Specificity of DNA-associated nuclear antigens in HeLa cells and distribution during the cell cycle. Chromosomal nonhistone protein:DNA complexes prepared from synchronized HeLa cells were used to immunize white rabbits. The antisera reacted specifically in complement fixation tests with chromatins isolated from HeLa cells and not with those from a number of other human and animal tissues. Specificity to this cell type was also demonstrated by immunocytochemical reaction. Both immunochemical tests revealed that the specific antigens are continuously present throughout the cell cycle. The immunological activity was dependent upon chromosomal nonhistone protein(s) being bound to DNA. Our findings are consistent with these chromatin antigens being stable nuclear components [complexes of chromosomal non-histone protein(s) with DNA] characteristic of cellular differentiation.


In vitro synthesis of murine pre-alpha-fetoprotein. Murine alpha-fetoprotein was synthesized in a wheat germ cell-free system in the presence of radioactive amino acids under the direction of alpha-fetoprotein messenger RNA isolated from mouse yolk sacs. The radiolabeled alpha-fetoprotein was isolated by immunoabsorption, and the amino acid residues at the NH2 terminus were determined by radioactive sequencing techniques. In a comparison to the NH2-terminal sequence of circulating alpha-fetoprotein, the in vitro-synthesized alpha-fetoprotein was found to contain an extra peptide 20 amino acids long linked at the NH2 terminus, the sequence of which is: (formula: see text). The molecular size, the hydrophobic nature, and the other properties of the peptide are consistent with the "leader" or "signal" piece found in the precursors of many other secretory proteins. This suggests that alpha-fetoprotein, the synthesis of which is limited primarily to fetal development, is produced in the form of the precursor as are secretory proteins in the adult tissues.


Rat alpha-macrofetoprotein (acute-phase alpha 2-macroglobulin) during hepatocarcinogenesis. Rat alpha-macrofetoprotein (AMF) and alpha-fetoprotein (AFP) are secreted by the fetal liver and become elevated in serum during hepatocarcinogenesis and in animals bearing hepatocellular carcinomas. It has been suggested that these fetal plasma proteins may be influenced by related control mechanisms. The experiments presented herein examined the early responses of these plasma proteins during hepatocarcinogenesis using the hepatocarcinogens acetylaminofluorene and diethylnitrosamine. Under these conditions, AFP serum concentrations were elevated within a few days of exposure to acetylaminofluorene, whereas AMF serum concentrations remained essentially normal. AFP became elevated after a number of weeks of exposure to diethylnitrosamine. In either regimen, AMF became elevated only later when large primary hepatocellular carcinomas were found. The time of appearance of AMF after transfer of an AFP-secreting Morris hepatoma indicated that AMF was elevated only in animals with extremely large, necrotic tumors. Thus, it appears that elevation of serum AFP resulted from either exposure to hepatocarcinogens or production by hepatocellular carcinomas, but that the elevations of serum AMF levels resulted from inflammatory injury or necrosis of tumor tissues.


Additive effects of bleomycin and neocarzinostatin on degradation of DNA, inhibition of DNA polymerase beta, and cell growth. The interactions of the two antitumor protein antibiotics, neocarzinostatin (NCS) and bleomycin (BLM), were studied on subcellular and cellular levels. BLM and NCS were found to remove thymine from double-stranded DNA. Combination experiments using BLM and NCS together in an assay with isolated DNA revealed an additive effect in splitting. Under limiting concentration conditions, BLM and NCS induce alkali-labile sites in DNA without a subsequent cleavage of the chain. After transfer of BLM- or NCS-treated DNA into an alkaline solution, strand scissions occur. Combination of BLM and NCS results in an additive DNA-cleaving effect, which indicates that the splitting reactions initiated by BLM or NCS are not influenced if the two antibiotics are applied in combination. The DNA polymerase beta is inhibited by BLM (at higher concentrations) and by NCS in a competitive way with respect to DNA. The inhibition constant of BLM and NCS in a combination experiment was found to be the result of the sum of the inhibition constants of BLM and NCS. Using L5178Y mouse lymphoma cells, it was found that cells incubated with both BLM and NCS show "unbalanced growth." The dose-response curves from BLM and NCS have identical slopes; they are characteristic for compounds which selectively inhibit DNA synthesis. By use of isobolograms, it could also be clearly shown that BLM and NCS interact additively.


Enhancement of bleomycin activity against Lewis lung tumors in mice by local hyperthermia. The cytotoxicity of the drug bleomycin in vitro has previously been shown to be enhanced by hyperthermia. This report demonstrates in vivo a more than additive interaction between local tumor hyperthermia (43 degrees, 60 min) and bleomycin (15 mg/kg s.c.) against s.c.-implanted Lewis lung carcinomas in mice. Local hyperthermia was produced by the application of 2450-MHz microwaves to the region of the tumor without induction of significant whole-body hyperthermia. The combined drug and heat treatments were applied to tumors on Days 4, 7, and 10 following implantation. The response of the tumors to simultaneous treatment was a 17-day growth delay compared with controls, whereas the local hyperthermia and bleomycin individually resulted in only 3- and 4-day growth delays, respectively. If the two treatments were given either 4 or 24 hr apart only an additive effect on growth delay was observed.


Effects of systemically administered bleomycin or adriamycin with local hyperthermia on primary tumor and lung metastases. Exposure of cells in tissue culture to bleomycin or Adriamycin during 43 degrees C hyperthermia increased cytotoxicity dramatically compared to exposure at 37 degrees C. This study was designed to test whether this interaction was useful in tumor-bearing animals. C3H mice bearing the KHT tumor were treated with bleomycin (7 or 15 mg/kg) or with Adriamycin (2.5 or 5 mg/kg) with or without local heating of the tumor to 43 degrees C for 30 minutes by 13.56 MHz radiofrequency fields. The effects were assessed by growth delay (mean tumor diameter doubling time) and cure rate. In separate experiments, BALB/c mice bearing EMT6 tumors were treated identically, but tumors were excised 2 hours after treatment and tumor cell survival was assayed by colony formation. Antitumor effects of systemic bleomycin were potentiated by local hyperthermia. The two modalities had to be administered close together in time to observe the potentiation, suggesting a true interaction. There was a "threshold" for bleomycin potentiation in vivo between 42 degrees C and 43 degrees C, just as observed in tissue culture experiments. The antitumor activity of Adriamycin was not potentiated in vivo in these tumor systems except in cell survival experiments at doses higher than those compatible with survival of the host. The toxicity of drug combined with heat was greater than that of either modality alone. Hyperthermia did not adversely affect the incidence or severity of spontaneous lung metastases from KHT tumors. In fact, groups treated with heat and bleomycin had less severe lung metastases than groups treated with bleomycin alone. We conclude that local heating of tumors may be a useful adjunct to systemic bleomycin therapy. In vivo potentiation of Adriamycin by heat, however, could not be demonstrated in these tumor systems.


Evaluation of cyclocytidine in children with advanced acute leukemia and solid tumors. Cyclocytidine, a slow-release form of cytosine arabinoside, was evaluated in 69 children with advanced acute leukemia and solid tumors. One child with acute lymphocytic leukemia attained a complete remission. This child had received intrathecal cytosine arabinoside prior to the cyclocytidine. Eighteen of the 31 patients with acute lymphocytic leukemia/acute undifferentiated leukemia who did not respond received two or more courses of the drug. There were no responses in 15 children with acute myelogenous leukemia, in 11 children with neuroblastoma, or in 11 children with various solid tumors of childhood. A dose of 600 ng/m2/day for 10 consecutive days is tolerated in children.


A new method for degradation of the protein part of glycoproteins: isolation of the carbohydrate chains of asialofetuin. A new method has been developed for degrading the protein part of several glycoproteins, whilst leaving the carbohydrate portion virtually intact apart from partial degradation at the reducing end. The method is based upon stabilization of the glycosidic linkages of the sugar residues by trifluoroacetyl groups and subsequent cleavage of the peptide bonds by transamidation. The two reactions are carried out in a mixture of trifluoroacetic anhydride and trifluoroacetic acid. After O- and N-detrifluoroacetylation, the carbohydrate portion can be isolated and re-N-acetylated. The applicability of the method is demonstrated by the isolation from asialofetuin of the carbohydrate chains that are attached by N- and O-glycosyl links.


Post-extrasystolic left ventricular peak pressure with and without left ventricular failure. 18 patients without valvular pathology, coronary artery disease, or idiopathic hypertrophic subaortic stenosis were haemodynamically and angiographically investigated in order to analyse the effects of a ventricular extrasystolic beat upon the post-extrasystolic left ventricular peak pressure. In eight normal patients (group I), the post-extrasystolic peak pressure (P.ES.P.P.) was lower than that of the pre-extrasystolic beat; in 10 patients with symptoms of left ventricular failure (group II) the P.ES.P.P. significantly increased. The reasons are: 1) cardiac origin: stroke volume increased more in group II; 2) arterial origin. a) aortic compliance was lower in group II (this is probably related to the older age of patients in group II), and by decrease in end-diastolic aortic pressure was smaller in group II. Part of this arterial effect (2b) may probably be explained from the fact that post-extrasystolic compensatory pauses are equal in both groups, but the decay time of arterial pressure during diastole (assuming an exponential decay) is larger in group II. At the same age and with the identical aortic compliance only the two factors 1 and 2b play a part in the changes in P.ES.P.P.


