Enumeration of T cells, B cells and monocytes in the peripheral blood of normal and lymphocytotic cattle. The rosette-forming capacity of bovine peripheral blood lymphocytes (PBL) was determined with dextran and 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). Both dextran and AET-enhanced rosette formation; however, AET-treated SRBC detected a larger percentage of rosette-forming cells and thus was used in this study. The specificity of rosette formation by bovine thymus-derived (T) lymphocytes was shown by (1) demonstration of rosettes and surface-membrane immunoglobulins sIg) on different cells in PBL and nylon-wool fractionated lymphocyte populations and (2) rosette formation by a large percentage (83--90%) of thymocytes from three bovine foetuses and two 14-month-old heifers. A procedure was also developed to identify bovine monocytes by latex phagocytosis and 10--30% latex-ingesting cells were detected in PBL preparations isolated by Ficoll-Hypaque flotation. The frequency of sIg-bearing latex-ingesting, and sIg-bearing latex non-ingesting cells in bovine peripheral blood was also determined. These procedures were utilized to determine the distribution of T and bone-marrow derived (B) lymphocytes in peripheral blood of normal and lymphocytotic cattle. PBL from twenty normal cattle contained approximately 63% T and 11% B (sIg+ latex non-ingesting) lymphocytes. In peripheral blood of three cattle with persistent lymphocytosis, a prodromal stage of bovine leukaemia, the percentage of B cells was elevated approximately to 59% whereas T lymphocytes decreased to 35%, thus providing additional evidence that persistent lymphocytosis is a B-cell disease.


Identification of lymphocyte subpopulations in E-RFC-enriched and E-RFC-depleted cell fractions of fresh and cryopreserved lymphocytes. Fresh lymphocytes and frozen-stored lymphocytes were separated into E-RFC-enriched and E-RFC-depleted cell fractions by density gradient centrifugation of sheep red blood cell (SRBC) rosette-forming cells (E-RFC), since the ability to form rosettes is primarily a T cell characteristic. Subpopulations of lymphocytes were identified, demonstrating the presence of cell surface markers: T cell specific antigens (T+), receptors for SRBC on T cells (E-RFC), Fc-receptors (FcR) for IgG type antibodies, and surface Ig (sIg). Our results indicate that, although the E-RFC-depleted fraction contains virtually no cells capable of binding SRBC, there is still a considerable proportion of T cells present in that fraction, as detected with the anti-T cell antiserum. Moreover, data are presented to indicate that the E-RFC-enriched fraction does not consist exclusively of T lymphocytes. Since this separation procedure is used frequently for the identification of the nature of effector cells in cell-mediated (CMC) and antibody-dependent cellular cytotoxicity (ADCC) assays, the identification of T cells in purified lymphocyte fractions by means of SRBC rosette formation may lead to a false conclusion as to the nature of the effector cells.


Folinic acid rescue for vincristine toxicity. Inadvertent vincristine sulfate overdosage was treated with folinic acid. The sequence of development of the signs and symptoms associated with known vincristine toxicity was accentuated; however, the duration of these toxicities was markedly compressed in time when compared with previous reports. The known pharmacologic and biologic activities of vincristine sulfate and presumed mechanism of counteractivity of folinic acid is presented.


Acute central cervical spinal cord syndrome treated by acupuncture and electrical stimulation (AES). Twelve cases of acute central cervical spinal cord syndrome were treated by acupuncture and electrical stimulation. The paraesthesia, as well as movements of the hand and upper extremities, were greatly improved immediately after each treatment.


Analysis of cohort effects from cross-sectional data. A statistical model and computer program are described to analyze data fro the existence of cohort effects. The analysis leads to the separate assessments of cohort, time and age influences on a series of age-specific rates that are available in cross-sectional form. The assessment of these three factors can possibly yield insights into specific disease etiologies.


Histiocytosis 'X' of the central nervous system. Clinical and pathological report of a case with predominant cerebellar involvement. The case is presented of a child with early manifestations of Hand-Schüller-Christian disease limited to exophthalmos, and rapid final progression towards a clinical picture dominated by intracranial hypertension and cerebellar involvement, caused by massive histiocytic infiltration of the cerebellum, combined with discrete subdural deposits of xanthomatous tissue and minor deposits in other parts of the central nervous system and other organs.


Electrical stimulation of the nervous system for pain control. Transcutaneous electrical stimulation appears to be a valid technique for the treatment of many pain states. Its use in chronic pain is limited and it appears to be much more likely to be effective in the relief of acute painful states. Nevertheless, since it provides a simple way to treat a significant number of patients whose pain would otherwise by intractable, it has been a valuable addition to the armamentarium of the physician dealing with chronic pain. Peripheral nerve stimulation is an excellent way to relieve pain of peripheral nerve injury origin and certain painful, poorly understood, vasopastic or reflex sympathetic states. Spinal cord stimulation has been revived by the advent of percutaneous stimulators. The technique is currently the best available for the treatment of the patient suffering from the chronic low back syndrome with severe arachnoiditis, for whom no definitive therapy is available. Brain stimulation has been relegated to therapy for pain of central nervous system origin. It is a most promising technique though its application appears to be limited at this point to a few specific problems. The seriousness of potential complications has kept it from being widely applicable to date. There is little information concerning the mechanism whereby these various techniques are effective. Transcutaneous and peripheral nerve stimulation might have their effect through peripheral mechanisms or through a gating mechanism in the posterior horn (Melzack and Wall 1965; Campbell and Taub 1973). Spinal cord stimulation could act through a retrograde effect upon a dorsal horn gate or have more central actions. Brain stimulation in the opiate receptor system may be effective through activation of this system. The mechanisms of action of stimulation in the sensory system centrally are certainly not well understood (Bloedel 1974).


Effects of cerebellar stimulation on epilepsy, the EEG and cerebral palsy in man. Eighteen of the first 29 patients with intractable epilepsy treated by chronic cerebellar stimulation (CCS) demonstrated a marked suppression of seizures. Sixty-eight of 100 patients with cerebral palsy showed clinical improvement after CCS. Electroencephalographic studies in three epileptic patients revealed a significant (P less than 0.001) reduction in number and duration of paroxysmal EEG discharges during epochs when the stimulator was on; prolonged effects were seen at stimulation rates of 200 c/sec and 10 c/sec (monophasic capacitively coupled stimuli). "Rebound" increases in numbers and durations of paroxysmal discharges occurred after cessation of CCS: immediate "rebounds" occurred within the next 5 min; such rebound effects were also seen in the frequency of clinical seizures. CCS at voltages well above threshold for the production of changes in H reflexes, late motor responses (V1 and V2), and evoked potentials resulted in increased "rebound" effects after cessation of stimulation and such effects were seen clinically and neurophysiologically in epileptic and cerebral palsy patients. Variability in the effects of CCS on seizures and the EEG may have been due to technical factors such as positions and impedances of electrodes, output of the stimulator, effects of anticonvulsant medication and patient differences; there was no clinical or physiological evidence of any undesirable neurological effect of CCS. In one patient, onset of CCS was frequently associated with cessation of polyspike and wave discharges; such results raise the possibility of triggering CCS from paroxysmal discharges in the EEG (contingency feedback) but rebound effects may complicate such therapy.


Changes in insulin and prolactin responsiveness of goat mammary tissue during ontogenesis and pregnancy. Mammary tissue of goats in different stages of ontogenesis and pregnancy were organ cultured and their insulin and prolactin responsiveness was studied. Based on the synthesis of DNA in the presence of insulin and tissue secretory responses to prolactin, the responsive tissues were found in young goats up to 3 month of age (prolactin responsive only up to 24 days of age), in mature virgin goats at the end of breeding season, mature virgin goats with precocious lactation and in nulliparous goats from the 8th week of pregnancy. Unresponsive tissues were found in goats from 4.5 month of age to the maturity. The high number of antral follicles in goat ovaries was found to be coincident with high mammary insulin and prolactin responsiveness during ontogenesis. It appears that ovarian and placental steroids and placental lactogen render the goat mammary tissue responsive to insulin and prolactin in organ culture.


Effect of insulin on RNA and protein biosynthesis in liver mitochondria from normal and alloxan diabetic rats. In liver mitochondria from alloxan diabetic rats the biosynthesis of RNA (as measured by 14C-orotic acid incorporation and RNA polymerase activity) and protein (as measured by 14C-leucine incorporation) were decreased. In contrast, insulin (20 U kg-1) injected to intact or diabetic rats increased both these measures. However, no change of mitochondrial DNA biosynthesis (as measured by incorporation of 3H-thymidine) was found. It was concluded that in liver cells insulin plays an important role in regulation of biosynthesis not only of nuclear (cytoplasmic) but also of mitochondrial RNA and proteins.


Fibroblast and epidermal growth factors are mitogenic agents for cultured granulosa cells of rodent, porcine, and human origin. The mitogenic effects of epidermal growth factor (EGF) and fibroblast growth factor (FGF) on cultured granulosa cells of different species have been analyzed. EGF and FGF are potent mitogenic agents for rabbit, porcine, and human granulosa cell cultures. While guinea pig granulosa cell cultures respond to FGF, they were hardly effected by EGF. Rat granulosa cell cultures did not respond markedly to either EGF or FGF. Our results, therefore, demonstrate that the mitogenic effects of EGF and FGF are not restricted to granulosa cells of bovine origin and, with the exception of rat granulosa cells, cultured granulosa cells responded either to FGF alone or to both EGF and FGF with a marked increase in their rates of proliferation.


