitant prophylaxis with Rh immunoglobulin.


Studies on vertebrate kidney. V. PAS-positive lipids in kidneys of fishes from different habitats. Five fish species living in different habitats, i.e. fresh water, estuarine and marine, were studied for the distribution of PAS-positive materia in various regions of the kidney, 10 minutes' oxidation with 0-5 per cent. HIO4 being employed prior to treatment with Schiff's reagent. PAS-positive material was detected in different sites of the kidney, i.e. brush border of proximal tubules, proximal tubule cells' cytoplasm, distal tubule cells' cytoplasm, glomerulus, basal cell border of proximal tubules and the interstitial cells. Of these sites, the brush border of the proximal tubule of Scoliodon sorrakowah showed the presence of PAS-positive lipids. Elsewhere the PAS-positive reaction was due to carbohydrates. Free aldehyde groups were absent. In Tilapia mossambica and Labeo rohita, PAS staining was enhanced after chloroform-methanol extraction, particularly in the brush border of the proximal tubule. The significance of these findings is discussed.


Studies of vertebrate kidney. VI. Histochemical localization of phospholipids in kidneys of fishes from different habitats. Six fish species living in different habitats such as fresh water, estuarine, and marine were studied for the distribution of phospholipids in various regions of the kidney. Well known histochemical methods, including those for choline-containing phospholipids were employed. The distribution pattern of phospholipids in different parts of the kidney such as the brush border, basal border and cytoplasmic granules of the proximal tubule cells, the cytoplasmic granules of the distal tubule cells, glomeruli and interstitial cells is given. Choline-containing phospholipids are present in the brush border of the proximal tubules of all the species studied and in the basal border of some. In the discussion an attempt at correlation of this histochemical pattern with the excretory functions and osmoregulation in these fishes is given.


[Clinical experiences with an external heparinoid in high concentration of effective substance]. 396 in-patients and 117 out-patients were treated of study the activity and tolerance of a highly dosed organo-heparinoid in the forms of ointment and gel. Either form was tolerated well; a local allergic cutaneous reaction was seen only in 1 patient. The efficiency of the product, assessed after a classification by points, can be denoted as good for every indication tested.


[Treatment of burns].

[Treatment of burns]. Bibliographic data on pathophysiology, therapeutic methods and mortality rate of burn disease are compared with own clinical experiences with a standardized therapy. Within a period of 18 month 35 patients with burn disease of special therapeutic problems were treated besides the overall number of patients in the operative intensive care unit of the Klinikum Steglitz. As special devices for patients with burns still do not exist provisional solutions had to be developed. It is referred to the close connexion between prognosis and mortality, on the one hand, and the age of the patients and the degree of burns, on the other hand. Whereas shock treatment, medicamentous therapy and nutrition are largely standardized, there are strongly differing opinions on local treatment and surgical wound care. The routine application of gammaglobulines, antibiotics and heparin controversial, too.


[Differential diagnosis of congenital lipidoses by lipid analyses of body fluids, biopsy and autopsy tissue]. 1. Presentation of the commomly used procedures for the extraction and separation of total lipids, glycolipids and phosholipids from fresh and formalin-fixed organs tissues (brain, liver, spleen, kidney) as well as from serum, CSF and urine. II. Description of the qualitative and quantitative analysis of individual lipid fractions (glycolipids, gangliosides, phospholipids, neutral lipids) by thin-layer chromatograhy and photodensitometry. III. Results of investigations performed on biopsy material, autopsy material, serum and urine in the following diseases: 1. Infantile, juvenile and adult Gaucher's disease: accumulation of glucocerebroside in liver and spleen. 2. Infantile and adult Niemann-Pick disease: accumulation of sphingomyelin in liver, spleen, kidney and lung. 3. Fabry's disease: increased urinary excretion of trihexosyl-ceramide and dihexosyl-ceramide. 4. Infantile and adult metachromatic leukodystrophy: accumulation of sulfatides in the central and peripheral nervous system and kidney, increased urinary excretion of sulfatides. 5. Austin's variant of metachromatic leukodystrophy: besides an increase of sulfatides in the white matter of brain accumulation of glycolipids in the cerebral cortex. 6. Tay-Sachs disease (GM2-gangliosidosis): cerebral accumulation of GM2-ganglioside and trihexosylceramide (enzyme variant B), additional visceral accumulation (liver, spleen, kidney) of tetrahexosyl-ceramide = globoside (enzyme variant 0). 7. Infantile generalized GM1-gangliosidosis: cerebral (and visceral) accumulation of GM1-ganglioside and tetrahexosyl-ceramide. 8. Late infantile GM1-gangliosidosis: Cerebral accumulation of GM1-ganlioside and tetrahexosylceramide. 9. GM3-gangliosidosis (lactosyl-ceramidosis): neuronal accumulation of lactosyl-ceramide,  GM2-ganglioside and GM3-ganglioside. 10. Refsum's disease: demonstration of phytanic acid esters of cholesterol in serum.


A community-oriented center for severely and profoundly retarded children. A center established three years ago to provide local residential services for severely and profoundly retarded children has developed programs that give the residents a high level of interaction with direct-care staff members and with the community. At the Elisabeth Ludeman Center, children live in groups of eight or fewer in four-bedroom homes served by neighborhood centers. In various community-oriented programs, high school students are trained for jobs as half-time technicians. Ludeman residents are placed half days in a community development center, part-time services are provided at Ludeman for clients of other agencies, and community members come to the center as volunteer instructors or as users of Ludeman facilities. A key part of the center's program is an audiovisual service used extensively for inservice training and staff communication.


Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis. The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.


Oxidase (donor: oxygen oxidoreductase) activity by peroxidase and alpha2-macroglobulin interaction. The interaction between peroxidase (donor: hydrogenperoxide oxidoreductase, EC 1.11.1.7) and human alpha2-macroglobulin has been studied by employing starch gel electrophoresis and spectrophotometric assay analysis.


A new type of inversion of a human Y chromosome. Using chromosome banding techniques, a phenotypically normal male was found to have an abnormal banding pattern of the Y chromosome. By the constitutive heterochromatin staining method, a darkly stained band was located on the short arm and the proximal region of the long arm. The quinacrine staining method also showed a similar abnormal banding pattern: a brightly fluorescing band was seen on the short arm and the proximal region of the long arm. By the conventional Giemsa staining method, however, no specific morphological abnormality was detected in the aberrant Y. On detailed karyotype analyses no recognizable abnormality of banding patterns of any other chromosome was found aside from the abnormal Y. The abnormality was determined to be a complex inversion of the Y chromosome, which is described as 46,X,inv(Y)(pter leads to p11::q11 leads to q12::cen::q12 leads to qter).