Relations between fibrillar centres and nucleolus-associated chromatin in Ehrlich tumour cells. By means of diaminobenzidine staining method, the relations between fibrillar centres and nucleolus-associated chromatin are analyzed in Ehrlich tumour cell nucleoli. There is a continuity between fibrillar centres and condensed intra-nucleolar chromatin. The meaning of these connections is discussed.


[Application to the study of connections in the CNS of the retrograde axonal transport of an iron-dextran complex]. The retrograde axonal transport of an iron-dextran complex was observed in neurons of the substantia nigra and of the intralaminar nuclei of the thalamus, after previous injection into the striatum. The histochemical demonstration of iron is simple and rapid, and can be combined with that of horseradish peroxidase, under precise conditions in the sequence of reactions. The iron-dextran complex revealed to be a valuable material for neuronal connectivity studies in the central nervous system.


[Identification of actin microfilaments in the neurosecretory axons of rat neural lobe]. Incubation of rat neural lobes with heavy meromyosin (HMM) after prolonged glycerination, induced characteristic arrowhead decoration of a number of microfilaments at different levels of the neurosecretory axons. In non terminal sections of axons the labelled microfilaments showed preferential relationships with microtubules in addition to occasional contacts with the axolemma and various axonal organelles. In axonal endings, they were mainly associated to microvesicles and appeared to be anchored on the axolemma facing the perivascular space at the level of membranous densifications.


Correlation of function and morphology of neonatal rat and embryonic chick cultured cardiac and vascular muscle cells. To develop morphological criteria which can be applied systematically for the identification of isolated cardiac and vascular muscle cells in mammalian and avian primary cultures, we have correlated structural and staining properties with excitability, contraction, and norepinephrine sensitivity of isolated muscle cells. The primary cultures of cardiac and vascular muscle contained muscle cells and nonmuscle cells. The muscle cells could be clearly identified by action potentials, contractility, and Masson's trichrome stain characteristics, similar to those of cells from intact source heart and blood vessels. Furthermore, the muscle cells were highly responsive to norepinephrine, showing unequivocal increases in contraction frequency. The sensitivity to norepinephrine was found to be very high (ED50 = 2.3 X 10(-9) M) Phase-contrast observation was sufficient to identify muscle cells only when those cells were contracting. There were no unequivocal morphological characteristics that distinguished between quiescent muscle cells and nonmuscle cells in the absence of histochemical staining. Ultrastructural examination by scanning electron microscopy failed to distinguish between muscle and nonmuscle cells. Histological staining was, therefore, the only reliable nonfunctional identification process that separated muscle cells from nonmuscle cells. Primary cultures, containing nonmuscle as well as muscle cells, are an important experimental preparation because the cellular heterogeneity probably minimizes muscle cell loss of function and phenotypic changes. The correlation we have established between cell staining and function will facilitate exploration of single cell properties, which together constitute hearts and blood vessels.


Equivalence between international units and mass units of alpha-foetoprotein. Report of a collaborative study. A study aimed at establishing a correspondence between the international unitage which presently defines the activity of the WHO alpha-foetoprotein (AFP) standard and mass units of AFP was carried out in collaboration with 11 laboratories from seven countries. Various methods of assay, including radial immunodiffusion, enzymo-immunoassay, Laurell rocket immunoelectrophoresis, radioimmunoassay and radio-rocket immunoelectrophoresis, were used. The mean value of 11 individual estimates indicated that one international unit (I.U.) was worth 1.17 ng of AFP (90% confidence interval = 1.06-1.28). Since there was no objective basis on which to evaluate the relative purity of AFPs from various sources, it was felt that each purified AFP should be given equal weight; accordingly when only results from the six laboratories which purified their reference material were taken into account, one I.U. was found to be worth on average 1.21 ng of AFP (90% confidence interval = 1.02-1.43). It is proposed that the official correspondence between international and mass units be based upon that equivalence. It is interesting to compare these results with those of the first collaborative study in 1974; at that time, one I.U. was found to be worth 1.19 ng of AFP on average which is very close to the 1.21 ng figure. The 90% confidence interval was 0.93-1.52. Although there is better agreement between laboratories in the second study, the distribution of individual equivalences is still quite heterogenous, and it is therefore recommended that the present international unitage continue to be used.


Cellular cooperation in lymphocyte activation. II. Cooperative response of human peripheral T and B lymphocytes to rabbit anti-human beta 2-microglobulin. In the present study we attempted to clarify the effects of anti-beta 2-microglobulin (a-beta 2m) on lymphocyte activation. Neither a-beta 2m IgG fraction nor F(ab')2 had a mitogenic effect on either highly purified T or B lymphocytes alone, while their mitogenic effect was observed when T and B lymphocytes were appropriately reconstituted. When T lymphocytes were reconstituted with mitomycin C (MMC) treated B lymphocytes, a negligible decrease in the response to a-beta 2m was observed compared to the response of an untreated mixture to a-beta 2m. On the other hand, when B lymphocytes were reconstituted with MMC-treated T lymphocytes, the response was markedly diminished. It was found, moreover, that the response of T lymphocytes separated by a semipermeable membrane from MMC-treated B lymphocytes was not enhanced, while a mixture of T and MMC-treated B lymphocytes in the same chamber showed a marked response. These results lead to the conclusion that the cells responding to a-beta 2m are mainly T lymphocytes whose response is strongly enhanced by B lymphocytes, and that for the mitogenic effect of a-beta 2m direct cell-to-cell interaction between T and B lymphocytes is necessary.


Inhibition of human neutrophil migration by aggregated gammaglobulin. Heat-aggregated human gammaglobulin has been shown to inhibit the random migration of human neutrophils in serum-containing medium. This inhibition was not due to metabolic exhaustion or deactivation of the cells, since migration in the presence of aggregated gammaglobulin and casein as a chemotactic stimulus was not inhibited. The inhibition of migration was not mediated by a negative chemotactic gradient produced as a result of complement activation, and could be demonstrated in complement-depleted serum. Sera obtained from patients with rheumatoid arthritis with evidence of circulating immune complexes were able to significantly inhibit neutrophil migration, indicating that this phenomenon may be a useful means for the detection of circulating immune complexes. It is suggested that aggregated gammaglobulin or immune complexes can inhibit the chemokinetic effect of serum on neutrophils by a reversible interaction with the neutrophil surface, and that this inhibition could contribute to the accumulation of neutrophils at sites of immune complex deposition in vivo.


Cephradine-induced interstitial nephritis. A patient developed acute interstitial nephritis following cephradine administration. The clinical illness preceding the interstitial nephritis and the eosinophilia in peripheral blood suggested an allergic reaction. Immunological studies demonstrated an allergy to cephradine and penicillins which was IgG2-mediated. It is probable, therefore, that allergy to cephradine caused the renal lesion from which there was full clinical and biochemical recovery.


Osteosarcoma complicating Paget's disease of the spine with neurologic complications. An osteosarcoma with neurologic manifestations complicated Paget's disease of the lumbar spine in a 63-year-old man. Initially without demonstrable metastasis, the patient was treated by decompressive laminectomy followed by radiation and adjuvant chemotherapy (COMPADRI-1 regimen). While such treatment may not be curative, there are significant palliative benefits.


Characteristics of renal handling of human immunoglobulin light chain by the perfused rat kidney. 1. The renal handling of purified human immunoglobulin light chain has been studied with an isolated perfused rat kidney preparation. 2. Human immunoglobulin light chain was freely filtered and largely reabsorbed. Fractional reabsorption was characteristic for each of four light chains and varied between 56% and 86%. No renal tubular maximum for human light chain was obtained. 3. Light chains at concentrations up to 10 times those seen in human myeloma were without effect on glomerular filtration rate or sodium and potassium reabsorption in experiments lasting up to 2 h. 4. Filtered and reabsorbed light chain returned ultimately to the perfusion medium, indicating a unique property of the tubular handling of this protein. None of the inhibitors tested (ouabain, frusemide, acetazolamide, probenicid) influenced light chain reabsorption. 5. The results are taken to indicate that light chain reaches the site of the transport enzyme, Na+,K+-dependent ATPase, at concentrations which vary with the nature of the light chain. This may provide a mechanism for renal damage in patients with myeloma, after prolonged exposure.


Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells. Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.