The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics. The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.


Thymic regeneration after dexamethasone treatment as a model for subpopulation development. Three mouse thymocyte subpopulations with differing sensitivity to dexamethasone were characterized by size and density. Distribution patterns were determined for thymocytes during normal development and during regeneration after treatment with dexamethasone. From these, the subpopulation proportions were calculated and cell population kinetics were evaluated. Medium-sized low density cells (type III) were least sensitive to dexamethasone and showed a relatively slow regeneration rate. The small cells of intermediate density, "early cortical" cells (type II), exhibited the most rapid regeneration rate, while the growth rate of the small cells of high density (type I) was intermediate. Cells of intermediate and high density were relatively sensitive to dexamethasone. The ratio of the phytohemagglutinin to concanavalin A (Con A) response increased after dexamethasone treatment with the increase in the proportion of type III cells. Con A responses were attributed to type II and type III cells. A tentative multipathway model for intra-thymic T cell development is presented. Large cells, not confined to a certain trajectory in the density gradient, may represent a pool of proliferating cells of the other three types.


Antibody-independent activation of complement by human peripheral B lymphocytes. Normal human peripheral blood lymphocytes were shown to activate complement in normal human serum (NHS). This activation led to C3 fixation on the cell membrane, which in turn was visualized by fluorescence. The reaction occurred in the absence of detectable amounts of antibodies, since an agammaglobulinemic patient's serum also supported complement activation, and the results were unaltered by absorption of normal human serum. Trypsin treatment of lymphocytes to remove possible complement activators bound to the cell membrane, did not have any effect on the complement activation by lymphocytes. Complement membrane fluorescence was abolished in the presence of EDTA or ethyleneglycolbis(aminoethylether) tetraacetate (EGTA) supplemented by Mg++ions indicating that the classical pathway of complement activation was involved in the reaction. Experiments performed with T and B-purified populations showed that the phenomenon was confined to the B cell fraction. Complement deposition did not diminish the number of EAC rosettes, and viability remained unchanged after exposure of lymphocytes to normal human serum.


Ontogeny of susceptibility of mouse splenic B cells to tolerance induction in vitro by TNP-D-GL. The susceptibility of mouse spleen cells to hapten-specific tolerance induction of a primary in vitro thymus-independent antibody response was examined. Both the induction of tolerance by 2,4,6-trinitrophenyl-D-glutamic acid-D-lysine (TNP96D-GL) and of antibody formation (elicited by TNP-Brucella abortus) in neonatal spleen cell cultures were unaffected by anti-Thy-1.2 plus complement treatment. Spleen cells from neonatal mice were only slightly more sensitive to TNP96D-GL tolerance induction than were cells from adult mice. The difference in susceptibility to tolerance induction was not nearly as great as that predicted by "clonal abortion"-type theories of B cell tolerogenesis.


Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice. Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.


Influenza virus-specific T cell-mediated cytotoxicity: integration of the virus antigen into the target cell membrane is essential for target cell formation. This study deals with the requirements for target cell recognition by influenza A virus-specific cytotoxic T lymphocytes (CTL). H-2-identical cells were incubated with infectious or UV light-inactivated influenza A virus expressing either cleaved or uncleaved hemagglutinin (HA). Thereafter, the treated cells were tested in a 4-h 51Cr assay for susceptibility to CTL-mediated cytolysis. Regardless whether the influenza virus was infectious, virions expressing cleaved HA were efficient in target cell formation. In contrast, cells incubated with either active or UV-inactivated virions expressing uncleaved HA were not lysed by virus-specific CTL. Yet, after mere trypsin-mediated cleavage of the HA of cell-absorbed viroins, strong cytolysis could be observed. On the other hand, solubilization of the envelope lipid bilayer by ethylether abolished the capacity of the remaining HA to induce target cell formation. The results clearly suggest that mere absorption of virions to the membrane of cells, which is performed by virus with uncleaved HA, is insufficient for target cell formation. For this, both cleaved HA and an intact envelope appear to be crucial. We conclude that fusion of the virion into the cell membrane is essential for target cell formation.


Specific binding of T lymphocytes to macrophages IV. Dependence on cations, temperature and cytochalasin B-sensitive mechanisms. Peritoneal exudate lymphocytes (PEL) from immune guinea pigs adhere to macrophages carrying the relevant antigen and are thereby stimulated to proliferate in culture. The resultant PEL represent a population highly enriched with regard to their capacity to specifically rebind to antigen-pulsed macrophages. We have studied the mechanisms underlying specific binding of lymphocytes to macrophages by examining the effects of physical and chemical modifications of the two cell types. Specific binding was inhibited by fixation of cells, metabolic inhibitors, low temperatures, cytochalasin B and divalent cation depletion. After specific binding has taken place, cation depletion, but not cytochalasin B or low temperatures, disrupts binding. These observations indicate that specific binding occurs by a series of discrete events that can be operationally distinguished.


Detection and partial characterization of a new murine T-lineage antigen: evidence for its enzymatic modulation. A molecule with an apparent molecular weight of 21,000 daltons was found to be a predominating differentiation antigen of peripheral rat T lymphocytes. This antigen is also expressed on the cell surface of thymus lymphocytes though partially in masked form. The observation that these determinants could be unmasked by neuraminidase treatment in vitro suggests that enzymatic processes may play an important role in antigenic transformation along the differentiation pathway of T axis lymphocytes. Since this structure was found to be displayed also by peritoneal exudate macrophages, it is referred to as T lymphocyte-macrophage antigen. The finding that an identical antigen could be identified in the mouse suggests its more common biological significance.


Peanut agglutinin. II. Characterization of the Thy-1, Tla and Ig phenotype of peanut agglutinin-positive cells in adult, embryonic and nude mice using double immunofluorescence. The Thy-1, Tla and Ig phenotype of peanut agglutinin (PNA)-binding cells was characterized in various strains of mice. In the thymus, PNA was found to bind principally but not exclusively to the Thy-1+ Tla + Ig-- steroid-sensitive cortical thymocytes. Thy1 + Tla -- Ig -- steroid-resistant cells are not labeled with PNA. In other lymphoid organs, PNA bound to a minority of T or null cells but generally not to B cells. During ontogeny, PNA + and PNA -- T lineage cells appear simultaneously in the liver at day 10 of gestation, in the thymus at day 11 and in the spleen at day 18. No evidence was found for a maturation from PNA + to PNA -- cells. Prethymocytes present in nude mice were also divided into a PNA + and PNA -- population.


Possible role for glycosphingolipids in the control of immune responses. Preparations of gangliosides from bovine brain contain material which acts as a strong mitogen on murine spleen cells. This material is highly lipophilic and co-purifies with the ganglioside fraction. It contains saccharides of a similar composition to those found in monosialogangliosides, as well as a spinogsine base and an appreciable amount of peptide. The common brain gangliosides GM1, GD1a and GD1b, on the other hand, are not mitogenic and act as suppressors of the mitogenic activity of bacterial lipopolysaccharide on murine spleen cells. Both the mitogenically active and suppressive fractions of bovine brain glycosphingolipid were found to act exclusively on B lymphocytes. Since gangliosides and related compounds are components of plasma membranes and of amphipathic nature, they may passively migrate between the lymphocyte subpopulations and thus act as physiological modulators of immune responses.


Birefringence signals and tension development in single frog muscle fibres at short stimulus intervals. The early large birefringence signal and mechanical activity were studied together in isolated single fibres of frog skeletal muscle with double stimulation at short stimulus intervals (2-60 msec) at room temperature and at 4--6 degrees C. In all fibres tested, extra tension and additional birefringence signal in response to the second stimulus appeared simultaneously and suddenly upon increasing the stimulus interval. The shape of the stimulus-interval versus tension-development curve makes it highly improbable that subthreshold calcium release occurs at shorter stimulus intervals; therefore, tension development reliably reflects Ca-release in these experiments. In contrast to the report by Suarez-Kurtz and Partker, birefringence signal and calcium release are shown not to be dissociated by double stimulation. This result supports the hypothesis that the early large birefringence signal is an intrinsic indicator of calcium release from the sr during EC-coupling in skeletal muscle.


Inhibition or augmentation of PHA-induced lymphocyte transformation by factors of calculated lymphoblastoid cell lines. Stimulation of human peripheral blood lymphocytes by phytohemagglutinin (PHA) was found to be suppressed or augmented by the addition of supernatants or cell dialysates of cultured lymphoblastoid cell lines.


Lymphocyte responsiveness to urinary glycosaminoglycan in systemic scleroderma. From the urine of patients with systemic scleroderma, we previously isolated a glycosaminoglycan, which could induce a scleroderma-like change in the skin of mouse. In the present work, the cell-mediated response to urinary glycosaminoglycans was examined by lymphocyte transformation test. The specific response was observed in lymphocytes of patients with scleroderma, when the above scleroderma-inducing glycosaminoglycan was added. By contrast, lymphocytes of patients with SLE or dermatomyositis and of normal persons hardly showed a response to this glycosaminoglycan. On the other hand, the glycosaminoglycans from normal urine could not stimulate lymphocytes of patients with scleroderma or healthy persons.


