Selective electrode for dibenzyl dimethyl ammonium cation as indicator of the membrane potential in biological systems.

The electrode sensitive to dibenzyl dimethyl ammonium (DDA+), which is considered to be an indicator of the membrane potential, was constructed by using tetraphenyl borone (TPB-) embedded in dichloroethane. Rapid and Nernstian responses were exhibited against DDA+ solutions ranging between 10(-2) and 3 - 10(-6) M in concentration. High selectivity for DDA+ was observed in the presence of various inorganis salts, ADP, ATP, oxidizable substrates and sugars. The electrode developed here was used to measure the DDA+ uptake in Streptococcus faecalis and the results agreed with those reported by Harold, F.M. and Papineau, D. ((1972) J. Membrane Biol. 8, 27-44 and 45-62). While they determined the DDA+ concentration in the medium by measuring the absorbance of the filtrate treated with the ion-exchangers, the electrode can measure directly the DDA+ concentration in the bacterial suspension without any any pretreatment. It was also shown that the electrode can measure the DDA+ uptake in mitochondria during energization.


Ribosomal RNA genes in Bacillus subtilis. Evidence for a cotranscription mechanism.

The analysis of the transcriptional mechanism of the ribosomal RNA genes in Bacillus subtilis was undertaken by a study of the rRNA chain elongation in the presence of rifampicin. The residual RNA synthesis after the addition of rifampicin and [3H] uridine to exponentially growing cells has shown that 56% of the radioactivity incorporated into total RNA belongs to the unstable fraction and 44% to the fraction containing mature rRNA and tRNA. Such study allowed an estimation of the half-life of messenger RNAs as being approximately 2 min. The analysis of the transcription pattern of the ribosomal RNA genes, as measured by the amount of radioactivity found in the ribosomal subunits, was complicated by a contamination of the 30 S subunits by 50 S subunits. A contamination of approximately 15% was estimated by polyacrylamide gel electrophoresis and competitive hybridization. The ratios of incorporated radioactivity at zero time when drug and label were concomitantly added ranged between 5.4-6.0, after correction for this contamination. The decay of the 23 S rRNA followed a straight line which became parabolic in its final portion. These results, and theoretical considerations on the lag of rifampicin action and on the variance of the specific activity of the nucleotide pool at the very early times of the experimental observation, favor the interpretation that the 16 and 23 S rRNA genes in B. subtilis belong to the same transcriptional unit, being cotranscribed, in that order, by the same molecule of RNA polymerase. The transcriptional times of the 16 and 23 S rRNA genes were estimated as being 30 and 60 s, respectively.


Heterologous transfection with bacteriophage phiX174 DNA. Unusual heterogeneous products.

Hydroxylamine-resistant infectious materials (HARIM) synthesized in natural non-host and progeny phage low productive bacterial spheroplasts upon transfection with bacteriophage phiX174 DNA were found to be unusually heterogeneous in their forms. Using Pseudomonas aeruginosa as a source of HARIM, it was shown that they have the following unusual features. (1) Almost all of the HARIM are denser than normal single-stranded (SS)- and double-stranded replicative form (RF)-DNAs of phiX174 found usually in the phage-infected host cells. (2) A great part of these heavy HARIM (approximately 84%) contain a variable length of single-stranded RNA associated with their infectious elements. (3) For most of the HARIM (approximately 80% of total molecules as the infectious elements of the heavy HARIM), the infectious elements are phiX-RFI-DNA. The wide-spread system for phiX-HARIM synthesis was shown to be present in many gram-negative bacterial cells.


Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.


Purification of the intestinal receptor for intrinsic factor by affinity chromatography.

The intestinal receptor for the intrinsic factor vitamin B-12 complex has been solubilized and then purified from the guinea pig ileum using a double structured affinity resin comprised of intrinsic factor coupled to vitamin B-12 which, in turn, was covalently linked to Sepharose 4B. The receptor purified approximately 57 000-fold from the crude homogenate, appears to be a homogenous protein which may be composed of two subunits which separated when the preparation was subjected to polyacrylamide disc gel electrophoresis. Ethylenediaminetetraacetic acid induced dissociation of the complex between the purified receptor and intrinsic factor-B-12 could not be reversed by the addition of excess Ca2+, unlike the effect of EDTA with semipurified receptor or crude ileal homogenates. Calcium reversed the EDTA effect only after the mixture was subjected to extensive dialysis suggesting that the chelating agent interacts directly with the receptor protein. Intrinsic factor-vitamin B-12 competively inhibited the binding of intrinsic factor-[57Co] vitamin B-12 to the purified receptor whereas vitamin B-12 free intrinsic factor did not, even at a 100-fold greater concentration.


Human pituitary thyrotropin. Characterization of five glycoproteins with thyrotropin activity.

Human pituitary thyrotropin prepared by chromatography on hydroxyapatite or on SP-Sephadex was fractionated into five active components by preparative poly-acrylamide gel electrophoresis. The potency of the five components was 4-9 units human Research Standard A/mg. Examination of the components by analytical electrophoresis and by immunological methods revealed no heterogeneity. Ultracentrifugaiton of the three major components showed homogeneity with sedimentaiton coefficinets in the range of 2.4-3.0 S and a value for the molecular weight of about 33 000. Amino acid and carbohydrate analyses indicated close similarites between the five components.


Human pancreatic lipase: a glycoprotein.