Radioimmunoassay of alpha-fetoprotein in children with primary intracranial tumors. Serum alpha-fetoprotein (AFP) was measured in 10 cases of primary brain tumors in children (4 cases of medulloblastoma, 4 cases of germ cell tumor and 2 cases of astrocytoma). As a result, elevation in AFP was observed only in a case of embryonal carcinoma that showed partial mixture of germinoma. The absence of AFP elevation in 3 other cases of pure germinoma (atypical teratoma; pinealoma) agrees with reports which describe that, in the germ cell tumor of the gonads, a rise in AFP is not observed in pure seminoma but is found in embryonal carcinoma and endodermal sinus tumor (yolk sac tumor). The fluctuations in AFP in serum and cerebrospinal fluid are considered to be of significance in the diagnosis and treatment of primary intracranial malignant germ cell tumors.


Proconvulsant, convulsant and other actions of the D- and L-stereoisomers of allylglycine in the photosensitive baboon, Papio papio. The effects of the intravenous or intracerebroventricular injection of the stereoisomers, and the racemic mixture, of allylglycine (2-amino-pent-4-enoic acid) have been studied in baboons, Papio papio, with photosensitive epilepsy. Enhancement of the natural syndrome of photosensitivy epilepsy is seen 1-12 h (maximally at 3-8 h) after L-allyglycine, 100 mg/kg, intravenously, or D,L-allyglycine, 200 mg/kg, intravenously. Such enhancement is seen with a slower onset, and to a lesser, and more variable, extent after D-allyglycine, 500-750 mg/kg, intravenously. Brief focal or generalised seizures occurred (in the absence of intermittent photic stimulation) after L-allyglycine, 150-200 mg/kg, intravenously. This effect is similar to that previously observed after D,L-allyglycine, 300-400 mg/kg. D-Allyglycine, 780 mg/kg, intravenously produced episodes of vertical nystagmus with increased extensor motor tone, but no 'spontaneous' seizures. Intracerebroventricular injection of L-allylglycine, D-allyglycine or D,L-allyglycine, 100 mg in 1 ml saline, did not modify the natural syndrome of photosensitive epilepsy. D-Allylglycine, or D,L-allyglycine, 100 mg intracerebroventricularly, after 1-2 h gave rise to a syndrome with vomiting, sustained vertical nystagmus, and intermittent extensor spasms. The results are interpreted in terms of regional differences in the metabolism of the two isomers to active compounds that can inhibit glutamic acid decarboxylase. D-Allylglycine is active only at the brain stem and cerebellum because D-amino acid oxidase is largely confined to these brain areas.


Coupling between neuronal activity and focal blood flow in experimental seizures. Local blood flow, ECoG and single cortical neurone activity were recorded simultaneously from single microelectrodes in 17 cats. Seizures were induced by repeated intravenous injections of pentylenetetrazol (PTZ, 10-20 mg/kg) or by local application of 1 M Na-penicillin. Seven to 20 sec after appearance of burst activity in cortical neurones and ECoG, focal flow increased up to 300% of control. The extent of this flow increase was significantly correlated with the change in firing rate of the neurones. With cessation of seizure activity the flow returned to or below control values. Forty to 70 mg/kg PTZ caused status epilepticus with high voltage rhythmic discharges lasting 30 min-2 h. In 3 cats with status, the flow decreased below control despite persisting seizures, indicative of uncoupling between activity and flow. The delayed coupling between activity and flow during drug-induced seizures indicates a metabolic mediator. Uncoupling observed in cases with long lasting seizures may be due to brain oedema following increased permeability of the blood-brain barrier.


The influence of cortisone on EEG and seizure activity in the baboon Papio papio. The ability of cortisone to modify EEG and seizure activity was investigated in the baboon, Papio papio. Acute intramuscular doses (0.5-4 mg/kg) caused a dose-dependent increase in seizure response to a flashing light stimulus. This increase in seizure response was apparent in both seizure duration and the spread of convulsive activity. Along with enhancement of seizures, cortisone was found to cause marked changes in the EEG, ranging from the appearance of interictal paroxysmal activity to alterations in spectral characteristics of the wave forms. Increases in slow waves appeared concomitant with a decrease in fast activity in the 18-25 c/sec range. Since previous studies have indicated that seizure proclivity in the Papio papio is maximal at the time of the day when cortisol excretion rates peak, these findings lend further evidence to the idea that corticosteroids may be involved in the thythmic variation of seizure activity in the baboon.


EEG, pattern-evoked potentials and nerve conduction velocity in a family with adrenoleucodystrophy. In a family with 8 children one case of adrenoleucodystrophy (ALD), verified by autopsy, 2 cases with clinical signs of ALD and four other clinically healthy subjects with pathological ACTH tests were found. As an initial sign in the EEG, temporo-occipital slowing occurred. Further EEG studies showed spread of the primary local abnormalities, indicating diffuse brain dysfunction during progression of the disease. Terminal EEG flattening may be characteristic for late stages of the disease. No correlation could be established between lateralization, paroxysms and actual stage of the disease. Abnormal EEGs occurred not only in clinically and subclinically affected male patients, but also in a girl, who might be a carrier of ALD. Visual evoked potentials showed an increased latency of the P2 wave in the clinically, and in one subclinically, affected subjects. Motor nerve conduction velocity was reduced in only 2 patients. Antidromic sensory nerve conduction velocity of the median nerve was normal in all patients.


Hippocampal theta in rats under urethane: generators and phase relations. Recordings were made from the directly visualized dorsal hippocampus of urethane-anesthetized rats with silver ball and tungsten microelectrodes. Theta of about 5 c/sec was elicited by reticular stimulation and was located in two broad strata in and subjacent to CA1. A superficial generator yielded 200-250 microV theta from the alveus, stratum oriens and stratum pyramidale; a null-theta and phase-reversal point was located in stratum radiatum; a deep generator yielded 300-500 microV theta in stratum lacunosum-moleculare of the hippocampus and distal stratum moleculare of the dentate gyrus, with maximum amplitude near the fissure. No theta was found in CA3. Cross-correlation analyses between generators yielded coefficients on the order of -0.80 while within a generator--especially the deep one--coefficients could reach +0.95. Phase analyses showed the two generators were about 180 degrees out of phase, with the deep generator leading. Septotemporal movement along the upper generator yielded no phase shifts; similar movement along the deep generator showed slight lead ing in septal portions. Subiculofimbrial movement in both generators showed a complex pattern of phase shifts: generally, subicular sites led fimbrial sites, but an inversion of 10 degrees-20 degrees occurred in the millimeter anterior to the center of the hippocampus. Anatomical, phase and other datal led to consideration of a possible retrohippocampal role in supplementing the well-known pacemaker in the medial septum.


Visual evoked potentials in monkeys.

Visual evoked potentials in monkeys. Visual evoked potentials (VEPs) were recorded from 2 cortical sites in stump-tailed macaques. VEPs recorded from striate cortex were basically consistent between animals (especially at low light intensity), remained remarkably stable over time, and compared favorably to VEPs reported by other investigators. We concluded that the VEP recorded from the striate cortex of day-active monkeys consists of 5 major peaks within the first 250 msec. The potentials recorded from post-central gyrus were simpler and more individualized and did not show intensity-related latency changes or increases in inter-subject variability. However, amplitudes of potentials recorded from both electrode placements increased with light intensity apparently reflecting the amplitude of individual potentials rather than the variability of these potentials from which the average VEPs were derived.


In-phase brain activities during arousal and sleep. EEG of sensorimotor area, EMG of neck muscles and unit activity in lateral geniculate (LG) nucleus, recorded in freely moving cats, were analysed for the presence of correlations between them. The 3 activities were first integrated with a 2 sec time constant, then a correlation coefficient was assessed between time-linked points of the 3 records for periods of 40 sec. The average vlaues of the correlation coefficients were found to increase from arousal through slow synchronized sleep (S sleep), reaching the highest value in REM sleep. The average of the values in REM was found to be significantly different from those in arousal and S sleep. These differences were statistically significant for the correlation coefficients between EEG and LG activities, as well as of EMG and LG. On the contrary, the correlation between EEG and EMG, which may be thought of as having more direct anatomical connections and a higher degree of physiological dependence, did not change as a function of the different physiological states.


Event-related potentials recorded from young and old adults during a memory retrieval task. Six healthy old and 8 healthy young subjects each received a series of trials in a memory retrieval task devised by Sternberg (1966). On each trials, the subject received a memory set of 1-4 digits and was then shown a test digit. The subject's task was to press one of two response buttons indicating whether the test digit was a member of the memory set for that trial. Response time (RT) was found to be an increasing, linear function of the number of items held in memory. The slope of the RT function was a composite measure of the time necessary to process each additional item in memory while the intercept was a measure of stimulus encoding and response processes that do not depend on memory set size (see Sternberg 1966, 1969, 1975). We found that the latency of P3 to the test stimulus also increased with increases in memory set size, although the slope of the P3 was less than that for RT. We have suggested that the intercept of the P3 slope reflects the time it takes to encode the test stimulus before the evaluation of the stimulus starts, while the slope reflects the amount of time per digit needed to evaluate the set. We have suggested that the difference between RT and P3 slopes represents the additional time per digit which the subjects waits before making a response, due to low confidence occurring with more difficult task conditions (i.e., when set size = 4). We further suggest that the intercept of the RT-P3 latency slope is a reflection of pure response processes. Time estimates of these processes are made for young and old subjects.