T cell activation of primed rabbit blood lymphocytes by antigen. Activation of primed rabbit PBL by homologous antigen in the early proliferative phase (on days 3--5) mainly involves lymphocytes which neither secrete specific antibody nor contain immunoglobulin in their cell membrane. This stimulation is antigen-specific, and evidence is given that the major proportion of these cells are T lymphocytes. The B cells forming Av-CHO-specific PFC were studied by autoradiography on days 6 and 12 of culture. Since incorporation of radioactive thymidine was found in the majority of PFC, these cells are also in proliferation.


The effects of four arthropod diets on the body and organ weights of the leopard frog, Rana pipiens, during vitellogenesis. Wild-caught adult Rana pipiens females were captured in midsummer and fed diets of crickets, flies sowbugs or wax moth larvae during a three-month period of active vitellogenesis. The cricket diet supported the most extensive body weight gain during this time and promoted a prolonged period of weight increase in an additional long-term study. Synchronous growth of the oocytes occurred in all four groups, but the ovaries and oviducts of cricket-fed animals were significantly larger than those of frogs on the other three diets. The significantly higher liver weights of frogs fed wax moth larvae may have reflected an augmentation of hepatic energy stores. Fat body weights were also highest in this group of animals. Frogs fed crickets and wax moth larvae possessed larger fat bodies than did the midsummer control animals killed immediately after their arrival in the laboratory. In contrast, frogs fed flies and sowbugs had smaller fat bodies than did the initial controls, suggesting that animals on these diets had utilized fat body lipid during vitellogenesis. Gastrocnemius and final body weights were lowest in frogs fed wax moth larvae. These findings may have reflected the nutritional content of the diet or the reduction in appetite frequently noted in these animals during observations of feeding behavior.


Chemoarchitectonics in a reptilian cerebral hemisphere--acid phosphatase and 5'-nucleotidase. Variations on the distribution of acid phosphatase and 5'-nucleotidase were observed among the cerebral nuclei of the Uromastix hardwickii. Most of the nuclei revealed more intensely positive reaction for 5'-nucleotidase than the acid phosphatase. However, an identical enzymatic pattern, demonstrating intense activity for the two enzymes, was observed in the cortical centers excepting the primordial hippocampus. The possible causes of the intensity and the functions of the enzymes were established in relation to the nuclei and fiber bundles.


[Antipyretic activity of SL-573 (II) (author's transl)]. Antipyretic activity of SL-573 was not influenced by age and sex difference in rats. The combined effect of other drugs on antipyretic activity of SL-573 was examined, using several drugs which might be clinically applicable. Cefazolin sodium, ampicillin sodium, codeine phosphate, hydrochlorothiazide and haloperidol did not show any significant effect on antipyretic activity of SL-573. Diazepam itself showed antipyretic activity, and its combined use with SL-573 resulted in an additive effect. SL-573 also showed antipyretic activity in mice with fever induced by yeast, as was seen in rats. SL-573 diminished the hyperthermic response to bacterial endotoxin and leucocytic pyrogen in rats, but not to 2, 4-dinitrophenol. Additionally, SL-573 did not inhibit the bacterial endotoxin-induced production of leucocytic pyrogen and its release in saline medium. SL-573, therefore, is considered to be a centrally acting antipyretic. Intraventricular injection of prostaglandin E2 and arachidonic acid induced a hyperthermia in mice. SL-573 clearly inhibited arachidonic acid-induced hyperthermia, but not prostaglandin E2-induced hyperthermia. Since SL-573 is known to inhibit prostaglandin biosynthesis from arachidonic acid, the prostaglandin biosynthesis inhibition may be one of the main mechanisms of antipyretic action of SL-573.


The acute effects of 5HTP, fluoxetine and quipazine on insulin and glucagon release in the intact rat. 5-hydroxytryptophan (5HTP), the immediate precursor of serotonin, induces a release of insulin and glucagon in the intact rat. These effects of 5HTP, which have previously been shown to be blocked by L-aromatic amino acid decarboxylase inhibition, were also prevented by methysergide (a serotonin receptor antagonist). Quipazine (a serotonin receptor agonist) did not alter pancreatic hormone release. Fluoxetine, a serotonin neuronal reuptake blocker did not effect insulin secretion and had a slight glucagon stimulatory effect, however the effects of 5HTP on insulin and glucagon release were not potentiated by fluoxetine pretreatment. Alpha and beta-adrenergic receptor blockade did not alter the pancreatic effects of 5HTP.


Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes. The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.


Deae-dextran and T-cell rosette formation. Use of DEAE-dextran (a polycation) increases and stabilizes the rosettes formed between sheep red cells and human peripheral blood lymphocytes. Under its influence, reproducible stable rosettes are formed after one hour of incubation in an ice bath instead of the usually required 18 to 24 hours. We have shown that rosettes are specific for T-cells and not due to "co-rosetting" of nonrosette forming cells into the T-cell rosette clusters. Only the cells from normal controls consistently show an increase in rosette formation hence routine use of this 'stabilizer' in clinical immunology is not recommended.


A suppressor cell in the response of rabbit T cells to Con A. The response to Concanavalin A is regulated by a helper cell, described elsewhere (4), and by a suppressor cell, described in this paper. This suppressor cell is an adherent T cell with Fc receptors. Evidence for properties of the suppressor cell was obtained by two types of experiments: 1) regulatory cells gave more help if Fc-bearing subpopulations were removed from them, i.e. the suppressor cell could not be removed with Degalan beads, coated with anti-allotype antibody, but not with beads, coated with F(ab')2 fragments of the antibody; 2) help for T cells, in their response to Con A, was augmented when T cells were eliminated from the regulating adherent cell preparation. Thus, the response of spleen T cells to Con A is regulated by two adherent cells, a B helper cell and a T suppressor cell. We have previously shown that the response to phytohemagglutinin (PHA) is regulated by an adherent helper cell (4). We have found no evidence, in the present study for an adherent suppressor cell which participates in the T cell response to PHA.


A method for preservation of lymphocyte rosettes in agarose. A simple, reproducible method for preservation of lymphocyte rosettes in agarose-gel as supporting medium had been developed. The method allows many test samples to be stored and examined under similar conditions, and rosette-forming cells to be identified by the morphologic features.


Two T lymphocyte populations as effector cells fro IgG dependent cytotoxicity. The characteristics and proportions of human lymphocyte populations capable of lysing IgG coated target cells were studied. Monolayer of target erythrocytes coated with rabbit IgG were employed to detect and isolate the lytic effector lymphocytes. Normal peripheral blood effector lymphocytes were shown to express IgG receptors, and a total of four cell types possesing different surface markers were identified. An average of 68% (range 51-80%) of the effector lymphocytes had T lymphocytes antigen and were capable of SRBC rosette formation, and 32% of the lymphocytes lacked T lymphocyte markers. Two populations of these T lymphocytes were identified: one possesses SRBC and IgG receptors, and the other possesses SRBC, IgG and complement receptors. The proportion of the former was estimated as 53.2% and the latter as 14.8% of the total effector cell population. Two other cell types were distinguished among the non-T lymphocytes. One type of lymphocytes bear IgG receptors (21.6%) and the other has both IgG and complement receptors (10.4%). The cytolytic capacity of the two T lymphocyte subpopulations is suggested to be an important reaction of the immune reactive T lymphocytes during the immune response.


Cellular parameters of the immunological memory induced by lysozyme-LPS mixtures and complexes. The primary antibody response to the protein antigen lysozyme can be enhanced many fold by complexing the antigen with lipopolysaccharide (LPS). This complex, as well as lysozyme/LPS admixtures, is also capable of inducing a state of lysozyme specific immunological memory. Investigation of this memory state has revealed that administration of lysozyme either admixed or complexed with LPS results in priming of both B and T lymphocytes. Adoptive transfer of secondary responsiveness from animals primed in either fashion was further found to be T cell dependent. The implications of these findings are discussed.


Inhibition of the E-rosette formation of T-lymphocytes by aggregated human IgG and restoration by levamisole. Aggregated human IgG significantly inhibited the E-rosette formation of T lymphocytes of healthy subjects and cancer patients. The aggregated IgG induced inhibition of rosette forming cells depended on the time of preincubation at 37 degrees. Levamisole, an anti-anergic chemotherapeutic agent, could partly but significantly prevent the aggregated IgG induced inhibition of the E-rosette formation.


The accumulation of immunoblasts in extravascular tissues including mammary gland, peritoneal cavity, gut and skin. The migration of [125I]UdR labelled mesenteric (MLN) and peripheral T immunoblasts (PLN) has been followed in mice with multiple sites attractive to immunoblasts. The sites studied were the inflamed gut (produced by Trichinella spiralis infection), inflamed skin, the peritoneal cavity and the mammary glands of lactating mice. PLN were capable of assembling in all of the four sites and in the presence of both inflamed gut and inflamed skin, PLN accumulated preferentially in the skin. MLN, in contrast migrated only to the gut and mammary glands and not to the skin or peritoneal cavity, and could not be diverted from one site to another. Time course experiments revealed that whereas PLN migrate rapidly through the inflamed gut, they are retained in the skin for at least 3 days.


Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes. A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell.


Suppression of IgE-antibody production by non-polypeptide dialysable thymic factor. The effect of dialysable thymic extract on the IgE-antibody production in rats was studied. A marked suppression of IgE-antibody production determined by mast cell degranulation and passive cutaneous anaphylaxis tests was found in the immunized rats injected with dialysable thymic extract. The treatment of this preparation with immobilized pronase affects neither its suppressive activity, nor does such treatment affect the activity of purified factor, isolated from the crude extract. The prolonged administration of purified preparation to rats leads to an increase in thymus weight. When syngeneic thymocytes incubated with purified non-polypeptide thymic factor were administered to immunized rats a significant suppression of IgE-antibody production was observed compared to that occurring in untreated ones. On this basis, it was concluded that a decrease of IgE-antibody production may be due to a direct stimulation of T-suppressor cells.