The 'intrinsic adjuvanticity' and immunogenicity of trinitrophenylated lipopolysaccharide. One thymus-independent immunogen, trinitrophenylated lipopolysaccharide (TNP-LPS) has been studied in vivo and in vitro in C3H/He and C3H/HeJ strains of mice. C3H/HeJ mice have been shown to be low responders, and C3H/He mice high responders to this immunogen. This 'low responder' status of C3H/HeJ mice has been demonstrated to be consequent to an intrinsic defect present at least at the level of B cells. It was demonstrated that high responder cells or 'in vivo milieu' could not restore this deficit to C3H/HeJ cells. It is proposed that the adjuvanticity, mitogenicity and polyclonal activating capacity of LPS are all fundamental to its property of acting as a thymus-independent carrier for the TNP determinant. This observation is discussed from the point of view that the T-cell independence for an antigen cannot derive solely from its multivalency but must depend upon the intrinsic adjuvanticity of that molecule.


Anti-thymocyte serum may enhance or suppress the response to the same antigenic determinant. Treatment of BALB/c mice with heterologous anti-thymocyte serum (ATS) increases their response to the C-polysaccharide of a rough strain of Pneumococcus, R36A, but suppresses their response to a vaccine of the whole organism. Phosphorylcholine is the major antigenic determinant for both the C-polysaccharide and vaccine. ATS also suppresses the response to phosphorylcholine when it is coupled to various protein carriers. In so far as was examined, ATS had no effect on adherent cells or the capability of adherent cells and antigen to stimulate an antibody response in non-adherent cells. The contrasting effects of the antiserum can best be accounted for by assuming that it acts on at least two different populations of thymic-derived cells.


Secretory component of the guinea-pig.

Secretory component of the guinea-pig. Free secretory component (FSC) was purified from guinea-pig milk by gel-filtration and immunoabsorption on anti-SC antibodies. Guinea-pig FSC cross-reacted with antisera to human FSC, and the reverse. Guinea-pig and human FSC resembled each other in molecular size, electrophoretic mobility and heterogeneity, as well as by the existence of antigenic determinants restricted to the unassociated form of the molecule. Guinea-pig FSC associates in vitro with guinea-pig IgM. Distribution of disulphide links is required to set free guinea-pig FSC from secretory IgA.


Common antigenic structures of HL-A antigens. VI. Common antigenic determinants located on the 33,000 Dalton alloantigenic fragment portion of papain-solubilized HL-A molecules. HL-A 33,000 Dalton fragments were isolated from three papain-solubilized HL-A preparations carrying different HL-A allospecificity by the dissociation induced in the reaction with rabbit antibodies to HL-A 11,000-Dalton fragment (i.e. human beta2-microglobulin). By assaying the binding activity with rabbit antisera against papain-solubilized HL-A molecules and against each of the HL-A component fragments and also with a battery of HL-A alloantisera, the 'antibody-dissociated' 33,000-Dalton fragments were shown to retain not only the HL-A alloantigenic determinants but also the HL-A common antigenic determinants that are native to the 33,000-Dalton fragment portion of undissociated HL-A molecules and to be devoid of the cryptic HL-A common antigenic determinants that are found on HL-A 33,000-Dalton fragments obtained by dissociation with chemical reagents including acid. However, on exposure to acid, the antibody-dissociated 33,000-Dalton fragments were found to lose their HL-A alloantigenic and common antigenic characteristics and gain the cryptic HL-A common antigenic determinants. The greater part of the HL-A common antigenic determinants found here appears to be primate cross-reacting determinants.


Multiple antigenic sites on an eicosapeptide. I. Precipitin studies in the goat. Purified peptide 105-124, an antigenic determinant from the carboxy terminus ribonuclease, was found to form an immune precipitate with antibody to that region prepared by affinity chromatography from goat hyperimmune antiserum to reduced carboxymethylated ribonuclease (CM-RNase). Cm-rnase also gave an immune precipitate with the antibody. Purified antibody to another region of similar size (40-61) did not form a precipitate with CM-RNase but did co-precipitate in the presence of antibody to peptide 105-124 and CM-RNase. The precipitin reaction between antibody to peptide 105-124 and CM-RNase was inhibited by two synthetic derivatives, peptides 118-124 and ala114-RNase 114-124. Stoichiometry of the precipitin reactions of antibody to 105-124 with CM-RNase or peptide 105-124 suggested an antigen valency of three or more. Consistent with this both peptides 105-124 and ala114-RNase 114-124 elicited immediate cutaneous reactions but 118-124 did not. These findings suggest that the eicosapeptide 105-124 is multivalent since at least three antibodies can react simultaneously with it.


Hapten and carrier specificities of cellular and humoral responses to highly substituted dinitrophenyl-human gamma-globulins in guinea-pigs. Delayed hypersensitivity (DH) and antibody reactions to the carrier and to the hapten have been studied in guinea-pigs immunized with different doses of highly substituted dinitrophenyl-human gamma-globulins (DNP56-HGG) in Freund's complete adjuvant (FCA). The results confirm that DH reactions are specific for the carrier while antibody-mediated reactions are specific for the hapten in the early stages of the immune response. Later in the response, however, DH reactions to the hapten as well as a transient humoral reaction to the carrier could be observed. T cells specific for the hapten and B cells specific for the carrier are therefore triggered after a single infection of a highly substituted hapten-carrier conjugate in FCA. Their regulatory functions in the immune response to hapten-carrier conjugates are discussed.


Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants. The polysaccharide antigen preparations from serotype a and serotype d strains of Streptococcus mutans contained both a serotype-specific antigenic determinant and a common a-d antigenic determinant, as demonstrated by agar gel diffusion studies and a quantitative cross-precipitin assay. The chromatographically purified antigens were isolated by a method which depended on their serological specificity to determine if these two antigenic determinants were located on the same molecule. The a and d polysaccharides were recovered from specific antigen-antibody complexes and characterized with respect to their immunological specificity and chemical composition. Agar gel diffusion tests demonstrated that, in both the a and d preparations, the serotype-specific antigenic determinant and the common a-d antigenic determinant were present in one molecule.