A comparison of EEG activity in the left and right cerebral hemispheres by power-spectrum analysis during language and non-language tasks. In 10 female subjects, power-spectrum analysis was performed on the alpha activity elicted during the resting state, 4 right hemispheric tasks, and 3 left hemispheric tasks. The data were treated in 3 ways: approach 1, comparing the right and left hemispheric alpha activity; approach 2, comparing the right and left hemispheric alpha activity adjusted for the resting state; and approach 3, comparing the right and left hemispheric alpha activity adjusted for the previous task. Approaches 1 and 2 revealed few significant differences in the alpha activity of the hemispheres, but approach 3 provided data that better fit the theory of decreased power spectrum of attenuation of activity in the activated hemisphere. Thus, approach 3 may be useful in developing an electroencephalographic test for determining cerebral dominance for language.


Contingent negative variation and evoked potential amplitude as a function of inspired nitrous oxide concentration. Nine subjects were given to breath 10%, 20%, 30% and 40% nitrous oxide in oxygen. The inhaled gas concentrations were monitored by a mass spectrograph. At each gas concentration the CNV was measured in a standard long S1-S2 CNV paradigm to a click and tone with a mandatory button press at S2. The results confirmed the previous observations of Lader and Morris (1974) that the cortical evoked potential decreases rapidly with increasing concentrations of nitrous oxide. The CNV, however, against prediction remains surprisingly well preserved up to the borders of unconsciousness. In this situation there is little correlation between CNV amplitude and attention, arousal or reaction time.


A functional measure of brain activity: brain stem transmission time. Surface-recorded auditory nerve and brain stem responses are being used routinely for diagnostic purposes in man. When interest is in auditory diagnosis, the electric response threshold is of primary importance. However, when used in neurological diagnosis, the wave form of the response is important. As a measure of one aspect of response wave form, this paper suggests the use of brain stem transmission time (BTT), defined as the time interval between the first earlobe-negative wave (response of the auditory nerve--the 'input' to the brain stem) and the earlobe-positive wave from the region of the inferior colliculus (the 'output' of the brain stem). The paper shows that BTT is longest in neonates, approaches adult values at the age of about 3 years, is relatively independent of click intensity, conductive hearing loss (middle ear lesion), click rate (except for high rates) and click frequency (filtered clicks). The finding that in a given age group, BTT is generally independent of most stimulus conditions, makes it a useful functional test of brain stem activity.


n-Hexane-induced changes in visual evoked potentials and electroretinograms of industrial workers. Visual evoked potentials (VEPs) and averaged extraocular electroretinograms (ERGs) were recorded from 15 workers occupationally exposed to n-hexane for 5-21 years and from 10 healthy control persons. The amplitude of the VEP components was clearly smaller among the exposed subjects with the exception of N0, which tended to be larger. In addition, the latencies of P1 and N1 were longer among the exposed workers, while that of P2 was slightly shorter. The peak-to-peak amplitude of the ERGs was also diministed among the exposed subjects. The changes were interpreted to indicate cerebral dysfunction, probably conduction block in intracerebral axons. n-Hexane is an aliphatic hydrocarbon found in gasoline and used in various industrial applications. It has been shown to cause axonal neuropathy of the dying-back type in both experimental animals and humans. According to the present findings the central nervous system is alos susceptible to the toxic effects of n-hexane.


[Insomnia and memory. Apropos of a case of striato-nigral degeneration]. Sleep and memory were investigated in a well educated patient suffering from striato-nigral degeneration. Despite almost total insomnia (only 591 min of stages 1 and 2, without any stage 3, 4 or paradoxical sleep during a continuous 4 day recording) it was not possible to objectify significant short or long term memory impairment.


A method for continuously monitoring the electrical threshold of single intraspinal nerve fibers. A method is described for the determination and continuous monitoring of the electrical threshold of single primary afferent fibres within the spinal cord of the cat. The centre barrels of multibarrel micropipettes, filled with 3.6 M NaCl, were used as stimulating electrodes, impulses in single afferents were recorded peripherally and a firing index of 50% was maintained by using a feedback circuit to regulate the amplitude of the stimulating pulses (0.2-0.3 msec duration, less than 2 microA).


[Psychoses, immunity and neurospecific antigens. Current knowledge and perspectives]. Investigations performed by various workers during the last ten years suggest that some forms of mental illness are associated with a variety of immunological reactions. Significant variations in immunoglobulin ratios have been observed in patients with psychotic illness, and cellular and humoral cytotoxic activities against cerebral tissue have been demonstrated in vitro. At the molecular level, several subjects can be proposed for investigation into immunological phenomena in neuropathology. Thus, the neurospecific antigens, which have been isolated by biochemical methods, but the physiological role of which is largely unknown, open news ways to the experimental exploration of psychopathology.


Purification and properties of RNA-dependent DNA polymerase from cytoplasmic A-type particles of murine mammary tumor virus. 1. The RNA-dependent DNA polymerase associated with cytoplasmic A-type particles of murine mammary tumor virus was isolated to near homogeneity by a procedure which includes dissociation of proteins from RNA by centrifugation in a step gradient of cesium chloride, followed by an affinity chromatography on poly(rC)-agarose column. Two species of DNA polymerase were separated by the chromatography: enzyme I in 0.55 M NaCl and enzyme II in 0.80 M NaCl eluate, respectively. 2. The purified DNA polymerases consist of two major polypeptides, with molecular weights of 94,000 and 42,000, as the intrinsic subunits. Both enzyme protomers with a sedimentation coefficient of 6.3--6.4 S and a molecular weight of 115,000--120,000 associate to form active oligomers in low-ionic-strength buffer. 3. Both enzymes catalyzed the hydrolysis of RNA in RNA . DNA hybrids as well as the RNA-dependent synthesis of DNA; these are the intrinsic activities of the reverse transcriptase from B-type particles of murine mammary tumor virus as well as from avian and mammalian C-type oncornaviruses. The general catalytic properties are similar to those of the enzyme from B-type particles. Compared with DNA polymerases I, DNA polymerase II exhibited a high affinity for all the template-primers tested and, in addition, a high preference for (rC)N . (dG)12--18.


Bone marrow culture in vitro. A technique for analysis and permanent recording of cellular composition. The in vitro cloning of haematopoietic progenitor cells derived from blood or bone marrow is now an established technique for the study of normal and abnormal blood formation. In semi-solid agar the results are conventionally recorded as the number of clusters or colonies that grow on the plate under controlled culture conditions. However, the demonstration of detailed morphology within these cellular aggregates remains unsatisfactory. Aspiration techniques are cumbersome and invariably disturb cellular relationships within the supporting matrix while supravital staining is limited by variable uptake of dye by the agar. We describe a method in which the entire cell-containing layer is removed from the Petri dish, fixed, and after mounting on a glass-slide, is air-dried. This preparation stains well with a wide variety of biological dyes, is minimally influenced by background colouration of the culture medium and excellent demonstration of morphologic detail is possible. A permanent record of the cellular composition of the culture is easily obtained by mounting the stained agar disc.


In vitro erythropoiesis. II. Cytochemical enumeration of erythroid stem cells (CFU-e and BFU-e) from normal mouse and human hematopoietic tissues. A procedure which provides more accuracy in the identification and scoring of in vitro derived erythroid colonies (i.e. BFU-e and CFU-e) from normal mouse and human marrow and spleen cells has been developed. Up to now identification of erythroid colonies has been based on staining with acidified benzidine which consistently displays "background staining" of granulocyte/macrophage colonies. The staining reaction with these reagents is such that identification of erythroid colonies is less than precise. The modified benzidine procedure described herein eliminates the staining reactivity of granulocyte/macrophage colonies and specifically reacts with only those cells that contain hemoglobin. Furthermore, individual colonies and/or cells may readily be counterstained and examined by high resolution light microscopy.


Inhibition of blastic transformation and immunoglobulin release in lymphocytes cultured in the presence of alpha-fetoprotein (AFP). Blastic transformation of phytohemagglutinin-stimulated lymphocytes in cultures containing various concentrations of homogenous alpha-fetoprotein (AFP) was studied. It was found that AFP in concentrations of 4-12 microgram/ml inhibits both blastic transformation and immunoglobulin release (IgA, IgG) in lymphocyte cultures. It is suggested that AFP plays an important role in immunoregulation during the fetal life.


Repeated light- and electron microscopic studies of the small-bowel mucosa in Whipple's disease. The small-bowel mucosa of a patient with Whipple's disease in the florid state and one year after starting antibiotic treatment, in complete clinical remission was examined by the author. As a result of treatment, light-microscopic changes led to total regression. Electronmicroscopically, in the florid state, destruction of small-bowel epithelial cells was observed, mostly in the Lieberkühn's crypts. The changes of epithelial cells regressed after one year of treatment. In the lamina propria, a large amount of macrophages containing characteristic inclusions were found which persisted even after treatment. The composition of inclusions has, however, changed. The macrophages were cells poor in organelles and of a primitive differentiation. Presumably, this morphological picture is associated with the digestion incapability of cells and this may lead to the development of "acquired storage disease".