Lymphocyte emperipolesis revisited. II. Further characterization of the lymphocyte subpopulation involved. We have previously shown that lymphocyte emperipolesis, the phenomenon whereby lymphocytes wander freely within the cytoplasm of larger host cells, is almost totally dependent on host cell pre-sensitization with IgG type antibody. In addition, the particular lymphocyte subpopulation involved was partially characterized as an Fc receptor bearing, non-T cell. In this study we have attempted to further define the emperipoletic lymphocyte subpopulation using cell fractionation procedures and correlation analysis. Significant correlation was found between emperipolesis and both Fc receptor bearing, surface immunoglobulin negative (Fc + SIg-) cells and antibody dependent lymphocyte mediated cytotoxicity; observations which suggest that the so-called K cells are the emperipoletic effector cells. The significance of these findings in relation to antibody dependent cytolysis is discussed.


The production of a soluble human T-lymphocyte derived factor which substitutes for helper T lymphocytes in the in vitro production of immunoglobulin. A soluble factor(s), human T-lymphocyte derived help factor (HHF), generated following pokeweed mitogen (PWM) activation of irradiated human peripheral blood T lymphocytes was shown to substitute partially for T-lymphocyte helper activity in the T-lymphocyte dependent PWM-stimulated synthesis of immunoglobulin by B cells. The degree of help provided was proportional to the number of cultured irradiated T-lymphocytes producing factor as well as the amount of factor added to the B-cell culture. The helper effect was equally provided by HHF syngeneic and allogeneic to the B lymphocyte. Although there was little stimulation of total protein synthesis, the synthesis and secretion of IgG, IgM and IgA were all stimulated three- to ten-fold by this factor. The B cells required a minimum of 40--55 h exposure to the factor from the initiation of the culture for an increase in Ig synthesis on day 5 to be observed. Addition of HHF to B cells pre-incubated with PWM for different time intervals showed that a maximum helper effect was exerted when the factor was added on day 0. Addition on day 1 provided less than 20% of maximum help. The factor did not promote significant increases in either B-cell or T-cell cell division.


Contact sensitivity and the DNA response in mice to high and low doses of oxazolone: low dose unresponsiveness following painting and feeding and its prevention by pretreatment with cyclophosphamide. Cyclophosphamide was used to assess the role of suppressor cells in the contact sensitivity reaction. A single painting with 300 microgram and 30 microgram oxazolone produced poor contact sensitivity reactions (ear swelling). Cyclophosphamide (200 mg/kg) 2 days before painting increased the response to the lower doses but had less effect on the response to 3 mg oxazolone. A single feed with 10 mg oxazolone caused strong contact sensitivity while lower doses (10-1000 microgram) caused poor responses. Cyclophosphamide increased the response to the lower doses but not to the highest dose of oxazolone. These results suggested that the poor response to painting and feeding lower doses of oxazolone was due to a suppressor system which was sensitive to cyclophosphamide. A different result was obtained when contact sensitivity was measured by arrival of radioactively labelled cells. Cyclophosphamide had the greatest effect on cell arrival when high doses were fed. This indicates that ear swelling and cell arrival measure separate aspects of the contact sensitivity response. The lower doses of oxazolone, which caused little contact sensitivity, reduced the response to a standard immunizing dose. This low dose unresponsiveness occurred after either painting or feeding (Chase-Sulzberger phenomenon). It did not occur in mice treated with cyclophosphamide before the first exposure to oxazolone. This suggested that the low dose unresponsiveness was due to suppressor cells. The response to oxazolone was also assessed by DNA synthesis in the regional lymph nodes. A small dose of oxazolone (30 microgram) caused a peak of DNA synthesis on day four while a high dose (3 mg) caused a peak on day three. Pretreatment with cyclophosphamide depressed the response to 30 microgram although it increased contact sensitivity. The secondary response was smaller than the primary on days 3, 4 and 5 after immunization but larger on day two. The depression but not the increase was prevented by cyclophosphamide and was probably due to a suppressor system.


Failure to block lymphocyte Fc receptor with anti-B lymphocyte sera (anti-DRW) in man. Sera harvested from multiparous women or from allograft recipients who had rejected their kidneys and containing various anti-DRW specificities, were studied on peripheral blood or lymph-node lymphocytes bearing or lacking the relevant B-cell alloantigen, for their EA or EAC-rosette-inhibition activity. Inhibition of C3 or Fc-receptor occurred independently of their anti-B lymphocyte cytotoxicity pattern. The same conclusion was reached when lymphocytes were either macrophage depleted or B-lymphocyte enriched. These results do not suggest a relationship in man between B-lymphocyte (DRW) alloantigen and Fc or C3 receptor structure.


Functional maturation of neonatal spleen cells. Maturation of neonatal spleen cells was studied in vitro with a cell population restricted with respect to functional properties. It is shown that the onset of the immune response to SRBc in post-natal mice was delayed because B and T cells were incompetent. It appears, however, that the development of these two cell populations does not occur in parallel. Since the addition of adult macrophages failed to overcome the incompetence of neonatal B cells in the presence of a T cell replacing factor, it is suggested that the late appearance of immune competence is due to the inability of B cells to process a T-cell signal.


Characterization of the receptor for IgM present on human B lymphocytes. The presence of a receptor for the Fc of IgM (muFcR) was demonstrated on the pathological B cells of all of sixteen patients with hairy-cell leukaemia and most, but not all, of twenty-four cases of chronic lymphocytic leukaemia, by a rosette method employing ox erythrocytes sensitized with purified IgM (EAm). This muFcR was also demonstrated on a small population of normal human mononuclear cells from peripheral blood. Pathological B cells with this receptor (Bm) simultaneously expressed a different and distinct receptor for the Fc of IgG, and were detectable without preincubation in medium containing foetal calf serum (FCS). The muFcR on B cells was blocked by Fc5mu and IgM, but not by F(ab')2mu fragments, or by IgG, whether monomeric or aggregated. Monomeric IgM and IgM bound to its antigen blocked much more effectively than pentameric IgM. B cells also possessed surface immunoglobulin and the Ia-like P29, 34 antigen, and an antiserum to this antigen blocked the muFcR. The muFcR on B cells differs in a number of ways from the  muFcR reported on T cells, and these differential characteristics are discussed in some detail. The muFcR was rapidly shed and resynthesized when washed Bm cells were maintained in medium not containing FCS and the general importance of this phenomenon in any study of muFcR is considered. It is suggested that Bm cells are memory cells and that the muFcR plays a part in the immune response.


Characterization of rat spleen-cell populations. I. Cell interactions in the regulation of in vitro response to concanavalin A. Spleen cells from BN rats were separated in a discontinuous density gradient. Cells from different fractions were shown to be functionally diverse, light cells enhancing DNA synthesis and cell division of the dense cell fractions, while dense cells suppressed DNA synthesis and cell division of light cells. Both these effects were also present in the absence of cell stimulation, were proportional to the number of modulating cells added to the cultures, and totally independent of the magnitude of response of controls. In Con A-stimulated cultures, mitogen dose also influenced the intensity of help and suppression. Both these effects are blocked by cell treatment with cycloheximide and can be mediated by cell supernatants. A T cell seems to be responsible for both helper and suppressor effects.


Thymus independent anti-horse erythrocyte antibody response and suppressor T cells in the Mexican axolotl (Amphibia, Urodela, ambystoma mexicanum). Anti-horse erythrocyte (anti-HRBC) antibody synthesis was studied in normal, early thymectomized and adult thymectomized axolotls. The kinetics of the responses were similar to those described in the same species for antibody synthesis against bacterial or viral antigens. Booster injections did not induce any characteristic anamnestic responses. Early and adult thymectomized axoltls gave in three experimental groups higher anti-HRBC responses than controls. It is concluded that HRBC acts in the axolotl as a thymus-independent antigen. The enhanced response in early as well as in adult thymectomized animals can be interpreted by the presence of a suppressor T-cell activity on anti-HRBC synthesis. These results do not exclude possible thymus-dependent responses for antibody synthesis in the axolotl, although such responses were not demonstrated in urodele. The questionable lacking of some functional T-cell subsets in urodele is discussed as a working hypothesis.


The effect of neonatal thymectomy on the level of salivary and serum immunoglobulins in rats. The effect of neonatal thymectomy on the level of salivary and serum immunoglobulins was studied in conventional rats. Salivary SIgA levels were significantly decreased from weaning (day 20) through the termination of the experiment, while salivary IgG levels were generally unaffected by neonatal thymectomy. Serum IgA was detected at a later interval after birth in samples from thymectomized rats than from normal and sham-thymectomized rats and the levels of serum IgA in the thymectomized rats remained 4-5 times lower throughout the 115 day experimental period. Serum IgG was decreased by approximately 40% from days 28 through 115 after birth in the thymectomized animals, while serum IgM was apparently unaffected by the neonatal thymectomy. These findings indicate that the synthesis of salivary SIgA is markedly influenced by the presence of thymus-derived cells and it is suggested that these cells may control and/or modify immune responses in oral secretions.