Biological activity of fragments derived from the extracellular slime glycolipoprotein of Pseudomonas aeruginosa. Glycolipoprotein, obtained from the extracellular slime layer of Pseudomonas aeruginosa, was purfied and subjected to chemical and enzymatic treatment in an attempt to assign certain of its biological activities to chemical moieties comprising the glycolipoprotein molecule. Treatment of the glycolipoprotein with phenol, although removing all detectable protein, yielded a fragment capable to exerting the biological activities associated with the untreated glycolipoprotein (leucopenia, lethality, inhibition of phagocytosis, antigenic specificity). Acetic acid treatment resulted in a fragment composed mainly of carbohydrate and a small amount of protein, but no detectable lipid. This fragment was devoid of leucopenic and lethal activity, but retained antigenic specificity and the ability to inhibit phagocytosis. The fragments release from the glycolipoprotein after treatment with phage 2-depolymerase were low-molecular-weight products and were devoid of the biological activities associate with the glycolipoprotein.


Effect of the quality of the lipopolysaccharide on mouse virulence of Salmonella enteritidis. The cell wall O antigen is known to affect mouse virulence of Salmonella. We have shown earlier that S. typhimurium expressing somatic antigen 9,12 is less virulent than its 4,12 sister transductants in mice after intraperitoneal inoculation, suggesting that the 4,12-type O antigen is connected with high virulence in mice. In this report we show, in accord with this suggestion, that when the naturally occurring O-9,12 S. enteritidis is made O-4,12 by transduction, its virulence is increased. The difference between O-9,12 and O-4,12 sister transductants is highly significant, with P less than 0.001.


Purification and serological characterization of a type-specific antigen of Streptococcus equisimilis. A microtiter complement fixation (CF) procedure was developed for use in detection of antibodies in sera of swine and rabbits vaccinated with Streptococcus equisimilis. Crude ultrasonic as well as acid-extracted preparations contained CF antigen, but the ultrasonic procedure resulted in a higher yield of active antigen. Evidence of serotype specificity with varying degrees of cross reactivity was detected with the CF procedure when representative strains of four different serotypes of S. equisimilis were compared by using their respective unadsorbed antisera. Fractionation of crude sonic extract or acid extract by centrifugation, precipitation with ammonium sulfate, and chromatography on carboxymethyl cellulose yielded a purified, type-specific antigen that reacted only with the homologous antiserum in the CF test and formed a single band by immunodiffusion. Complement-fixing antibodies in immune swine sera were predominately immunoglobulin G.


Immunologically specific production of interferon in cultures of rabbit blood lymphocytes: association with in vitro tests for cell-mediated immunity. Lymphocytes of animals with delayed hypersensitivity produce mediators of cellular immunity when challenged in vitro with specific antigen. Among these are macrophage migration inhibitory factor (MIF) and interferon (IF). Nonspecific mitogens also induce the production of these lymphokines. In the following study leukocytes and column-purified lymphocytes of the same peripheral blood sample from tuberculin (purified protein derivatives [PPD])-sensitive rabbits were concurrently cultured in medium alone or with PPD. Supernatants of 1- and 4-day lymphocyte cultures were assayed for MIF. Supernatants of 1-, 2- to 4- and 5- to 7-day leukocyte cultures were assayed for IF by inhibition of cytopathic effect of vesicular stomatitis virus on rabbit kidney cultures. In the presence of PPD, normal lymphocytes did not produce MIF, but lymphocytes from sensitized animals did (8/8 animals), after 1 and 4 days of culture. Leukocytes from normal animals produced little or no IF when cultured with or without PPD. Leukocytes from sensitized animals cultured in medium alone produced little IF. However, when cultured with PPD they produced significant amounts of IF on day-1 (6/8 animals) and day-2 to day-4 (4/8) animals. There was no correlation between relative amounts of MIF and IF produced by cultures of respective cells from individual animals. Rabbit IF produced or released in vitro appeared in significant and maximum amounts by 24 h coincident with the time release of significant amounts of another mediator of cellular immunity, MIF.


Cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans. Crossed immunoelectrophoresis with absorption in situ was used to distinguish the cytoplasmic antigens unique to the mycelial or yeast phase of Candida albicans from cytoplasmic antigens shared by both phases. The soluble cytoplasmic extracts of each growth phase had at least six distinct antigenic constituents not shared by the other phase. This technique is recommended for the analysis of closely related antigenic complexes.


Defense mechanisms against bovine herpesvirus: relationship of virus-host cell events to susceptibility to antibody-complement cell lysis. The interaction of infectious bovine rhinotracheitis virus and susceptible host cells was examined to determine whether an infected cell could be destroyed by humoral immune mechanisms before or after the transmission of virus to susceptible adjacent cells. Viral antigens were detectable on cell membranes at 6 h postinfection, but cells were not susceptible to antibody-complement lysis until 10 h postinfection. Intracellular infectious virus was also detectable at 10 h postinfection, and transmission to adjacent cells by the intracellular route began at this time. Extracellular virus was not detectable until 12 to 13 h postinfection. By the continual addition of antibody and complement, virus dissemination could be reduced more than 50-fold. These results support the hypothesis that the humoral immune mechanism may be involved in the recovery from herpesvirus infections.


Antigen-binding cells in human fetal liver. Antigen-binding cells (ABC) could be detected regularly by autoradiography among haemic cells in the liver of human fetuses ranging in age from 8 to 24 weeks. For radioiodine-labeled thyroglobulin, the antigen mainly used in these studies, counts of ABC ranged from 5.0 to 24.3 per 1,000 cells scanned. There was a trend for counts of ABC in liver to be highest at 10-12 weeks of fetal life. Binding of labeled thyroglobulin was inhibited by excess unlabeled thyroglobulin, but not by other protein antigens. Artifacts due to binding of antigen to normoblasts, which comprised 90% of the haemic cells in fetal lines, and to cells with 'sticky' surfaces were excluded as far as possible. There was no response by fetal liver cells to phytohaemagglutinin. Although there was only minimal inhibition of binding by anti-immunoglobulin sera of known potency, the ABC in human fetal liver were assumed to correspond to immunoglobulin-bearing precursors of B cells described by others in the liver of the fetal mouse.


Inhibition of sensitization of T-cells by alpha-fetoprotein. A two-phase model of allograft immunity was studied. In the first phase, specific immune T-cells were generated by incubation of responder cells with mitomycin-C treated allogeneic stimulator cells of the same H-2 type as mouse mastocytoma tumor cells. In the second step, the ability of the sensitized cells to kill Cr51-labelled P-815 mastocytoma cells was assayed. Alpha-fetoprotein (AFP) was shown to inhibit the generation of immune cytotoxic T-cells at low concentrations (1-100 ng/ml) when added at the beginning of the first phase. When added at the end of the first phase or in the second (killing) phase, AFP was found to have no significant effect on cytotoxicity, indicating that it did not inhibit the killer T-cell once it was generated.