Shape change of blood platelets induced by myelin basic protein. Myelin basic protein (MBP) isolated from bovine spinal cord caused a marked shape change reaction of human blood platelets which was not accompanied by the release reaction and not inhibited by methysergide and spiroperidol. Only those basic proteins, including MBP, which had previously shown to exert neuronal depolarisation also induced the shape change reactions. Therefore, these findings may extend the use of platelets as neuronal models.


An alkaline protease in the delayed hypersensitivity skin lesions in the guinea-pigs. An alkaline hemoglobinolytic protease was extracted from the delayed hypersensitivity skin lesions induced by bovine gamma-globulin as an antigen in the guinea-pig. The enzyme was heat-labile and inhibited by thiol-blocking reagents. The mol.wt. was more than 100,000 and optimal pH around 9.


Cell type-specific neural cell surface antigens. Immunological methods have served to define several cell surface antigens that are differentially expressed among neural cell types and are developmentally regulated. These antigens have served as useful markers for cell identification and isolation of several neural cell types. The molecular nature and functional properties of almost all of these antigens are presently unknown.


Incorporation of 35S-sulfate into yolk platelets of Xenopus laevis embryos. A study using electron microscope autoradiography. Inorganic 35S-sulfate was injected into Xenopus laevis embryos before first cleavage to study incorporation of the label into the yolk platelets in order to localize glycosaminoglycan synthesis. Electron microscope autoradiography of embryonic thin sections from blastulae and gastrulae revealed that the primary site of label incorporation is at the edge of the yolk platelets, and, to a lesser extent, in their interiors. Autoradiography of isolated yolk platelets, lacking unit membranes, indicated the absence of label. Thus, edge associated label comes from the yolk platelets membrane, and interior label is solubilized in the glycerol-water gradient during yolk platelets isolation. Ruthenium red staining of yolk platelet in situ shows haavy deposits of the dye on the yolk platelet membrane surface facing the cytoplasmic surface. The crystalline main body of isolated yolk platelets does not take up the dye. It appears that continuous synthesis or sulfation of glycosaminoglycan occurs primarily at the outer surface yolk platelet membranes during early development, providing a novel site for this process.


[Phospholipids and blood coagulation].

[Phospholipids and blood coagulation]. After a simplified survey of the chemistry of phospholipids (PL) their presence in the lipid part of thromboplastin is dealt with, the significance of this structure being stressed. In addition, the condition, mode of action and synthesis of PL in blood platelets are extensively discussed and the impact of the composition of thrombocytic PL in the membrane and granular fraction by plasma lipid substances is referred to. The relations between single forms of PL and those proteins activating coagulation are represented and the conditions for the development of such activating complexes are referred to. Subsequently the coagulation stimulating properties of PL, thromboplastin, thrombocytes, and artifically produced PL are compared and the attempt is made to draw certain conclusions from sometimes contradictory results. Finally, the anticoagulative effect of phosphatidylserin is referred to.


Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera. Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.


Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts. Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.


Elevation of an acid ribonuclease in serum of patients with chronic granulocytic leukaemia. Ribonuclease (Ribonucleate nucleotide 2'-transferase E.C. 2.7.7.17) activity in serum of patients with chronic granulocytic leukaemia measured at pH 4.5-6.0 amounts to more than three times of that in serum of healthy subjects. At pH 6.0-8.0 the elevation of ribonuclease activity in serum of patients with chronic granulocytic leukaemia is less pronounced and amounts to about two times of that in normal ones. Using chromatography on CM Sephadex C-50 column, serum ribonuclease of both normal and chronic granulocytic leukaemia patients was separated into five distinct fractions. In serum of healthy subjects ribonuclease fractions denoted I-V contribute to 10; 21; 29; 22, and 18 percent of the total ribonuclease activity. In the serum of patients with chronic granulocytic leukaemia a decrease in ribonuclease fraction III to merely 17 percent and an increase in contribution of fraction IV to 32 percent of total ribonuclease activity could be observed. The comparison of each individual concentration of fraction in normal and leukaemia patients serum reveals, that ribonuclease fraction IV will increase about 3 times. A less pronounced increase could also be found for fractions I, II and V. However, ribonuclease fraction IV may be supposed to carry more than 50 percent of the whole extra load of ribonuclease present in the serum of chronic granulocytic leukaemia patients.


Recovery course in mouse spleen and bone marrow after continuous irradiation. The paper deals with the recovery process of some parameters in the spleen and bone marrow till day 60 after continuous irradiation with a daily dose of 476.5 mGy (50 R), 957 mGy (100 R) and 4785 mGy (500 R) up to the total accumulated dose of 9570 mGy (1000 R). The recovery process in the spleen and bone marrow are relatively significant and completed till day 28 or 60 respectively after irradiation.


Progenitor white cells and serum inhibitors in patients with bone marrow aplasia. In patients with various blood diseases, especially with bone marrow hypoplasia following parameters were determined: presence of antibodies against autologous bone marrow, number of granulocyte-progenitor cells in short term bone marrow cultures and stimulating or inhibiting factors in patient's serum influencing the proliferative activity of bone marrow colonies. The results as well as possible clinical aspects are discussed.


[Relationship between the capacity to be stimulated of lymphocyte subpopulations and the RAI staging in patients with chronic lymphocytic leukemia]. By means of the incorporation rate of 3H thymidine into the lymphocytes of patients with chronic lymphatic leukaemia the possibility of stimulating them by using different mitogens was checked and compared with normal persons. The examination covered 11 patients treated with extracorporeal irradiation of the blood (ECIB), 5 patients treated with a chlorambucil therapy, and 10 untreated patients who were classified according to the staging system proposed by RAI. The lymphocytes of the peripheral blood were stimulated as mixed and isolated T and B-lymphocytes in the microculture by using the mitogens PHA, PWM, ConA, and LPS. In all CLL patients there was a diminished stimulation rate of a mixed lymphocyte population. A relation existed between the seriousness of the stage and the diminution of the incorporation rate of 3H thymidine. A corresponding correlation could not be identified in untreated CLL patients. Isolated T-lymphocytes revealed better results of stimulation than the total population. As to their function B-lymphocytes showed a dependance on the kind of therapy. In the mixed lymphocyte culture of normal persons the best findings could be observed after stimulation with PHA, that is also valid for CLL patients. PHA, PWA, ConA, and LPS were suitable as substances stimulating B-lymphocytes with different efficacy in normal persons and CLL patients. Both collectives showed the best results in the T-lymphocyte culture after stimulation with LPS.


[Pre- and postoperative erythrocyte volume distribution curves in children with cyanotic heart defects]. The behaviour of the volume distribution curves (VVK) of red blood cells in children with cyanosed organic heart defect is reported by taking preoperative and postoperative observations as a basis. MCV determined from the volume distribution curve amounts to 80.3 micron3. If the volume distribution curves of the preoperative patient group are compared with the postoperative patient group (4 months) by means of the criteria DSmax, DS, inclination and dispersion, no significant changes can be identified. Therefore, shifting maturity divisions cannot be assumed in children with cyanosed organic heart defects, even in those with a preoperative hypoxia of a high degree.


[Sialic acid content and IgG binding of the glycocalyx of preserved erythrocytes]. Fractions of light and heavy erythrocytes were separated by centrifugation from blood samples banked in ACD-AG medium at 4 degrees C for periods up to 6 weeks. Both light and heavy erythrocytes have lost about 4,9% of their content of sialic acids during banking for 6 weeks. This reduction is in accord with a 6%-decrease of their agglutination by means of alcian blue. It is, however, a variance with the inhibition of agglutination by anti-IgG sera. The present findings provide evidence for the role alterations of the red cell membrane may play in the process of recognition and phagocytosis of banked erythrocytes. With regard to these alterations we suggest two types of rapid elimination of transfused banked erythrocytes: a) Primary elimination refers to cells primarily loaded with immunoglobulins such as to get recognized and phagocytized by macrophages. b) Secondary elimination accounts for rigid erythrocytes suffering from additional degradation while retained in the spleen prior to their loading with immunoglobulins and ensuing phagocytosis. Secondary elimination is considered a process more relevant to reutilisation of banked blood.


Some aspects for the further improvement in cell cryopreservation. According to theoretical considerations and to results of own experimental investigations in heart muscle fragments of adult rats the authors are of opinion that more attention should be attributed to the cryoprotective medium as a whole, i.e. not only to the proper cryoprotectant, but to the other components of the cryoprotective medium as well. The suppose that the still unsatisfactory results of cell preservation, including blood and bone marrow cells, can be essentially improved by optimizing the composition of the basal medium.