Relationships among differentiated T-cell subpopulations. IV. Preferential induction of delayed footpad reaction to xenografts and its radioresistant nature. Immune response in mice to xenogeneic cells was characterized by positive delayed footpad reaction and negative macrophage migration inhibition. Mice immunized with allogeneic cells exhibited negative delayed footpad reaction and positive macrophage migration inhibition. Cytotoxic T lymphocytes were detected only in mice immunized with allogeneic tumour cells. Delayed footpad reaction against xenogeneic cells was radioresistant and the immune lymphocytes responsible for such a reaction were presumed to have some relation to xenograft rejection.


Induction of suppression and help during in vitro immunization of mouse spleen cells. Both specific and non-specific suppressor cells are induced during the in vitro culture of mouse spleen cells in Mishell-Dutton-type cultures. The induction of T cells capable of providing helper activity does not require the presence of antigen during the culture period. In a mixture of helper and suppressor cells, as generated during the culture of spleen cells in the presence or absence of antigen, suppression is dominant if tested on normal spleen cells. This same cell mixture can reveal potent T-cell help if added in low numbers to T-cell depleted B cells in the presence of antigen. Partitioning of this cell mixture in a microculture system reveals potent T-cell help. Up to 1/600 recovered viable cultured mouse spleen cells can provide T-cell help for an anti-SRBC response. Evidence points to a coupled induction of non-specific help and suppression.


Cyclosporin A: in vivo and in vitro suppression of rat T-lymphocyte function. The immunosuppressive effect of cyclosporin A (CS-A) was investigated in RIC-Sprague-Dawley rats. In vivo, CS-A totally abolished the formation of antibodies to the hapten dinitrophenyl (DNP) in rats immunized with DNP-keyhole limpet haemocyanin. In vitro, the effect of CS-A was investigated in spleen cell cultures stimulated by concanavalin A, phytohaemagglutinin or lipopolysaccharide. The suppression due to CS-A was more pronounced in cultures set up with cells from rats fed the drug than in spleen cell cultures from control animals supplemented with serum containing CS-A. Purified by filtration through Degalan-rat Ig-anti IgG columns, T lymphocytes from CS-A treated rats were no longer suppressed by CS-A serum in contrast to purified T cells obtained from control rats. Thus, CS-A seems to interfere with the mitogenic triggering of a subpopulation of T lymphocytes resulting in a functional clonal deletion.


Suppression of antibody responses by cells from mice painted with picryl chloride. T cells from mice painted with picryl chloride inhibit secondary IgG anti-TNP antibody responses of normal mice to the sensitizer. Like other suppressor T cells produced after painting which inhibit DNA synthesis and the generation of cytotoxic T cells, these cells could be produced in adult thymectomized mice but not by mice treated with high doses of cyclophosphamide (250 mg/kg). The cells had to be injected within 48 h of a primary painting to inhibit the response to challenge 2-3 weeks later. This associated with their ability to inhibit DNA synthesis in draining lymph nodes after a primary painting. Double transfer experiments using spleen and lymph node cells failed to show any further activation or induction of suppressor function after challenge with antigen. As judged by the ability of anti-theta treated cells from suppressed mice to function as anti TNP primed B cells in adoptive responses to TNP-KLH no defect in B-cell memory was found. When, however, the ability of painting with picryl chloride to prime for challenge with TNP-KLH was used as a measure of B-cell function in situ it was found that the cells could inhibit responses. Responses to primary and secondary injections of TNP-KLH were not inhibited.


Open field behavior after chronic self stimulation. Long term exposure of adult male Sprague Dawley rats to rewarding brain stimulation produced alterations in open field behavior in comparison to nonstimulated controls. Initial activity was increased while movement latency was decreased for the experimental animals. Grooming and defecation were essentially unchanged by stimulation. These findings suggest a selective and tonic influence of brain stimulation upon exploration.


Effects of L-5-hydroxytryptophan on monoamine and amino acids turnover in the Lesch-Nyhan syndrome. In a patient with the Lesch-Nyhan syndrome we found decreased spinal fluid 5-hydroxyindole acetic acid (5-HIAA), the major metabolite of serotonin, and decreased homovanillic acid (HVA), the major metabolite of dopamine, indicating a decrease in monoamine metabolism. Administration of 5-hydroxytryptophan and carbidopa produced an increase in spinal fluid 5-HIAA, indicating that it might be possible to correct the serotonin deficiency in this syndrome, but there were no changes in the marked mental retardation and neurological deficits. Self-mutilation appeared to be suppressed by therapy but the effectiveness of the drugs decreased with time. There were also changes in the spinal fluid concentration of amino acids that might affect brain protein synthesis. These changes were corrected during administration of 5-hydroxytryptophan and carbidopa.


Features of the damage produced by proflavine on transforming deoxyribonucleic acid. Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.


The surgical management of massive gastro-intestinal hemorrhage due to stress ulcer following surgery. In this paper, thirty-six cases complicated by gastrointestinal bleeding after various operations were reviewed. The mortality rate was 47.2%. In 20 surgical cases, the mortality rate was 35.0%. Especially in the cases of post-intracranial surgery, a remarkably better result was obtained by surgical than by conservative treatment. With reference to the better results of surgical treatment, we suggested that the surgical indication depended on a stressor due to the original postoperative phase, which induced a stress ulcer. In post-CNS surgery, operative treatment should not be performed in a comatose patients. As regards jaundiced patients, those with low improved bilirubin levels should be preferred to those with infected bile ducts. In cases of abdominal surgery, sepsis and functional failure of the liver and kidney must be taken into consideration. From our clinical experience, subtotal gastrectomy combined with truncal vagotomy appears to be a more satisfactory treatment for stress ulcers than gastrectomy or vagotomy alone.


Immunoelectron microscopic localization of penicillinase in Bacillus licheniformis. Penicillinase was localized in log-phase cells of Bacillus licheniformis 749/C by labeling with ferritin-anti-penicillinase immunoglobulin G conjugate. Mildly fixed homogenized cells, isolated subcellular fractions, and frozen thin sections were labeled. The label was distributed in discrete patches in the cell envelope. The patches extended from the inside part of the membrane to the outside part of the wall. The inside part of the membrane was labeled more extensively than the outside part. The cytoplasm also bound some ferritin-immunoglobulin G conjugate. Immunoelectrophoresis and biochemical assay of cytosol material suggest that the cytoplasmic antigenic sites are a protease-sensitive form of penicillinase.


Hydrogen formation in nearly stoichiometric amounts from glucose by a Rhodopseudomonas sphaeroides mutant. Rhodopseudomonas sphaeroides produces molecular H2 and CO2 from reduced organic compounds which serve as electron sources and from light which provides energy in the form of adenosine 5'-triphosphate. This process is mediated by a nitrogenase enzyme. A mutant has been found that, unlike the wild type, will quantitatively convert glucose to H2 and CO2. Techniques for isolating other strains capable of utilizing other unusual electron sources are presented. Metabolism of glucose by the wild-type strain leads to an accumulation of gluconate. The isolated mutant strain does not appear to accumulate gluconate.


Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides. A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.


The activation of microsomal steroid 21-hydroxylase by cytosol from the cortex of bovine adrenals. At low microsome concentrations, the addition of cytosol from bovine adrenal cortex markedly accelerates the rate of hydroxylation of 17-hydroxyprogesterone at C-21. The detection of this effect was made possible by the development of a new, rapid, and sensitive procedure for the measurement of the initial rate kinetics of steroid 21-hydroxylase. This procedure is based on the fact that the reactant, 17-hydroxyprogesterone, possesses solvent partition properties which are different from those of the product, cortexolone. The specificity of the assay was confirmed by the isolation of only one product which was identified as cortexolone by radiochemical techniques. This assay procedure has great sensitivity and makes possible the accurate determination of the Michaelis constant at low enzyme concentrations. The Km for 17-hydroxyprogesterone with saturating amounts of TPNH was found to be 0.3 micronM. At the low microsome concentrations permitted by this assay, the addition of cytosol has a profound effect upon the rate of hydroxylation. The rate is markedly accelerated although the Km for the substrate is not altered. Neither the substrate nor the carbon monoxide-induced difference spectra are changed by the addition of cytosol, suggesting that activation by cytosol dose not affect the catalytic unit of the 21-hydroxylase complex.


Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.


Combined 17 alpha- and 18-hydroxylase deficiency associated with complete male pseudohermaphroditism and hypoaldosteronism. A 15-year-old phenotypic female with XY genotype presented with male pseudohermaphroditism, gynecomastia, hypokalemic alkalosis, and hypertension. Being raised as a girl, the patient failed to menstruate and developed no sexual hair. At laparatomy no Müllerian structures were found but testes were removed which showed histologically atrophy of seminiferous tubules and abundant Leydig cells. Deoxycorticosterone (2.0 mg/24 h) and corticosterone (117 mg/24 h) secretion rates were extremely elevated, whereas those of aldosterone (17 micrograms/24 h), deoxycortisol (22 micrograms/24 h), and cortisol (23 micrograms/24 h) were almost unmeasurable. The excretion rates of 18-OH-deoxycorticosterone (less than 0.25 microgram/24 h) and 18-OH-corticosterone (3.0 microgram/24 h) were subnormal in spite of high plasma ACTH levels (200 pg/ml), and deoxycorticosterone (32.2 micrograms/24 h) and corticosterone (269 micrograms/24 h) excess. Gonadotropins were elevated. Deficient 17 alpha-hydroxylation was suspected because of: 1) low levels of 17 alpha-OH-progesterone; 2) diminished excretion rates of both 17 alpha-OH-progesterone and pregnanetriol; and 3) reduced concentrations of dehydroepiandrosterone, androstenedione, and testosterone in testicular tissue. We conclude that this male pseudohermaphrodite with complete female phenotype had 17 alpha-hydroxylase deficiency. The enzymatic defect was linked with an associated 18-hydroxylase deficiency.