Secondary specific immune response in vitro to MSV tumor cells. The interactions which occur between antigenic tumor cells and normal or immune lymphoid cells in a 3-day in vitro culture, have been studied with a murine sarcoma virus (MSV)-induced tumor. The 3H-thymidine incorporation of lymphoma cells growing in suspension, and the radioactive-chromium release of freshly sampled lymphoma cells regularly added to the culture, have been compared to determine the part played by immune lymphoid cells in cytolysis and cytostasis of the tumor-cell population. The cytolytic activity increases in the culture from day 0 to day 3. It is due, predominantly, to T-cells, and remains specific to antigens shared by MSV tumors and related lymphomas. This activity would be difficult to detect unless freshly sampled ascitic cells were used as targets, since the lymphoma cells spontaneously lose a part of their sensitivity to immune cytolysis during in vitro culture. The method used in the present experiments is a secondary chromium release test (SCRT), which measures the invitro secondary stimulation of cytotoxic T-lymphocytes (CTL) by tumor cells. In the absence of stimulatory cells, the CTL activity would have rapidly fallen in vitro. The cytostatic activity also increases during the 3 days in vitro, in parallel to the cytolytic activity: it is due to non-T-cells and remains mainly non-specific. The significance of these data for the interpretation of invitro demonstrated cell-mediated anti-tumor immune reactions is briefly discussed, as well as their relevance in the in vivo role of immune CTL.


Antigenic specificities of the cell-mediated anti-tumor reactions in the MSV system studied by the secondary chromium release test. The SCRT and the inhibition test have been used to determine the specificity of cell surfacr antigens reacting with anti-M-MSV cytolytic lymphocytes. The method provides very sensitive and specific results. Some discrepancies exist between the results of SCRT and those obtained by the in vitro inhibition by tumor cells of cell-mediated immune cytolysis. The main point is that allogeneic cells are stimulatory in SCRT, whereas they are not reactive in the inhibition test. Several hypotheses are discussed to explain these discrepancies. In all the experiments, a strong secondary stimulation of cytotoxic lymphocytes was obtained in vitro when FMRGi (+) cells were used as stimulators, whatever the nature and the histocompatibility antigens of these cells. This suggests that an antigen of the "FMRGi system" is regularly involved in the cell-mediated anti-MSV reaction. However, other antigenic specificities of different natures are probably also concerned due to the antigenic complexity of these tumors.


Selective and non-selective lymphocytotoxicity in human melanoma: observation on the effect of long-term culture and fetal bovine serum on target-cell sensitivity to lymphocytes. In vitro cell mediated cytotoxicity (CMC) assays have been conducted in a human melanoma system with a 3H-proline retention technique. Melanoma target cells from long-term cultures ("cell lines") are found to exhibit increased susceptibility for lymphocyte cytotoxicity in comparison to the same target cells from short-term culture. The higher sensitivity of the "cell line" derived target cells is seen with lymphocytes, irrespective of diagnosis of the donor. In parallel experiments with the target cells grown in medium supplemented with fetal calf serum (FCS) and AB+ human serum (from a normal male doner), the melanoma target cells grown with FCS do not show any enhanced cytotoxicity, suggesting no causal relationship of such enhanced sensitivity of "cell line"-derived target cells to "heterologous melanoma antigens" that might have been acquired by the target cells following the use of FCS in tissue culture. In controlled assays of in vitro CMC, lymphocytes from melanoma patients (14/44) exhibited selective cytotoxicity (destruction of only one target-cell type) against the melanoma target cells, whereas only 3/97 control lymphocytes (other malignancies and normal donors) showed such melanoma-selective cytotoxicity. This difference is statistically significant at p less than 0.001. Non-selective cytotoxicity (destruction of two or more unrelated target cell types) was seen with lymphocytes from 9/44 melanoma patients, 13/51 patients with other malignancies and 8/46 normal donors. No correlation of selective cytotoxicity could be established with donors' age, sex, stage of disease, therapy or history of blood transfusion. Such a correlation may emerge as our series becomes larger. Despite the lack of any correlation between selective cytotoxicity and disease status, our study reaffirms the existence of selective cytotoxicity by melanoma patients' lymphocytes against melanoma target cells.


Androgens and estrogens in the plasma and prostatic tissue of normal dogs and dogs with benign prostatic hypertrophy. The endogenous concentrations of certain androgens and estrogens have been quantified in the plasma and prostatic tissue from normal dogs and dogs with benign prostatic hypertrophy (BPH). In the tissue and plasma from both dog populations, the level of estrone was higher than that of estradiol. The concentration of testosterone in the tissue and plasma of both normal dogs and dogs exceeded the concentration of either estrogen. Prostatic levels of dihydrotestosterone greatly exceeded that of testosterone. A comparison of normal dogs and dogs with BPH revealed that in both the plasma and prostatic tissue the concentrations of estradiol and estrone were significantly elevated (P less tha 0.05) in the BPH dogs. Plasma and prostatic levels of testosterone did not differ between the two groups; however, the concentration of dihydrotestosterone in hyperplastic glands was approximately 4 times greater than normal.


3alpha-Reduction of dihydrotestosterone in human normal and hypertrophic prostatic tissues. The activity of 3alpha-reduction of dihydrotestosterone was located in both the particulate and the soluble fractions of human prostatic tissues. Activity of 3alpha-reduction was higher than that of 3beta-reduction under the incubation conditions employed; therefore, the main reductive metabolic pathway of dihydrotestosterone seemed to be the formation of 5alpha-androstane-3alpha,17beta-diol in human prostatic tissues. There was no significant difference between normal and hypertrophic prostatic tissues when the activity of 3alpha-reduction of dihydrostertosterone was compared on a wet weight basis.


Quantitation of adenovirus soluble antigens by crossed immunoelectrophoresis: application to serological characterization of mutants. The two-dimensional immunoelectrophoresis technique was applied to adenovirus type 2 soluble antigens. Electrophoresis of adenovirus-infected HeLa cell extracts in adenovirus antibody-containing agarose gel permitted us to assay for hexon, assembled penton, free penton base and free fiber simultaneously in one single experiment. This technique was routinely used to characterize the adenovirus type 2 temperature-sensitive mutants.