[Protective effect of selenium compounds in the freeze preservation of erythrocytes]. The influence of sodium selenite, selenomethionine and sodium selenite with tocopherols in combination on the survival of cryopreserved erythrocytes was investigated. Percent hemolysis is marked decreased after a three-hour incubation of the whole blood with addition of selenomethionine as well as sodium selenite with tocopherole in combination before cryopreservation. The protective effect is attributed to the antioxydative and membrane stabilizing effectiveness of selenium and tocopherol.


[The binding of dyes of fibrinogen].

[The binding of dyes of fibrinogen]. Examinations performed by means of the agargel-electrophoresis, immunoelectrophoresis, discelectrophoresis, and chromatography on gel in human plasma and preparations of fibrinogen revealed that fibrinogen will form complexes with 18 examined dyes of different chemical structure, from a total amounting to 46. The findings indicate a marked affinity of fibrinogen towards small molecules in general. In nearly all complex forming colour materials, fibrinogen could be identified by immunodiffusion in the colour-blood supernatant liquid. The physiological significance of the binding capacity of fibrinogen is discussed.


Short heparin thrombin clotting time, increased fibrinogen and platelet aggregates count in coronary disease: a probable hypercoagulable state. Several indices of plasmatic and platelet coagulability (H.T.C.T., AT III, fibrinogen and Wu-Hoak test) were studied in 82 patients with acute and chronic coronary artery disease. The results were compared with those obtained in a control group. The most interesting result is the consistent shortening of H.T.C.T. as compared to the control group, particularly in patients with acute myocardial infarction. H.T.C.T. was always inversely correlated with the fibrinogen level. Those data suggest an important influence of fibrinogen levels on H.T.C.T., but this observation does not rule out the possibility that the heparin neutralizing activity (PF 4) will also influence the test. No positive correlation between H.T.C.T. and AT III could be observed. The elevated levels of AT III in acute myocardial infarction did not confirm the existence of a consumption of AT III due to chronic intravascular coagulation in these patients. The Wu-Hoak test increased only in patients with acute coronary disease, never in the other groups. In conclusion, H.T.C.T. may be assumed to be a valid test for indicating the existence of a possible hypercoagulability state.


[A further case of Tn-polyagglutination]. Another case of Tn-polyagglutination is described where for 6 years those responses have been observed which are typical of the acquired erythrocyte changes, viz. mixed-field polyagglutination by normal adult sera of all blood groups, no response with anti-TAh, agglutination of a certain part of erythrocytes by anti-TnSs, anti-ADb, anti-AHP, agglomeration of the other part by protamin sulfate and A-like specificity. Papain treatment eliminates polyagglutination by human sera of adults. Sera of new-borns did not agglutinate Tn-erythrocytes. Mrs. B. B. 38 years old belongs to blood group 0 and shows latent signs of haemolysis as well as permanently lowered leukocyte and thrombocyte numbers. The difficulties in blood group serology in connection with polyagglutination are referred too.


Seasonal changes in the blood parameters of two major carps, Labeo rohita (Ham.) and Cirrhina mrigala (Ham.). Seasonal changes in blood properties of two Indian major carps, L. rohita and C. mrigala, were investigated. Haematocrit values, haemoglobin concentrations and erythrocyte numbers showed seasonal fluctuations, being high from September to November and again in April-May, and low from January to March. Males had generally higher blood values than the females.


[Surgical treatment of rectal neoplasms in the very old patient]. Efforts and results of curative and palliative treatment of rectum carcinoma in the very aged (more than 75 years old) are shown by a follow-up study including 212 patients operated in our clinic. Establishing an absolute indication for radical resection of the malignant tumor the contraindications in this collective are pointed out. There is practically no difference between the results of curative and palliative treatment of rectum carcinoma in the very aged regarding letality and morbidity if contraindications are strictly considered and accurate pre- and postoperative treatment is guaranteed.


Expanding psychiatry's role in child abuse treatment. Child abuse, a problem that has plagued societies for centuries, has been largely ignored by psychiatrists, the author asserts. He traces the relatively recent interest in the problem of other medical specialists such as pediatricians and radiologists and maintains that it is time for psychiatrists to become more involved. He explains how problems of confidentiality, the patient's lack of motivation for treatment, and other factors have discouraged many psychiatrists from treating child abusers. He demonstrates ways psychiatrists can treat both the abused child and his family if they are willing to use non-traditional services such as home intervention.


The role of religious institutions in evangelization. Religious institutions are called to make an exodus out of their own holy land into the true holy land of humankind. By reflecting on the Beatitudes and Jesus' humanity, Catholic-sponsored health care institutions can better understand the meaning of evangelization.


Group-specific component (Gc) 'subtypes' of Gc1 by isoelectric focusing in US blacks and whites. Isoelectric focusing was applied to the Gc polymorphism. In agreement with Constans et al., we found two common 'subtypes' of Gc1 that could not be identified by conventional electrophoretic procedures. They are labeled Gc1F and Gc1S. Gc1F has a slightly lower isoelectric point than Gc1S. In groups of US blacks the allele frequencies were for Gc1F; 0.732 and for Gc1S; 0.147. In whites these figures were 0.149 and 0.572. We also found GcAb in blacks with a frequency of 0.015. The concentrations in serum of Gc protein as measured by radial immunodiffusion did not differ according to phenotype.


Isolation and characterization of diploid clones from adult and newborn rat liver cell lines. A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and pseudodiploid clone derived from adult rat liver cell line were positive for alpha-fetoprotein.


Characterization of the WIDR: a human colon carcinoma cell line. We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.


[Immunoglobulins and other plasme proteins in late pregnancy (author's transl)]. Serum levels of immunoglobulins, alpha-2-macroglobulin and complement component C3 were measured from 490 pregnant women shortly before date and compared with the serum concentration of the same proteins in a group of female blood donors. Concentration of immunoglobulin G was found to be significantly (2 p less than 0.001) reduced during late pregnancy, while the serum level of immunoglobulin M in the group of pregnant women was significantly (2 p less than 0.01) increased. No difference between the serum concentration of immunoglobulin A could be observed between both groups. A significant increase (2 p less than 0.01) could also be observed regarding the serum levels of the complement component C3 and of alpha-2-Macroglobulin in the group of pregnant women. The findings are discussed with respect to the alterations during late pregnancy.


Interactions between human serum proteins and oral streptococci reveal occurrence of receptors for aggregated beta 2-microglobulin. A total of 31 strains of oral streptococci representing Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, and Streptococcus milleri were tested for possible binding of human immunoglobulins G, G1, G2, G3, G4, A1, A2, M1, and M2 and haptoglobin, hemoglobin, fibrinogen, and aggregated beta 2-microglobulin. Radiolabeled beta 2-microglobulin in aggregated form showed affinity for 20 of the 31 strains tested. Binding activity for the protein was found in strains belonging to all five species. The bacterial receptor was resistant to trypsin. Monomeric, unlabeled beta 2-microglobulin did not interfere with the binding of the aggregated form. Of the other proteins tested, only the immunoglobulin A1 protein showed positive binding, and that was only with a single strain of S. milleri. beta 2-Microglobulin is present on all nucleated cell membranes in vivo. The reaction between aggregated beta 2-microglobulin and oral streptococci is a new type of human-bacterium interaction which should be considered in studies of bacterial adherence.


Structure and serological specificity of the K13-antigenic polysaccharide (K13 antigen) of urinary tract-infective Escherichia coli. The primary structure of the K13-antigenic polysaccharide (K13 antigen) of Escherichia coli O6:K13:H1 was elucidated by composition, periodate oxidation, Smith degradation, and methylation analysis. The polysaccharide consists of a repeating sequence of 3-linked ribofuranose and 7-linked 3-deoxymannooctulosonic acid (KDO). About 50% of the KDO residues are O-acetylated at position 4 or 5. Measurement of the optical rotary dispersion indicated that in aqueous solution the K13 polysaccharide assumes a secondary structure in which the carboxyl groups of KDO are engaged. The serological specificity of the K13 polysaccharide is expressed through KDO and its O-acetyl substituent, the ribose unit being antigenically silent. There are two populations of anti-K13 antibodies one directed against the charged region of the KDO and the other against the O-acetyl groups.


Optical demonstration of Chlamydiae in cell cultures by means of fluorochrome 33,258 H. Fluorochrome 33,258 H, a bisbenzimidazole, was employed for demonstrating Chlamydiae in cell cultures, and proved to be particularly suitable for illustrating the unique intracellular reproduction processes of these organisms. The staining procedure is simple and permits a selective differentiation of chlamydial inclusions and their developmental processes in the cell. Contrary to other staining methods, all stages of the chlamydial reproduction cycle can be illustrated thus permitting the early demonstration of Chlamydiae in the cell. The procedure is suitable not only in experimental investigations on Chlamydiae but also in clinical diagnostics.