Role of tachycardia as an inotropic stimulus in man. We examined the inotropic effect of tachycardia in nine postsurgical aortocoronary bypass graft patients (with intact cardiac innervation) and nine cardiac allograft recipients (with denervated hearts). The changes in stroke volume (SV) and velocity of circumferential fiber shortening (VCF) which accompany sudden increases and decreases in atrial pacing frequency were determined by computer-aided fluoroscopic analysis of the motion of surgically implanted midwall myocardial markers. Because the first beat after a change in rate retains the frequency characteristics of the preceding rate, we compared the first posttachycardia beat with control beats and late tachycardia beats with the first tachycardia beat; afterload and preload for each pair of beats were similar. For an increase in heart rate of 50 beats/min, SV and VCF rose 79 and 64% from the first tachycardia beat to late tachycardia beats, and SV and VCF rose 8 and 35% from control beats to the first posttachycardia beat in the innervated group. Responses in the denervated group were not significantly different from those in the innervated group. The degree of the inotropic response was positively correlated with the magnitude of the increase in heart rate (r = 0.91). The decay in augmented contractility after decreasing the rate back to control levels fits an exponential relationship with a mean t((1/2)) of 1.7 s. Thus, in conscious man, increases in heart rate represent a positive inotropic stimulus, independent of other factors influencing ventricular performance and unaffected by neural innervation, and should be considered when changes in cardiac function are interpreted during serial studies or after drug administration.


In vivo inactivation of erythrocyte S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine in adenosine deaminase-deficient patients. The cytotoxic nucleoside 2'-deoxyadenosine is excreted in excessive amounts by individuals with genetic deficiency of adenosine deaminase, and may be in part responsible for the severe combined immune dysfunction from which they suffer. Earlier studies from this laboratory showed that 2'-deoxyadenosine causes the irreversible inactivation of the enzyme S-adenosylhomocysteine hydrolase by an active site-directed, "suicide-like" process. In this communication we have demonstrated similar inactivation of S-adenosylhomocysteine hydrolase in hemolysate and in intact erythrocytes, as well as a striking deficiency of S-adenosylhomocysteine hydrolase activity in the erythrocytes of three adenosine deaminase-deficient patients. In vivo suicide-like inactivation of S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine may contribute to the cytotoxicity of 2'-deoxyadenosine and to the immune dysfunction in adenosine deaminase deficiency.


Fecal blood loss and plasma salicylate study of salicylsalicylic acid and aspirin. Using a placebo-controlled methodology, 20 healthy volunteers housed in a clinical research facility for 23 days were studied for fecal blood loss and plasma salicylate levels after taking salsalate (salicylsalicylic acid) or aspirin. Daily dosages were 3000 mg salsalate or 3900 mg aspirin. Aspirin produced statistically significant gastrointestinal blood loss over control levels and over that produced by salsalate (P less than 0.01). Blood loss with salsalate was not different than that with placebo. Despite the intentional disparity of dosages between the two drugs, plasma salicylate levels were not statistically different. Side effects occurred at about equal frequency with either drug. Most prominent were headache and nausea. However, concomitant upper respiratory infection in 12 subjects rendered interpretation difficult.


Computed tomography in diabetes insipidus. Thirteen patients aged 1 to 59 years hospitalized for diabetes insipidus (DI) were investigated by computed tomography (CT). Apart from one craniopharyngioma, CT revealed in 8 out of 12 cases several modifications in the hypothalamic-pituitary region. In this group, CT demonstrated a small, round, isodense mass in the chiasmatic cistern that enhanced after contrast medium injection. Coronal and sagittal reconstructions helped pinpoint the lesion in the infundibulum and pituitary stalk, and, in one case, in the supraoptic nuclei. These findings, and the noninvasiveness of CT, make this technique one of the most accurate means of radiological investigation of DI and also of determining the efficacy of therapy.


Brain edema defined by cranial computed tomography. Cranial computed tomography permitted an in vivo classification of brain edema. The distinction of the four basic types of edema (vasogenic, ischemic, cytotoxic, and periventricular) was important in establishing the chronicity, extent, and pathologic basis of cerebral abnormalities. Vasogenic edema (neoplasm, abscess) was readily distinguished from cytotoxic edema by the predominant involvement of the white matter and associated abnormal enhancement following intravenous contrast medium injection. The diminished density of ischemic edema was visualized in both gray and white matter and occurred in a vascular distribution. An absence of enhancement in the cerebral capillary bed and attenuation or nonvisualization of the circle of Willis following the rapid bolus injection of iodinated contrast medium defined an arrest of cerebral circulation as might be seen in brain death. Periventricular interstitial edema was most often a marker of acute or subacute obstructive hydrocephalus.


Intracranial neurosarcoidosis.

Intracranial neurosarcoidosis. Neuroradiological findings in two cases of histologically confirmed intracranial sarcoidosis are presented, one with periaqueductal granulomata causing aqueduct obstruction. The granulomatous aggregation was of slightly greater attenuation than normal brain parenchyma and did enhance homogeneously following contrast medium administration. No direct signs of basal arachnoiditis could be found on computed tomography (CT) in the other case in which hydrocephalus was produced by a granulomatous infiltration of the fourth ventricle and cisterna magna. The neuropathological and pertinent clinical and laboratory manifestations of neurosarcoidosis are reviewed as a means of predicting recognizable CT patterns of the entity.


Subdural hematomas: an unusual appearance on computed tomography. It may at times be difficult to separate supra- from infratentorial and intra- from extraaxial pathology on the basis of axial transverse computed tomography (CT) scans. Coronal CT scans may be quite useful in more accurate localization of compartments. Three head injured patients with subdural hematomas were studied with axial transverse CT scans. In addition to the typical appearance of subdural hematomas, unusually shaped areas of increased attenuation were noted in the region of the tentorium cerebelli separate from the mass of the subdural collection. Coronal scans confirmed the supratentorial, extraaxial collection as an extension of the subdural hematoma into the supratentorial subdural space.


Low avidity as a cause of prozone phenomena. The incidence of prozones was studied, under various conditions, in a model indirect haemagglutination system and in a radioimmunoassay--both assays depending on the use of anti-immunoglobulin (anti-Ig). It was found that prozones were correlated with the presence of low avidity antibodies, and that these could compete with high avidity antibodies for the limited amount of anti-Ig available. It is proposed that the relatively rapid dissociation of low avidity antibodies allows them to form immune aggregates with the anti-Ig. With increasing size these aggregates would become more susceptible to being washed off. In this way low avidity antibodies could occupy the anti-Ig, and yet be relatively ineffective, either for haemagglutination or for the binding of radioactively labelled (or fluorescein-labelled) anti-Ig to the antigen.


Purification of rabbit B spleen cells by removal of adherent and of T cells. B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.


Identification and quantitation of B and T cells by cytofluorographic analysis. With cytofluorographic analysis (CFGA) of cells stained with the fluorescent dye acridine orange (AO), the major peripheral white cell populations--lymphocytes, monocytes, and polymorphonuclear cells--display different characteristics and appear as distinct populations which can be quantitated. In this study we present a method for determining percentages of human T and B cells lymphocyte subpopulations by CFGA and display of the data on a computer-generated 3-dimensional grid. Lymphocytes were depleted of either B, T, or both B and T cells by rosetting with erythrocytes and separated by centrifugation. The B cell and T cell depleted and non-rosetting cell subpopulations localized on constant, distinct areas of the display grid. The percentages of T and B cells in peripheral blood samples from 6 normals analyzed by CFGA did not differ from the results obtained by light microscope counting (LMC).


Ascites production in 9 rat strains.

Ascites production in 9 rat strains. A simple inexpensive, and rapid method for inducing large volumes of ascitic fluid in rats is described. The procedure involves intraperitoneal injections of Freund's adjuvant emulsion on alternate days. Antibody activity of the ascitic fluid approximates that found in the serum. Female rats consistently respond better than males. Members of all 9 strains of rats tested produced ascites using this procedure.


Fluid-phase immunoradiometric assay for the detection of qualitative abnormalities of factor VIII/von Willebrand factor in variants of von Willebrand's disease. Antigenic reactivity of F.VIII/WF in variants of von Willebrand's disease (vWd) was studied with both fluid-phase and solid-phase immunoradiometric assays. Two different (rabbit and goat) 125I-labeled specific antibodies against purified F.VIII/WF were used in both their divalent (IgG) and their monovalent (Fab fragment) forms. Dose-response curves obtained by reacting a constant amount of antibody with serial dilutions of plasmas from normal or homozygous vWd demonstrated the specificity of the test. The accuracy was significantly higher with 125I-Fab fragments of goat anti-F.VIII/WF antiserum that intact goat IgG or rabbit IgG or Fab fragments. The significant decrease of the slope of the dose-response curves obtained with plasma from variants of vWd has been interpreted as due to the presence of abnormal F.VIII/WF molecules with decreased antigenic reactivity. A similar anomaly was found in cryosupernatant prepared from normal plasma, paralleling similarities demonstrated between variants of vWd and cryosupernatant. Results of experiments performed by reacting constant plasma dilutions from control or variants of confirmed the decreased antigenic reactivity of variant F.VIII/WF.