A method for demonstration of synaptic densities in CNS. The synaptic densities in rat CNS were demonstrated using an alcohol solution of uranyl acetate at pH 5.0-6.0 of unosmicated material. A number of procedures were performed to establish the chemical characteristics of the structures stained by the method described.


Alkali burns of the rabbit cornea. II. A histochemical study of glycosaminoglycans. In alkali burned rabbit cornea the stainability of glycosaminoglycans in cold microtome setions was investigated. Staining by Alcian blue in 3% acetic acid, Alcian blue in various MgCl2 concentration and toluidine blue (pH 4.5) was employed. From the 1st to the 4th experimental day the intensity of reactions was decreased. This is most probably due to an increased hydration of the corneal stroma. On the 7th day hydration was markedly suppressed and reached nearly the normal level. In this time interval a decreased stainability of glycosaminoglycans was seen accompanied by a complete loss of staining in the marginal zone. On the 14th day the stainability in the traumatized area began to restore and in the marginal zone appeared. On the 32nd day the staining intensity of both areas was normalised, however when lower concentrations of MgCl2 were used; in the presence of higher concentrations of MgCl2 the decreased staining intensity persisted and points to a lower sulfatation of glycosaminoglycans. This was particularly remarkable in the area bordering the injured zone. This decrease runs parallel to the increased activities of acid glycosidases (especially of acid beta-galactosidase) which were reported previously.


Direct spectrophotometric observation of intracellular nitro-blue tetrazolium and its formazan by multiple internal reflectance infrared spectroscopy. A technique is described which permits the direct, infrared (IR) spectrophotometric observation of peripheral leukocytes by utilizing multiple internal reflectance (MIR) spectroscopy on zinc selenide prisms. The IR spectra of nitro-blue tetrazolium (NBT) and its derivative, nitro-blue diformazan (NBF), are described and examined and these spectra are compared with those of the intracellular NBT and NBF yielding several peaks which may prove to be analytically useful. Additionally, the presence of unreduced NBT in leukocytes is demonstrated, which microscopic and chemical "NBT tests" have not previously done.


The cellular distribution of aldolase isozymes in rat kidney and brain determined in tissue sections by the immuno-histochemical method. The use of the immuno-histochemical method permits the localization of aldolase isozymes in tissue sections. Upon incubating a section with a monomer-specific antiserum, isozymes containing that monomer remain in the section, whereas other cytoplasmic enzymes diffuse out of the section. If soluble antigen is added subsequently, it is bound by the tissue-bound antibody. These antibody fixed aldolases can then be stained by the use of a tetrazolium test linked to substrate hydrolysis. In this way it was demonstrated that isozymes of aldolase containing mostly the A monomer are predominantly localized in the distal tubules, the collecting tubules, the vessels and capillaries of the kidney, the ganglia, the Purkinje cells, the neurons, the white matter and the chorioid plexus of the brain. Aldolase containing mostly B-monomers were found in the proximal tubules. Aldolase isozymes particularly rich in C-monomers were seen in the nervus opticus, the pia mater, the vessels of cerebrum and the molecular layer of the cortex cerebelli.


Myoepithelial cells in human thymus: staining, polarization and fluorescence microscopic studies. Myoid cells in human thymus were studied around the turn of the century, and alterations in patients with cardiovascular disease were reported. It was therefore deemed of interest to reinvestigate these long forgotten cells. The configurational staining, polarization and fluorescence microscopic properties of smooth myofibrils in thymic epithelial cells were identical with those of classical myoepithelial cells, smooth muscle, and A bands of striated muscle. Cross-striated myoid cells could not be found in thymus of children. Myoepithelial cells formed a layer at the surface of thymic lobules; others were scattered throughout the cortex and medulla. In addition, the medulla contained seemingly hypertrophic myoepithelial cells. Hassall's corpuscles consisted of layers of myoepithelial cells. Hammar (1905) regarded epithelial cells with smooth myofibrils in human thymus as equivalents of the cross-striated myoid cells in lower vertebrates. The myoepithelial cells observed in this study are apparently identical with the smooth myoid cells of early anatomists; the hypertrophic myoepithelial cells correspond to the unicellular Hassall's corpuscles. The functions of these cells are not yet clear; the wide variations from case to case in the same age group indicate that the myoepithelial cells are affected by a variety of diseases.


The alkaline hydrolysis of nucleic acid for removal of RNA associated fluorescence in phenantridium related flow through cytofluorometry. Flow through microfluorometry of phenantridium and fluoresceinisothiocyanate stained HeLa cells showed the absence of extranuclear RNA related fluorescence after prolonged hydrolysis of cells in ethanolic barium hydroxide. DNA distribution were as good as in RNAse treated samples. Distribution pattern of cellular protein/DNA ratios yielded greater resolution power than those after RNAse digestion. The cellular protein content has not been affected. The ethanolic hydrolysis has the advantage to be of less expense and to allow cell conservations for further measurements with other well preserving fixatives. Thus, application of glutaraldehyde has been proved to be valuable.


[Isolated amyloid of the larynx (author's transl)]. A laryngeal amyloid in a 45-year-old man was removed from the sinus of Morgagni via laryngofissure at the Ear, Nose, and Throat Department of the University of Teheran. Aspects of the amyloid are discussed with regard to the literature.


Aminoglycoside 3'-phosphotransferase III, a new phosphotransferase. Resistance mechanism. The aminoglycoside phosphotransferase of Pseudomonas aeruginosa 21-75 was purified by affinity chromatography using dibekacin-Sephadex 4B or lividomycin A-Sepharose 4B followed by DEAE Sephadex A-50 chromatography. It had activities of both the known aminoglycoside 3'-phosphotransferases I and II, and transferred phosphate from ATP to the 3'-hydroxyl group of kanamycin A, ribostamycin and butirosin A and 5-hydroxyl group of lividomycin A. This enzyme was designated aminoglycoside 3'-phosphotransferase III. It showed strong substrate inhibition by kanamycin A and ribostamycin when their concentration exceeded 6 muM. Purification and characterization of this enzyme are reported.


Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores. The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.