Structure of tumor antigen on hybrid cells between mouse mammary ascites tumor and mouse fibroblast L cells. Somatic cell hybrids between mouse fibroblast L cells and MM2 mouse mammary ascites tumor grown in BALB/c mice were isolated and the structures of tumor-associated surface antigen of the hybrid cells, and parental MM2 and mouse L cells were investigated by the methods of radioiodination of membrane proteins, immunoprecipitation with a specific antiserum against tumor-associated surface antigens of MM2 tumor (anti-MM2 serum), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two molecules of 105,000 and 76,000 daltons were detected on the MM2 cell surface, but no MM2 tumor antigen was detected on the mouse L cells. On the hybrids between these two kinds of cells, in addition to the two MM2 tumor antigens, molecules of 48,000-51,000 and 12,000 daltons were observed. On Sendai-virus-infected mouse L cells only a molecule of 68,000 daltons was detected by the anti-MM2 serum, and furthermore this molecule was also detected by normal mouse serum, indicating that antibodies against Sendai virus were contaminating in both the anti-MM2 and normal mouse sera used, and thus the molecules detected on the hybrid cells were distinguishable from possible viral components of Sendai virus on the hybrid cells. The results indicate that somatic cell hybrids between mouse L cells and MM2 tumor grown in BALB/c mice expressed on their cell surface the molecules that were not exposed on either parent cell. The experiments comparing newly detected molecules with the H-2 antigen suggested that they were similar to H-2 in their electrophoretic pattern.


Reciprocal 8;14 translocation in EBV-negative B-cell acute lymphocytic leukemia with Burkitt-type cells. A cytogenetic study, including prophase-prometaphase chromosome analysis, of a patient with EBV-genome-negative acute lymphocytic leukemia of B-cell type with Burkitt-type cells is presented. All bone-marrow mitoses examined had a 14q+ marker chromosome which was identified as a reciprocal 8;14 translocation of exactly the same type as in Burkitt's lymphoma.


Cell lines with spontaneous secretion of pregnancy-associated alpha 2-globulin. Pregnancy-associated alpha 2-globulin (PA alpha 2G) was quantitated by radioimmunoassay in culture media from 34 exponentially growing human cell lines. Only 8/34 cell lines produced PA alpha 2G. The highest secretion was found in cell lines of histiocytic lymphoma origin while it was low in some carcinoma-derived lines. The results may support an assumption that PA alpha 2G is a normal cell product of B-lymphoid, monocytic and epithelial cells but may also simply indicate that ectopic PA alpha 2G production is especially common in tumors derived from such cells. The availability of PA alpha 2G-producing cell lines should facilitate studies of the immunoregulatory role of this protein.


Symbolism in the dreams of the blind.

Symbolism in the dreams of the blind. The present study compared a group of visually handicapped adults and normally-sighted college students with respect to extent of symbolic content in nocturnal dreams. The visually handicapped group comprised individuals who were partially sighted, congenitally blind and adventitiously blind. Each subject was required to keep a dream-diary for a period of two or four months. These diaries were then content-analyzed for frequency of symbolic instances, with symbols being identified in line with the criteria suggested by Hall (1966) and Hall and Nordby (1972). Although individual differences were marked and some visually handicapped subjects reported highly symbolic dreams, this group, on the whole, revealed less symbolism in dreams than did the normally sighted subjects. There was also a tendency for individuals blinded later in life (after age seven) to manifest more symbolic content in dreams than subjects blinded earlier (before age five). However, for the partially sighted subjects in the study, there emerged no clear relationship between age of onset of visual impairment and amount of symbolism in dreams. In general, the present findings were congruent with those of Blank (1958).


Vitreous body oxygen tension following experimental branch retinal vein obstruction. We obstructed either temporal or nasal superior and inferior retinal veins on one eye of rhesus monkeys with xenon photocoagulation. This resulted in large areas of nonperfused retina adjacent to normal retina in the same eye. Intraretinal neovascularization (new vessels in the retina) developed following the absorption of retinal hemorrhage and edema. We used microelectrodes to measure and compare vitreous body oxygen tensions over the nonperfused and over the normal retinal areas. There was no significant difference between the oxygen tension measurements in the same eye.


Ocular findings in systemic cutaneous basophil hypersensitivity. SCBH was induced by immunizing guinea pigs with a protein antigen and challenging 1 week later with a large intraperitoneal dose of the same antigen. Animals developed a delayed-onset, erythematous skin rash and dermal infiltration by basophils and eosinophils. The uveal tracts of these animals were infiltrated by eosinophils, as were several other internal organs. The eye is affected in SCBH, and as in other forms of ocular cell-mediated hypersensitivity, the eosinophil is a prominent cellular component of these reactions.


Effect of iontophoretic and topical application of antiviral agents in treatment of experimental HSV-1 keratitis in rabbits. Cathodal (-) iontophoresis of 9-beta-D-arabinofuranosyl-adenine 5'-monophosphate (vidarabine monophosphate; Ara-AMP) was performed once daily for 3 days for the treatment of experimental herpes simplex virus type 1 (HSV-1) keratitis in rabbit eyes, and the therapeutic efficacy was compared with that of topical treatment of Ara-AMP and idoxuridine (IDU) administered five times daily for 4 days. With the treatment initiated 24 hr after viral inoculation, Ara-AMP cathodal iontophoresis resulted in significant suppression of epithelial and anterior segment disease processes. Topical IDU (0.5%) or Ara-AMP (10%) also significantly improved the disease process when compared to the placebo-treated group; however, iontophoresis of Ara-AMP resulted in a more marked improvement. Slit-lamp examination indicated that iontophoresis did not cause any observable pathologic changes in corneal epithelium, stroma, conjunctiva, or iris of rabbit eyes. This experiment suggests that iontophoresis of Ara-AMP is a safe and effective approach for preventing the development of herpes simplex keratitis in rabbits.


Ultrastructural modulations of cell surface determinants in pathological processes. A novel approach for the ultrastructural localization of surface sialic acids is presented. Membrane-bound sialyl residues are chemically modified in situ by the covalent attachment of biotinyl residues, the latter of which are subsequently localized by ferritin-conjugated avidin. In contrast to previous methods, which have been based on electrostatic interactions, the above method does not affect cell surface charge. Consequently, the macromolecular configuration of the labeled sialoglycoconjugates is preserved. The method has been found to be more accurate in the quantitative evaluation and the topographical localization of membrane-based sialic acids both in normal and pathologically induced surface modulations. Modulations in cell surface sialic acid content and/or distribution have been demonstrated in beta-thalassemia and transformed lymphoid cells, and the consequences of such alterations are discussed regarding destruction vs. escape from the immune surveillance system.


Flow cytometry of human spermatozoa.

Flow cytometry of human spermatozoa. Methods are given for the preparation and staining of human spermatozoa for flow cytometric DNA measurements. Using agents for the reductive cleavage of disulfide crosslinks and suitable proteolytic enzymes an effective decondensation of the sperm chromatin and a DNA-proportional uptake of fluorochromes is achieved. Thus reliable and precise measurements of the relative DNA content of human spermatozoa are possible and the two subpopulations of haploid spermatozoa can be distinguished according to the difference in their DNA content.


The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain. Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3 +/- 0.11 and 0.8 +/- 0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.


[Topo-optical reactions on the membrane of the human red cell ghost (author's transl)]. Previous studies have proved that the thiazin dyes toluidine blue, azure A, azure B, 1.9-dimethyl methylene blue and the quinolin dyes N,N'-diethylpseudoisocyanine chloride, N,N'-6,6'-dichlorpseudoisocyanine chloride are suitable for topo-optical reaction on the membrane of the red blood cells. In the present study the applicability of the thiazin and quinolin dyes on the membrane of the human red cell ghost was examined. Optical analysis revealed that the thiazin dyes are bound in radial position to the membrane, while the quinolin dyes are bound parallel to the membrane's plane.


Immunohistochemistry on Bouin-fixed fetal tissue, stored for thirty years in ethanol. Human fetal tissue fixed with Bouin and kept for 30 years in ethanol is still useful in immunohistochemistry. The localization of beta-endorphin immunoreactive cells in the pituitary is given as example.