The relationship between initial creatine phosphate breakdown and recovery oxygen consumption for a single isometric tetanus of the frog sartorius muscle at 20 degrees C. A previous paper (Mahler, M. 1978 J. Gen. Physiol. 71:559--580) describes the time-course of the suprabasal rate of oxygen consumption (delta QO2) in the sartorius muscle of R. pipiens after isometric tetani of 0.1--1.0 s at 20 degrees C. To test whether these were the responses to impulse changes in the rate of ATP hydrolysis, we compared the total suprabasal oxygen consumption during recovery (delta[O2]) with the amount of ATP hydrolyzed during a contraction, measured indirectly as the decrease in creatine phosphate (delta[CP]O). If suprabasal ATP hydrolysis during recovery is negligible in comparison with that during contraction, delta[CP]0/delta[O2] should approximate the P:O2 ratio for oxidative metabolism, which has an expected value of 6.1--6.5. We found: formula; see text. We conclude that in this muscle at 20 degrees C: (a) after a tetanus of 0.2--1.0 s, delta QO2(t) can be considered the response to an impulse increase in the rate of ATP hydrolysis; (b) the reversal during recovery of unidentified exothermic reactions occurring during the contraction (Woledge, R. C. 1971. Prog. Biophys. Mol. Biol. 22:39--74) can be coupled to an ATP hydrolysis that is at most a small fraction of delta[CP]0; (c) the pooled mean for delta[CP]0/delta[O2], 6.58 +/- 0.55, sets an experimental lower bound for the P:O2 ratio in vivo.


Reversibility and mode of action of Black Widow spider venom on the vertebrate neuromuscular junction. Black widow spider venom (BWSV) stimulates transmitter release and depletes synaptic vesicles from muscles bathed in a sodium free medium containing 1 mM EGTA. However, frog neuromuscular junctions treated with BWSV in glucosamine Ringer's and post-treated with antivenin recover normal function. This suggests that probably the permanent block of neuromuscular transmission is due to changes in permeability of the nerve ending plasma membrane to cations such as Na+. When BWSV is applied in a medium lacking divalent cations and containing 1 mM EGTA, in most of the cases no effect is observed. We found that this inhibition can be overcome in three ways: (a) by adding divalent cations to the medium; (b) by increasing the tonicity of the medium with sucrose; (c) by raising the temperature of the medium. These results suggest that the lack of divalent cations influences the membrane fluidity. Moreover, in view of the report by Yahara and Kakimoto-Sameshima (1977. Proc. Natl. Acad. Sci. U.S.A. 74:4511--4515) that hypertonic media induce capping of surface receptors in lymphocytes and thymocytes, we think that these data further support the hypothesis that BWSV stimulates release by a dual mode of action; namely, it increases the nerve ending permeability to cations and also stimulates release directly via a process of redistribution of membrane components, a process which may also inhibit vesicle recycling.


Maternal T-lymphocyte response and gingivitis in pregnancy. The exacerbation of a chronic gingivitis during pregnancy was confirmed in a group of 24 pregnant patients who were observed at the 14th and 30th weeks of pregnancy and postpartum. This exacerbation occurred despite a fall in the amount of plaque accumulation and confirmed that during pregnancy some factor in addition to plaque is involved in the increased inflammation. Using in vitro methods, it was shown that during pregnancy there is a depression of maternal T-cell responsiveness and it is suggested that this depression of the cell-mediated immune response could be a factor in the altered responsiveness of the gingival tissues to plaque. However lymphocytes responses to Veillonella alkalescens ultrasonicate (a plaque antigen) were generally poor and it was not possible to demonstrate any significant difference between the altered responsiveness of maternal lymphocytes to Veillonella and those of nonpregnant controls.


Isolated pupillary paralysis in a case of herpes zoster. The clinical findings in a 16-year-old boy with unilateral isolated pupillary paralysis in a case of herpes zoster ophthalmicus is described because of the extreme rarity of the condition. The condition appeared to be due to a partial third nerve lesion affecting only the fibers subserving the light and near reflexes.


Potassium currents in frog ventricular muscle: evidence from voltage clamp currents and extracellular K accumulation. 1. The single sucrose voltage clamp technique was used to control the membrane potential of strips of frog ventricular muscle and to measure the membrane current. The extracellular K accumulation was estimated from the after-potential observed after the release of the voltage clamp. 2. Comparing the time course of the membrane current to the time course of the development of the after-potential at different membrane potentials, it was found that all slow current changes are related to changes in the K current across the membrane. 3. Based on measurements of membrane current and the after-potential, the total membrane current was separated into two fractions: (a) the K current which gives rise to K accumulation and (b) the residual membrane current which is unrelated to K accumulation. The current-voltage relation for the residual membrane current is linear or slightly inwardly-rectifying. Residual current is zero at the resting potential and increases to about 1 microamperemeter/cm2 at -20 mV. 4. The measured membrane currents and after-potentials indicate qualitative differences between the K currents which dominate below and above -20 mV. More negative to -20 mV the after-potential develops rapidly while at potentials positive to -20 mV the after-potential develops with some delay. 5. The current dominating below -20 mV is inwardly-rectifying. The current-voltage relation has a maximum (about 2 microamperemeter/cm2) and a region with marked negative slope conductance. The outward current in the region of negative slope conductance is increased with increasing [K]o. 6. A model for the inwardly rectifying K current is described. The model accurately reproduces the shape of the measured current-voltage relations and their modification by alterations in the extracellular K concentration. The model is also compatible with the observation that all slow current changes below -20 mV are directly related to K accumulation. 7. The K current which dominates at potentials positive to -20 mV is activated by a potential and time dependent process which is unrelated to extracellular K accumulation. 8. Q10 for the magnitude of the inwardly rectifying K current is about 1.35 while the Q10 for the rate of increase of the time dependent K current is about 3--4. 9. Cs blocks the inwardly recitfying K current but has little effect on the time dependent K current. 10. The changes in the action potential duration caused by increasing the extracellular K concentration or addition of Cs to the perfusate can be explained by the effect of K and Cs on the inwardly rectifying K current.


Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction. 1. The acetylcholine-sensitive ionic channels at the neuromuscular junction were studied in voltage-clamped single muscle fibres from a monolayer preparation of the cutaneous pectoris muscle from Rana pipiens. The experimental observations were of three types: (a) reversal potential as a function of external Na and Ca concentrations, (b) the single channel conductance (gamma) from noise analysis as a function of these same concentrations, and (c) gamma as a function of membrane potential. 2. The reversal potential in normal Na Ringer was -3.8 +/- 0.5 mV (+/- S.E. of mean, n = 22) and decreased approximately linearly as the logarithm of the outside Na activity as this activity decreased to 10% of normal. 3. The single channel conductance in normal Na Ringer was 27.5 +/- 0.7 pS (n = 28) and reached a limiting value close to 10 pS as Na was replaced with sucrose. 4. Increasing [Ca]o from 2 to 10 mM made the reversal potential more positive and decreased the single channel conductance. Mg caused similar effects. 5. Various theories that have been used to describe the mechanism of ion permeation through e.p.c. channels were tested. Constant field theory (eqns. (3), (4) and (5)), a modified Takeuchi approach (eqn. (6)), and a single barrier theory (eqns. (8), (9) and (10)) could not account for all of the experimental observations. 6. In particular, constant field theory, with no surface charge density, could account for the following: (a) the reversal potential measurements for solutions containing 2 mM-Ca (with PK/PNa = 1.2 and PCa/PNa = 1.02), (b) the single channel conductance values for solutions containing 2 mM-Ca and Na concentrations down to 20% of normal, (c) that gamma has little voltage dependence. 7. However, constant field theory, with no assumed surface charge density, could not account for the following: (a) the reversal potential observed for Ringer containing 80 mM-Ca, (b) the gamma values observed for very low Na concentrations, (c) the observation that increasing Ca from 2 to 10 mM in a solution containing 75% normal Na results in a decrease in gamma. 8. From the failure of the Takeuchi approach (eqn. (6)), it is argued that ion interactions must occur at e.p.c. channels because ion flux independence is the only asumption in the derivation of eqn. (6) without experimental verification. 9. The ion interactions at e.p.c. channels probably include both surface charge effects and competition for a binding site.


A dual effect of formaldehyde on the inwardly rectifying potassium conductance in skeletal muscle. 1. The inwardly rectifying potassium conductance of the membrane of frog sartorius muscle fibres is greatly reduced by treatment of muscles for 30 min with a solution containing formaldehyde (10 mM). 2. A transient increase in the conductance of the inward rectifier is observed early during formaldehyde action. 3. Analysis of the biphasic time course of the conductance changes, as determined under controlled voltage conditions, suggests that treatment with formaldehyde alters simultaneously, but in opposite ways, two factors that determine the conductance of the inward rectifier. 4. The linear component of the current-voltage relation, which dominates the relation at strongly positive potentials, is not affected while the above changes occur. But on prolonged exposure to formaldehyde the leak conductance increases. 5. The effects of formaldehyde on the inward rectifier are reversible on prolonged superfusion with normal Ringer solution. 6. The slight inward rectification remaining after most of the extracellular K is replaced by Rb, is similarly reduced by treatment with formaldehyde. 7. The results are interpreted in terms of the chemical properties of formaldehyde and present views of the mechanisms of inward rectification.