Antigenic determinants of bovine myelin encephalitogenic protein recognized by rabbit antibody to myelin encephalitogenic protein. The antigenic determinants of bovine myelin encephalitogenic protein were investigated by quantitative complement fixation and hapten inhibition using rabbit anti-monkey protein and anti-bovine protein and purified and characterized fragments of bovine protein. The two regions of bovine encephalitogenic protein containing determinants were sequences 1 to 43 and 90 to 170. One rabbit antiserum recognized a determinant(s) probably in residues 15 to 40 while for another rabbit antiserum the determinant of 1 to 43 resided in 1 to 20. The determinant(s) of residues 90 to 170 involved the region around the tryptophan at position 116. Fragment 44-89, which contains the major encephalitogenic determinant for the rabbit, was virtually devoid of any reactivity with the rabbit anti-encephalitogenic protein. It appears that portions of the protein other than the encephalitogenic site are responsible for stimulation of bone marrow-derived cells and antibody production. In demonstrating selected regions of the protein as sites for antigenic determinants, the present immunochemical studies also suggest that the protein might have a more folded conformational alignment than previously suspected.


Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein. Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein.


Proteoglycans in primate arteries. I. Ultrastructural localization and distribution in the intima. Proteoglycans were identified and localized histochemically and ultrastructurally in normal and hyperplastic arterial intimas in nonhuman primates (Macaca nemestrina). These regions were consistently more alcianophilic than the adjacent medial layers and this alcianophilia was absent after treatment with glycosaminoglycan-degradative enzymes. Ultrastructurally, the intimal intercellular matrix consisted of numerous, irregularly shaped, 200-500-A diameter granules possessing 30--60-A diameter filamentous projections, and these granules were dispersed between collagen and elastic fibers. The granules exhibited a marked affinity for ruthenium red and were interconnected via their filamentous projections. The ruthenium red-positive granules were intimately associated with the plasma membrane of intimal smooth muscle cells and attached to collagen fibrils and elastic fibers. The matrix granules were completely removed after testicular hyaluronidase or chondroitinase ABC digestion but only partially removed after leech hyaluronidase treatment. These results suggest that the matrix granules contain some hyaluronic acid and one or more isomers of chondroitin sulfate. In addition to the large ruthenium red-positive matrix granules, a smaller class of ruthenium red-positive granule (100--200-A diameter) was present within the basement membranes beneath the endothelium and surrounding the smooth muscle cells. Ruthenium red also exhibited an affinity for the surface coat of the smooth muscle cells. The potential importance of proteoglycans in arterial intimal hyperplasia is discussed.


Opaque deposits on gap junction membranes after glutaraldehyde-calcium fixation. When cloned hybrid cells (A/Bm-5) were grown to confluence and fixed in glutaraldehyde-calcium, electron-opaque deposits were observed on the cytoplasmic faces of plasma membrane. Deposits were most abundant at gap junctions. Deposits were often precisely paired, cell-to-cell, across the gap junctional membranes, and these paired deposits were frequently equivalent in size. This relationship was most often observed on long profiles of gap junctions, in contrast to the asymmetric distribution of larger deposits commonly found on short junctional profiles. Deposits were present with or without heavy metal staining but did not appear when calcium was omitted from the fixative. Fixation at room temperature yielded more and larger deposits than fixation at 0 degrees C. The significance of these observations is discussed with regard to the possible binding of calcium at fixed membrane sites or the precipitation of calcium by anions produced by enzymes located at the gap junction.


Perturbation of mammalian cell division. II. Studies on the isolation and characterization of human mini segregant cells. A method is described for the isolation, according to size, of mini segregants produced by the abnormal cleavage of reversibly arrested mitotic HeLa cells. Many of these mini segregants contain small amounts of DNA, as judged by Feulgen staining and chromosome analysis. After fusion with mitotic HeLa cells, the interphase chromosomes of the mini segregants are seen as either monovalent or bivalent prematurely condensed chromosomes (PCC), some of which are damaged. A proportion of isolated mini segregants synthesize DNA, RNA and protein. Fusion of mini segregants with interphase HeLa cells gives rise to cells with 'hybrid' karyotypes.


Morphological heterogeneity of HeLa cell mitochondria visualized by a modified diaminobenzidine staining technique. The diaminobenzidine (DAB) technique for the ultrastructural localization of sites of cytochrome c oxidase activity in animal tissues has been adapted to the visualization of mitochondria in animal cells growing in culture. The modified technique allows the staining of mitochondria in all cells in coverslip preparatins for light microscopy. Electron microscopy of thin sections of material treated by this method has revealed that all mitochondrial profiles within a cell (and only these) are stained and they exhibit a well preserved size and internal structure. Coverslip cultures of synchronized and unsynchronized HeLa (F-315) cells stained with the DAB reaction were examined under oil immersion. In the majority of the cells, mitochondria were recognized as discrete bodies in the thinner peripheral portion of the cytoplasm. This observation indicates that in a large proportion of HeLa F-315 cells, at least under the growth conditions used here, the mitochondrial complement is dividied into distinct organelles. This examination also revealed a considerable morphological heterogeneity of mitochondria, which exhibited an ovoid or short rod-like or, less frequently, long filamentous shape, with some evidence of branching. The variability in mitochondrial morphology appeared to be far more prounced between different cells than within individual cells; this cellular heterogeneity was not related in any obvious way to cell-cycle-dependent changes.


Further data on a histochemical reaction as applied to the thin-layer chromatography of sterols. A method for the characterization of sterols on silica gel G layers is proposed. After the chromatograms have been sprayed with a permanganate-sulphuric acid oxidative reagent and the reaction has been terminated with sodium hydrogen sulphite, the plates are sprayed with the colour-developing reagent (an acid solution of alcian blue or toluidine blue). The plates are also viewed under UV radiation (254 and 366 nm). Only 3 of the 28 sterol samples assayed (spot content 8 mug) did not show positive reactions after oxidation, which suggests that this step can be used as a "universal" detection method for sterols. After staining the plate, several sterols are shown to be easily differentiated from one another. The exposure of the plates to UV radiation assists characterization. In general, the reaction exhibits satisfactory sensitivity for the qualitative and differentiating detection of sterols; it is also rapid and easy to carry out.


The two-dimensional characterization of amino acids in actinomycin hydrolysates. Actinomycins, which belong to a family of chromopeptide antibiotics, consist of a hetero-tricyclic chromophore to which are attached two pentapeptide chains either identical or different in amino acid sequence. The classification of existing actinomycins and the identification of new actinomycins are dependent on the characterization and quantitation of the amino acids present in the peptide chains. A simple, fast and highly reliable two-dimensional separation technique employing electrophoresis in a formic acid/acetic acid buffer (pH 1.9) on thin layers of microcrystalline cellulose followed by thin-layer chromatography with an n-butanol:water:glacial acetic acid (50:40:10) solvent system in a direction perpendicular to the electrophoresis was developed to separate the amino acids contained in hydrolysates of the actinomycins. The separated amino acids were identified by two parameters, the chromatographic Rf value and the electrophoretic mobility calculated relative to some standard migrating compound.