Immunochemical comparison of myosin light chains from chicken fast white, slow red, and cardiac muscle. Antibodies were formed against the myosin light chains isolated from chicken fast skeletal, slow skeletal, and cardiac muscle and the antigenicities of the light chains were compared by double immunodiffusion and immunoelectrophoresis. It was shown that fast light chains are immunologically different from light chains of slow and cardiac myosin, while the slow and cardiac muscle light chains have similar immunological characteristics; that is, the light chains of apparent molecular weight about 27,000 daltons in SDS-acrylamide gel electrophoresis of slow and cardiac muscle are immunologically indistinguishable, and the other light chains of apparent molecular weight about 19,000 daltons of both muscles include a common antigenic site.


alpha-Fetoprotein gene expression. Control of alpha-fetoprotein mRNA levels in cultured rat hepatoma cells. The expression of alpha-fetoprotein (AFP) was infestigated in a cloned cell culture derived from Morris hepatoma 7777, which shows a density-dependent variation in the AFP synthesis rate. The rate of secretion of AFP was found to be governed by the level of cytoplasmic mRNAAFP. Saturation hybridization of pulse-labeled RNA to excess cloned cDNAAFP was used to illustrate quantitatively how mRNAAFP is regulated in these tumor cells. It was found that the mRNAAFP level is primarily determined by its rate of transcription and mRNAAFP declines to 40% of its maximum level, and it accumulates at 20% of its maximum rate. The half-life of mRNAAFP was found to be 40 h, 5 to 6 times that of poly(A)-containing RNA. This difference in stability, in cells doubling every 20 h, results in a 2 1/2-fold increase in the fraction of mRNAAFP above that expected from the relative transcription rate of mRNAAFP. During maximal synthesis of AFP, mRNAAFP accumulates in the cytoplasm at a rate 25 times greater than an average middle abundance mRNA and 1000 times greater than the average low abundance mRNA. These results and the relatively high translational efficiency of mRNAAFP explain how cells can optimize production of an abundant protein.


Distribution of myosin isoenzymes among skeletal muscle fiber types. Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.


Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes. Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.


Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast. Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.


Slowly migrating axonal polypeptides. Inequalities in their rate and amount of transport between two branches of bifurcating axons. Polypeptides in the dorsal root ganglion (L5) of the adult rat were radioactively labeled, and components slowly migrating in the sciatic nerve (peripheral axons) and dorsal root (central axons) were analyzed, using SDS-polyacrylamide slab gel electrophoresis and fluorography. In particular, the transport rates and amounts of six major polypeptides, i.e., the triplet (reference 15; with mol wts of 200,000, 160,000, and 68,000 daltons), alpha- and beta-tubulins and actin were compared between the two axon branches. In peripheral axons, fronts of the triplet, tubulins, and actin migrate at 2-3 mm/d, 9-13 mm/d and approximately 19 mm/d, respectively. The corresponding values in central axons are 1-2 mm/d, 3-4 mm/d, and approximately 4 mm/d, indicating an obvious asymmetry in the transport rate between the two branches of bifurcating axons. A greater amount of labeled triplet, tubulins, and actin each is found to migrate in peripheral than in central axons. Another striking aspect of asymmetry between the two branches relates to the tubulins/triplet ratio which is significantly higher in the peripheral branch. Considerable proportions of radioactivities associated with tubulins and actin in the ganglion are nonmigratory, which are thought to derive mostly from periaxonal satellite cells. In contrast, most if not all of the labeled triplet is migratory, suggesting a virtual absence of triplet polypeptides in satellite cells. The possible significance of peripheral-central inequalities in slow axoplasmic transport is discussed from the viewpoints of axon volume and axonal outgrowth.


Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA. Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.


Actin in spindles of Haemanthus katherinae endosperm. I. General results using various glycerination methods. We have studied actin-containing filaments in spindles in Haemanthus endosperm cells glycerinated by various methods; the actin-containing filaments were identified by their reaction with rabbit skeletal muscle heavy meromyosin (HMM) to form 'decorated' filaments. Actin-containing filaments in the spindle were seen in amongst microtubules in bundles (both non-kinetochore microtubule bundles and kinetochore microtuble bundles) and were also seen not associated with microtubules. There were very few extra-spindle actin-containing filaments in these cells. Actin-containing filaments seemed to interact with microtubules, because the filaments remained close to and parallel to microtubules even when the microtubules were sharply curved. Because of the close association between microtubules and actin-containing filaments we could not identify all the actin-containing filaments present in microtubule bundles: microtubules obscured actin-containing filaments. We studied Haemanthus endosperm cells as they were glycerinated. For some of these observations we used phase-contrast microscopy. Glycerination caused the cells to shrink, initially, and this was followed by rapid expansion, but the cells did not expand to as large a volume as before glycerination. Spindle structure was maintained despite these changes in cell size. Evidences for this are that relative chromosome positions were maintained during glycerination, that spindle birefringence was maintained during glycerination, and that individual chromosomal spindle fibres remained birefringent during glycerination. Electron-microscopic observations supported this in that kinetochore microtubule bundles and non-kinetochore microtubule bundle were maintained during glycerination, as was the helical arrangement of spindle ribosomes into polyribosomes. One-step glycerination procedures were used (cells were treated with mixtures containing 25% glycerol, Triton-X-100 and HMM), and such procedures might be of general use. Living cells were embedded in fibrin clots in making light-microscopic observations; this procedure, too, might be of general use.


Topographical variations in the structure of the smooth septate junction. Smooth septate junctions in the midgut of Musca domestica and in Malpighian tubules of both Musca and Rhodnius prolixus are described. Details of the structures revealed after standard fixation, fixation in the presence of the stain, lanthanum hydroxide, and after freeze-fracture are discussed in the light of models previously put forward to explain the interrelations of the images obtained by these different methods. The organization of the junction between cells of the midgut varies in the apical-to-basal axis. At the apical border the septa (or ridges in freeze-fracture replicas) are packed tightly and follow an undulating but strictly parallel course. This packing loosens towards the middle of the junction until, at its basal extremity, the septa (ridges in replicas) are widely separated and follow independent meandering courses. That these features are found both in lanthanum-infiltrated specimens and freeze-fracture replicas allows a correlation to be made between the septa and the freeze-fracture ridges. The functional significance of these smooth septate junctions is discussed.


Comparative serology of two clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron. Antiserum prepared in rabbits against Bacteroides fragilis showed numerous bands when reacted with B. fragilis antigen in Ouchterlony plates. This antiserum also reacted with Bacteroides thetaiotaomicron and showed one band of apparent identity with B. fragilis. The band of identity common for both organisms was lost if the antigen was heated at 80 degrees C for 30 min. Antisera prepared against B. thetaiotaomicron did not react with B. fragilis.


Concentration of dihydrotestosterone and 3 alpha-androstanediol in naturally occurring and androgen-induced prostatic hyperplasia in the dog. Previous studies have suggested that dihydrotestosterone accumulation in the prostate may be involved in the pathogenesis of prostatic hyperplasia in man and dog. However, the fact that the administration of 10 mg dihydrotestosterone/d to castrated, mongrel dogs (0.5 mg/kg body wt) causes little growth in the prostate, whereas identical doses of 3alpha- androstanediol regularly induce prostatic hyperplasia (> 14 g weight) has raised the possibility that the dihydrotestosterone accumulation may be the result rather than the cause of the pathology. To investigate the mechanism of this phenomenon, we measured the levels of dihydrotestosterone and 3alpha-androstanediol in prostates from 75 dogs. In both naturally occurring and 3alpha-androstanediol-induced prostatic hyperplasia, the levels of dihydrotestosterone were high (>5 ng/g), whereas in immature glands and glands from dihydrotestosterone-treated animals, levels were similar (2.1 and 2.6 ng/g, respectively). 3alpha-Androstanediol levels were no different in animals treated with dihydrotestosterone or 3alpha-androstanediol.Therefore, because exogenous 3alpha-androstanediol is a better precursor of prostatic dihydrotestosterone than exogenous dihydrotestosterone itself, the effects of treatment with larger doses (2.5 mg/kg per d) of dihydrotestosterone and 3alpha-androstanediol for 12 wk were examined. In these amounts, dihydrotestosterone was as effective as 3alpha-androstanediol in inducing the development of prostatic hyperplasia and in elevating prostatic dihydrotestosterone concentration. Because dihydrotestosterone accumulates in spontaneous prostatic hyperplasia, because the administration of sufficient amounts of dihydrotestosterone to the castrated dog can induce the development of prostatic hyperplasia, and because 3alpha-androstanediol induces the development of hyperplasia via conversion to dihydrotestosterone, we conclude that accumulation of dihydrotestosterone is the cause of canine prostatic hyperplasia.


Evidence for involvement of cyclic nucleotides in intrinsic factor secretion by isolated rabbit gastric mucosa. Secretion of intrinsic factor (IF) has previously been demonstrated in isolated rabbit fundic mucosa maintained in organ culture. We have now investigated the possibility that cyclic nucleotides may play a role in IF secretion. A phosphodiesterase inhibitor, 3-isobutyl methylxanthine (IBMX), stimulated IF secretion nearly fourfold while increasing tissue levels of both cyclic AMP (cAMP) and cyclic GMP (cGMP). Peak IF secretion in response to IBMX was not reached until after tissue cAMP levels were maximal. Dibutyryl cAMP and 8-Br-cAMP increased secretion by the same order of magnitude as did IBMX, whereas corresponding analogues of cGMP had no such effect. Histamine increased secretion of IF. In the presence of 40 microM IBMX, histamine elevated tissue levels of cAMP, but not of cGMP, and the stimulating effect of 10 microM histamine on IF secretion was potentiated. An H2 receptor antagonist, cimetidine, blocked the increases in IF secretion and tissue cAMP levels due to histamine, and the increase in IF secretion due to IBMX. These observations are consistent with a role for cAMP in the secretion of IF by isolated gastric mucosa.