The measurement and dynamic implications of thin filament lengths in heart muscle. 1. The lengths of the thin filaments in amphibian and mammalian cardiac muscle have been determined from electron micrographs of serial transverse sections. Thin filament lengths in frog atrial trabeculae range from 0.8 to greater than 1.3 micrometers, with a maximum possible error of 0.14--0.15 micrometer. In rat atrial tissue the span is from 0.6 to more than 1.1 micrometer, whereas in rat papillary muscle the breadth of the distribution is much narrower, from 0.9 to greater than 1.1 micrometer. Double overlap of thin filaments should, therefore, exist over a wide range of sarcomere lenghts. Thin filaments from opposite halves of a sarcomere accommodate each other by flexing up to an angle of about 2 degrees and moving from the trigonal position among the thick filaments to the centre of the region between two thick filaments. Such rearrangement probably contributes to the internal resistance to shortening in the muscle. 2. Except for the variation in thin filament lengths, the over-all morphology of the cardiac sarcomere is generally similar to that found in skeletal muscle. Thick filaments in heart muscle are uniform in length, and their profiles change along their lengths. They are generally round in the M band, triangular adjacent to the M band, round again in the overlap region, and either round or triangular near the tapered tips. The M bridges in rat cardiac tissue link neighbouring thick filaments to form a symmetric hexagonal array, whereas in the frog atrium, the M bridge connexions are incomplete and often form isolated triangular clusters. 3. Computed sarcomere length-developed tension curves were calculated using the thin filament length distributions and the assumptions basic to the sliding filament theory of muscle contraction. The curves for atrial tissue have plateau regions approximately as wide as the one-half micron variation in thin filament length. 4. Work done against the internal loads during systole may be stored as potential energy and released during diastole to produce sarcomeric re-extension.


Extracellular potassium accumulation in voltage-clamped frog ventricular muscle. 1. Application of voltage clamp pulses (1--10 sec) to frog ventricular strips causes temporary changes in the extracellular K concentration. 2. The changes in the extracellular K concentration can be estimated from (a) slowly decaying post-clamp after-potentials, (b) changes in the action potential duration, and (c) measurements with a K-selective micro-electrode. 3. The depolarization of the resting potential and the shortening of the action potential are present in approximately the same proportions during voltage-clamp induced extracellular K accumulation and during perfusion with a K-ricn Ringer solution but small consistent differences are noticed. 4. The measurements of the after-potential, the action potential shortening, and the K-electrode response were analysed as indicators of extracellular K+ activity and it was concluded that the after-potential provides the most convenient and reliable estimate of the absolute magnitude of the voltage-clamp induced extracellular K accumulation. 5. The depolarizing after-potentials decay more slowly than the hyperpolarizing after-potentials but it is found that this reflects the selectivity of the membrane to K+ concentrations as predicted by the Nernst or the Goldman equations. 6. Analysis of the redistribution of accumulated K+ from the decay of the after-potential suggests that the major part of the redistribution process can be described by a single time constant (2--4 sec). A much longer time constant is required for a smaller component of the 'tail' in order to bring [K]o to the normal resting state. 7. N-shaped relations similar to the 'steady state' current-voltage relation are obtained when the post-clamp after-potential, the action potential shortening, and the K-electrode response are plotted versus the clamped membrane potential. The maxima of these curves are located around -40 mV and the minima around -20 mV. 8. In spite of a significant outward membrane current (1--1.5 microamperemeter) in the minimum region (-20 mV), the post-clamp after-potential is often hyperpolarizing in nature suggesting extracellular K depletion. 9. These findings indicate that the K efflux is lower at -20 mV than at both higher and lower potentials and suggest that the N-shape 'steady state' current-voltage relation mainly reflects the voltage dependency of the K current. 10. A theory for K accumulation in a single compartment is presented which predicts that a simple linear RC-circuit may describe the electrical response of the preparation in a limited potential range around the resting potential. The extracellular accumulation space was estimated to be 13--16% of the total volume of the preparation. It is tentatively suggested that the accumulation space is equivalent to the subendothelial fraction of the extracellular space.


The effect of aprotinin on luteolytic and uterine contractile mechanisms in the pregnant rat at term. The effect of the kallikrein inhibitor aprotinin on luteal function, uterine activity and parturition was studied in primigravid pregnant rats. Luteal function was monitored by the determination of serum progesterone levels. Aprotinin given daily from Day 19 to Day 22 of gestation had no effect on progesterone concentrations compared to saline-treated controls, but indomethacin delayed the decline in progesterone levels over the same time period. Aprotinin treatment had no effect on fetal and placental weights from Days 19 to 22 of gestation. Aprotinin infusion in Day-22 pregnant rats resulted in a reduction in uterine motility (studied by continuous recording in conscious rats by means of an intrauterine microballoon) in 10/12 rats. Continuous infusion of aprotinin into rats which had been allowed to deliver one young resulted in a significantly prolonged duration of parturition compared to that in saline-infused controls. In one rat the delivery process was completely arrested and recommended only when the infusion was stopped. Aprotonin had no effect on either the spontaneous or oxytocin-induced uterine contractions of the isolated Day-22 pregnant rat uterus. It is concluded that the kallikrein-kinin system in the late pregnant rat does not appear to be involved in the luteolytic process but may play a functional role in the control of uterine and/or cervical function before and during parturition.


Hemophilus influenza septicemia and midtrimester abortion. A case of midtrimester septic abortion due to Hemophilus influenza is discussed on the basis that the pathogeneses of the septic abortion was due to the hematogenous spread of the organism orginating from upper respiratory infection. The absence of ruptured membranes and the elevated antibody titer at the onset of the pelvic infection were in keeping with this hypothesis. Additional data on the bacteriology of this organism and the probability of the occurrence of a type b organism with original ampicillin resistance are presented.


Sepsis and upper gastrointestinal hemorrhage due to duodenal-caval fistula: complications of delayed treatment of a penetrating abdominal injury (case report). Sepsis and upper gastrointestinal hemorrhage due to duodenal-caval fistula complicated delayed treatment of an abdominal gunshot wound. Gastric and duodenal decompression, external drainage of the duodenal repair, and caval ligation are preferred for delayed treatment of combined duodenal and infrarenal vena caval injuries.


Effects of transcutaneous nerve stimulation on the vesicourethral function in spinal cord injury patients. There were 17 spinal cord injury men with drug-resistant pain who were treated with transcutaneous nerve stimulation. Bladder and sphincter dysfunctions were evaluated by gas cystosphincterometry and rectal sphinctero-electromyography with the patient in 2 different positions. With the use of transcutaneous nerve stimulation for pain we noted no demonstrable urodynamic changes in acute and chronic paraplegics and chronic quadriplegics. However, there was an increase in the urethral pressures and in the rectal electromyogram spike potentials in acute and recent (less than 2 years post-injury) quadriplegics with postural detrusor-striated sphincter dyssynergia. Therefore, caution is necessary when transcutaneous nerve stimulation is used in this particular group of patients since it might be detrimental to the genitourinary tract.


New pathways in portal hypertension surgery. An individualized treatment of portal hypertension is advocated. The treatment is suggested to be based upon the presence of complications to the disease: bleeding oesophageal varices, insufficient cardia function, regurgitation and oesophagitis, hyperacidity, stomach and duodenal ulcer, ascites and hypersplenism. The choice of method of treatment of the patient depends on the presence of the symptoms. There are several methods available. These can be divided in methods directed against one symptom - unisymptomatic treatment - and methods directed against several symptoms - polysymptomatic treatments. The author advocates a more frequent use of decongestion operations and pexi operations. For acute control of bleeding varices it seems that sclerotherapy is the preferred choice at present.


Selective coronary-caval shunt using internal jugular autograft, in the treatment of portal hypertension. The selective coronary-caval shunt is considered to be one of the better procedures in the surgical treatment of portal hypertension. We have altered the previously described technique by using an internal jugular vein grafting which possesses some advantages. We have performed this shunt utilizing an internal jugular vein autograft in eight patients, who had at least one major hemorrhage from esophageal varices three to twelve months before operation. There were no postoperative deaths. All four patients are still alive three months to six years after shunting without bleeding episodes. In the X-ray and endoscopic studies the esophagogastric varices were noted to be markedly smaller in size than they were before shunting.


Inhibition of in vitro Friend murine leukemia virus infection of lipopolysaccharide-activated B-cells with concanavalin A. Stimulation with bacterial lipopolysaccharide (LPS) of splenic B-lymphocytes infected in vitro with Friend virus complex increased the number of cells with replicating murine leukemia virus (MuLV) [i.e., infectious centers (IC)] up to 100-fold. Concanavalin A (Con A) did not have such an effect. However, the addition of Con A to the LPS-stimulated cultures decreased the number of IC. The inhibitory concentration of Con A (2.5 microgram/ml) was eightfold less than that capable of neutralizing the in vitro infectivity of MuLV (20 microgram/ml). The effect of Con A was not mediated by T-cells; the inhibition of infection was comparable with use of whole spleen cell suspensions from normal BALB/c mice, with T-cell-depleted cell suspensions, or with spleen cells with congenitally athymic nude mice. However, specific removal of Con A from the surface of B-cells with alpha-methyl-D-mannopyranoside prior to the infection reversed the inhibitory effect entirely. It is suggested that the lectin interferes with MuLV on the membrane of B-cells.