Characterizaiton of two populations of human lymphocytes bearing easily detectable surface immunoglobulin. Two separate lymphocyte populations, each bearing easily detectable surface immunoglobulin, have been detected in human peripheral blood. The first, B cells, has surface determinants that are stable at 37degreeC, but are removed by pronase and regenerate in culture. The cells are nylon adherent and have a receptor for C3, and studies with unit gravity sedimentation indicate they are mostly small lymphocytes. B cells comprise 9.5% of the total lymphocytes, with the normal range from 3-16%. As many or more lymphocytes lack membrane-incorporated Ig determinants but have an Fc receptor that binds IgG1 and IgG3 in normal serum maximally at 4degreeC. This receptor for cytophilic IgG is removed by pronase but not by trypsin. The second population has been named L lymphocytes because of membrane-labile IgG determinants. L cells do not adhere to nylon, do not form rosettes with sheep erythrocytes sensitized with antibody and mouse complement, and are larger than small lymphocytes. These lymphocytes with cold-reactive Fc receptors for serum IgG do not form E-rosettes or respond to phytohemaggutinin. Since L cells do not have surface markers of T and B lymphocytes, it is likely that they comprise a separate population.


Immune responses to the cleavage-associated neoantigens of fibrinogen in man. Identification and characterization of humoral antibodies specific for cleavage fragments. Cleavage of human fibrinogen and fibrin by plasmin is associated with modification of native antigenic expression and the exposure of cleavage-associated neoantigenic sites on the derivative molecular fragments. In this study, the presence of humoral antibodies in man to cleavage-associated neoantigens has been demonstrated by primary antigen binding radioimmunochemical assays. Specific binding of radioiodinated human fibrinogen D fragment by serum immunoglobulins was demonstrated in 52 of 59 random normal human sera and was independent of immunoglobulin concentration. Binding was mediated by F (ab)2 fragments of IgG, and specificity for neoantigens was indicated by the capacity of the D fragment but not native fibrinogen to competitively inhibit the antibody. The population distribution of antibody to these cleavage-associated neoantigens indicated the presence of a major group of individuals (77%) with a mean antigen binding capacity of 11.8 pmol/ml serum. Two minor populations with : (a) low or undetectable binding capacities (less than 6.0 pmol/ml serum) and (b) exhibiting markedly elevated binding capacities (less 18.0 pmol/ml serum) were delineated. Independent of these features, sera could also be readily separated into two groups that differed with respect to relative antibody affinity. The antibodies in most sera exhibited marked heterogeneity of binding affinity, whereas a small group of sera contained antibodies exhibiting relative homogeneity of binding affinity. Specific antibody was rather equally distributed between the major immunoglobulin classes, and in no serum was the antibody restricted to a single immunoglobulin class. Antibodies capable of binding fibrinogen fragments X, Y, and D and fibrin D fragment were detected in most sera. The quantity of antibody differed for different fragments with X greater than Y congruent to D greater than fibrin D. The presence of antibody capable of binding any single fragment was statistically correlated with the presence of antibody capable of binding other cleavage fragments. No antibody to the E fragment was detected. Antibody to cleavage fragments was not demonstrable in sera containing fibrinogen or fibrin cleavage fragments. Demonstration of this humoral immune response to the products of the fibrinolytic systems provides a new interface between the coagulation and immune system.


Human antibody-dependent cellular cytotoxicity. Isolation and identification of a subpopulation of peripheral blood lymphocytes which kill antibody-coated autologous target cells. Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.


Mustard meal in dairy rations.

Mustard meal in dairy rations. Consumption of 0% mustard meal and 15% soybean meal, 7.5% mustard meal and 7.5% soybean meal, or 15% mustard meal and 0% soybean meal rations did not differ in palatability studies with 10 group-fed lactating cows when the mustard meal was treated with 3% caustic soda. Order of preference was for 0, 7.5, and 15% mustard meal rations when mustard meal was untreated. Twelve lactating cows were in each of two lactation trials to compare the three rations of untreated mustard meal. Milk, milk fat, and solids-not-fat, and milk protein did not differ for either trial. Protein-bound iodine of plasma for all cows were within the normal range. Three cows were placed on each of the three rations and received a minimum of 9 kg per day for 6 mo preparturition to determine goitrogenic effects. All cows gave birth to normal, vigorous calves. Limited organoleptic evaluations of milk indicated that untreated mustard meal may impart a detrimental flavor to milk, but a taste panel could not differentiate between milk from cows on the three rations of treated mustard meal. Twenty-one male and 43 female Holstein claves received either 0, 10, or 20% mustard meal starter rations from birth to 3 mo of age. Growth, feed consumption, or plasma protein-bound iodine did not differ.


Friendship and disaffiliation among the skid row population. Personal relationships of skid row men have been described in terms of disaffiliation and replacemtent of friends. Analysis of social relationships of Philadelphia skid row residents upheld the theory of replacement. There was, however, a decline in the number of friends reported after the age of 70, which generally coincided with 20 years residence in the area. The loss of relationships was more closely associated with length of residence in skid row than with age. Qualitative measures of personal relationships indicated no major association between sociability and length of residence in skid row but a small association with advancing age. The proportions of friendless men desiring either friends of casual acquaintances were not related to either age or length of residence. These findings fail to uphold the disaffiliation hypothesis of aging and run counter to Vesliard's postulate of loss of need for meaningful personal relationships with increased exposure to the life of an outcast.


Histochemical localization of glutathione in tissues. A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.


The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. I. Theoretical kinetics and experiments with films containing isolated deoxyribonucleic acid. Theoretical considerations on the expected kinetics of the course of the Feulgen-Schiff reaction show that the leveling off of the first part of the Feulgen hydrolysis curve can be explained by the gradual conversion of deoxyribonucleic acid (DNA) to apurinic acid (APA). In addition, depolymerization of DNA caused by the acid used for hydrolysis can account for the decline after a maximum is reached in this curve. With the aid of polyacrylamide model films containing DNA, a detailed study was made both of the process of purine liberation which results in the formation of APA and of the depolymerization processes which cause losses of stainable material. The liberation of purine bases was analyzed by ultraviolet absorbance measurements and by gel chromatography of the neutralized hydrolysing acid. APA concentration was monitored by following the loss of ultraviolet absorbance associated with the purine losses. The depolymerization process was followed by phosphorus determinations. The experimental results were found to be in accordance with the kinetics expected from the theoretical model.


The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. II. Investigations on films containing condensed or swollen chicken erythrocyte nuclei. As models for different states of chromatin compactness, nuclei from chicken erythrocytes were isolated and either osmotically swollen or kept as condensed as possible. Both types of nuclei were then fixed and incorporated into polyacrylamide films. Hydrolysis with 5 N HCl and staining with Schiff's reagent of these model films were studied using several parameters. The phosphate content of the films was analyzed as a parameter for the depolymerization losses and the staining with Schiff's reagent as a parameter for the apurinic acid (APA) content. The loss of ultraviolet absorbance from the films and the accumulation of ultraviolet absorbing substances in the hydrolyzing acid were monitored as parameters for the progress of hydrolysis. Conversion of the generated aldehyde groups to APA-Schiff chromophore is shown to take place with the same stoichiometry for both types of nuclei as well as for DNA in model films. It is further shown that the nuclei- and DNA-films are suitable models for investigating the influence of chromatin compactness on the course of the Feulgen-Schiff reaction. For the most compact form of chromatin studied, a very high reduction in staining intensity of up to 40% could be demonstrated after certain normally applied hydrolysis times. This is due primarily to a decrease with a factor of 2.3 of the depurination rate constants of these models (from 0.030/min to 0.013/min). Therefore prolonged hydrolysis periods are required to obtain the same APA concentrations, but then depolymerization processes cause losses of nuclear material. The differences in depurination rates could be explained by a decrease in [H3O]+ in the neighborhood of the purine-sugar linkages, caused by the presence of fixed positive charges form the protein components of the chromatin. These findings may explain the cytophotometrically determined differences in chromophore yield of 10-20% found in the nuclei of cells with different states of compactness of their chromatin. The descending part of the Feulgen hydrolysis curve represents the depolymerization of APA and loss by diffusion of the reaction products. In the Appendix, cytophotometric data of cells have been analyzed to show that this part of the hydrolysis curve may be used to estimate the acid stability of chromatin complexes. The depurination and depolymerization rates found closely correspond with the data obtained from the model films.


Atomic absorption spectrophotometry applied to photographic densitometry. For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.


Application of 125I-labelled soluble proteins in the histoautoradiographic detection of antigen and antibodies in the spleen of rabbits during primary immune response. An autoradiographic method for detecting soluble antigen (chicken serum albumin, CSA) and specific antibodies in the spleen of rabbits during a primary immune response is described. The method consists of incubating sections from the spleen with 125I-labelled IgG2 anti CSA (for demonstration of antigen) or with 125I-labelled antigen (for demonstration of specific antibodies). This treatment of histological sections combines the advantages and principles of the immunofluorescence technique with the possibility of evaluating the exact localization of the proteins by light microscopy in preparations stained with haematoxylin or methyl green-pyronin. The sensitivity of detection is very high: both antigen and antibodies could be demonstrated in the spleen follicles for as long as 42 days after the primary intravenous injection.


Effect of progesterone on collagen breakdown and tissue collagenolytic activity in the involuting rat uterus. Progestational agents were studied for their effects on collagenolytic activity and loss of collagen and wet weight from the involuting post-partum rat uterus. Administration of very large doses of progesterone (80-150 mg/day) significantly retarded uterine involution and loss of collagen. This was accompanied by a significant reduction in uterine collagenolytic activity. By 72 h post partum, uteri of rats treated with 150 mg progesterone/day had wet weights 30% above, collagen 85% above, and collagenolytic activity 45% below, those of the control uteri. Similar effects were produced by 17alpha-acetoxy-6alpha-methylprogesterone at the same dosage levels. However, the progestational agent 6-chloro-17alpha-acetoxypregna-4,6-diene-3,20-dione acetate had no effect in this system.


Induction of antiphosphorylcholine antibody formation by anti-idiotypic antibodies. Anti-idiotypic antibodies have been used to mimic antigen in the mouse antiphosphorylcholine response in order to investigate the induction of precursors of antibody-forming cells. We have shown that interaction of anti-idiotype antibody with receptor antibody molecules induces the formation of antibodies that are specific for phosphorylcholine and carry the idiotypic determinants. This induction is dependent on the recognition of carrier determinants on the anti-idiotype antibody by helper T cells. We conclude that receptor antibody molecules on the surface of the precursors of antibody-forming cells deliver the antigenic signal for the induction of these cells.


The pinocytic rate of activated macrophages. Peritoneal macrophages from mice injected 4 days previously with Brewer's thioglycollate medium have a pinocytic rate, in culture, of 190 ng horseradish peroxidase (HRP)/100 mug cell protein/h, compared to the rate of resident peritoneal cells of 53 ng HRP/100 mug cell protein/h. Mice injected with endotoxin or with only certain of the components of the Brewer's medium show an intermediate level of stimulation. The rate of unstimulated, endotoxin-stimulated, or thioglycollate-stimulated cells shows little change over several days in culture. The pinocytic rate of thioglycollate-stimulated cells can, however, be further increased by exposure of concanavalin A. Although cells may show transient increases in their pinocytic rate in many situations, a sustained increase in pinocytic rate is a sign of the "activated" state of macrophages.


Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. II. Determination of frequency and characteristics of idiotypic T and B lymphocytes in normal rats using direct visualization. Anti-idiotypic antibodies made against the antigen-binding receptors of T lymphocytes for a given antigen (Ag-B locus antigens in rats) can be shown to react with IgG antibodies of the same antigen-binding reactivity. Using such anti-idiotypic antibodies, normal Lewis T lymphocytes of B and T type can be visualized by the use of anti-(Lewis-anti-DA) antibodies. Visualization was made possible by the use of direct fluorescent antibody tests or by autoradiography. Using the first technique and naked eye observations 6.2% of normal Lewis T lymphocytes expressed idiotypic markers signifying anti-DA reactivity, whereas anti-DA-reactive B lymphocytes as measured by this approach was in the order of 1.1%. Autoradiography was purified normal Lewis T lymphocytes gave similar figures. When comparing the intensity of fluorescence at the single cell level using quantitative cytofluorometry anti-idiotypic antibodies reactive with T lymphocytes gave a similar degree of intensity as was obtained using anti-Ig antibodies against B lymphocytes.