Human lipase has been purified from pancreatic juice. The protein has a molecular weight of 48 000 and an N-terminal residue of lysine. It has been characterized as a glycoprotein containing 4.7 mol of glucosamine, 2.8 mol of mannose, 2.9 mol of fucose, 3.0 mol. of galactose and 1.1 mol of glucose per mol of protein. Two isolipases have been separated by polyacrylamide gel electrophoresis. Their isoelectric points are 5.80 and 5.85, respectively and both are glycoproteins. Immunological cross reactions have been obtained between human lipase and other mammalian lipases (porcine, bovine, ovine, canine and rat).


Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.


The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.


Effect of glutaraldehyde treatment on enzyme-loaded erythrocytes.

In principle, enzyme-loaded erythrocytes can be used as a vehicle for enzyme replacement therapy in lysosomal storage diseases. Glutaraldehyde treatment renders these erythrocytes more resistant to lysis without inactivating the enzymes that have been entrapped inside them. Glutaraldehyde treatment does not prevent ingestion of enzyme-loaded erythrocytes by macrophages in vitro so that these cells can be used to deliver enzymes to lysosomes. In vivo, the glutaraldehyde-treated cells are quickly removed from the circulation by the spleen or liver. The degree of glutaraldehyde treatment allows the erythrocytes to be targeted either to the spleen (low glutaraldehyde concentrations) or to the liver (higher glutaraldehyde concentrations).


In vitro stimulation of primate hemoglobin synthesis by L-thyroxine.

Bone marrow cells from adult and abortus primates (marmosets) were incubated in vitro to determine their responsiveness to L-thyroxine. 3H-leucine incorporation into purified globin chains was the parameter assayed to determine responsiveness. Bone marrow from spontaneously aborted animals consistently was stimulated by the presence of physiologic levels of L-thyroxine. Bone marrow cells from adult animals were unaffected by the hormone.


The effects of EDTA and EGTA on renin secretion.

1 The effects of the disodium salt of ethylenediamine tetra-acetate (EDTA) and 1, 2, bis, 2 aminoethoxyethane-NNN'N'-tetra-acetic acid (EGTA) on renin secretion and vascular resistance were studied in the isolated perfused kidney of the rat. 2 Both substances produced a significant increase of renin release 3 In the absence of calcium and magnesium, EDTA still increased rein release and there was now a considerable increase of perfusion pressure. 4 The rise of pressure but not the rise of renin was inhibited by the removal of potassium from the perfusate when EDTA was administered in the absence of calcium. 5 Propranolol and phenoxybenzamine had no effect on the vasoconstrictor action of EDTA. 6 EGTA was less effective as a renin releaser than EDTA until magnesium was removed from the perfusate. Furter, it had only a small effect on perfusion pressure in contrast to EDTA.


Effect of indomethacin and related drugs on the calcium ion-dependent secretion of lysosomal and other enzymes by neutrophil polymorphonuclear leucocytes in vitro.

1 Rabbit isolated peritoneal neutrophil polymorphonuclear leucocytes were depleted of calcium by exposure for 1 h to calcium-free bathing fluid at 4 degrees C. 2 Addition of calcium ions to the previously calcium-depleted calls during incubation at 37 degrees C stimulated the release of beta-glucuronidase and of lysozyme but not of lactate dehydrogenase. 3 Low concentrations of indomethacin, flufenamate or salicylate, such as those which occur in the blood plasma after therapeutic doses of these drugs, selectively inhibited the calcium-induced release of beta-glucuronidase. The slight release of this enzyme which occurred in the absence of added calcium ions was not altered by these drugs, neither was the  release of lactate dehydrogenase. 4 Release of lysozyme was inhibited by low concentrations of salicylate, amidopyrine or oxyphenbutazone, independent of the presence or absence of calcium ions. 5 Chloroquine, hydrocortisone or colchicine did not alter the release of leucocyte enzymes.


Deleterious effects of prolonged electrical excitation of striate cortex in macaques.

Macaques were trained to respond to electrical excitation applied through electrodes permanently implanted within or upon striate cortex. Threshold current for the animal to detect this stimulation was highly consistent from day to day and, in the absence of tissue encapsulation of the electrodes or deliberately inflicted damage, remained stable indefinitely, 38 months in the longest case so far. Stimulating continuously for 1-8h, however, produces an elevation of threshold, which may be permanent or temporary, depending upon a variety of conditions. A major cause of such injury is the hydrolysis commonly occurring consequent to passage of low-level currents between solutions and metal electrodes. Even when the hydrolytic reaction is eliminated by restricting the level of electrode polarization or by using capacitative stimulation with tantalum pentoxide electrodes, a rise in threshold often still occurs with protracted stimulation. With proper control in some instances, however, effective stimulation at 2-10 times the threshold level could be maintained indefinitely without apparent injury, e.g. in a blind monkey having a threshold of 290 muA that could respond immediately to an 80-muA diminution in 580-muA, 0.2-msec stimulus pulses which had been applied steadily for 1 h at 50 Hz.


Action of inhibitors on monoamine and diamine metabolism in the rat.

The effects of a series of inhibitors of monoamine oxidase (EC 1.4.3.4) and diamine oxidase (EC 1.4.3.6) and of two chelating agents were studied in rats, with respect to the catabolism of labeled pentylamine and putrescine to radioactive carbon dioxide. D-Tranylcypromine, clorgyline, and deprenyl inhibited oxidation of the monoamine, with essentially no effect on putrescine, under our test conditions. Aminoguanidine inhibited putrescine but not pentylamine oxidation. Iproniazid, isoniazid, and Lilly 51641 affected the catabolism of both amines. Pargyline inhibited putrescine oxidation, apparently in a reversible manner.


Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.


Rh isoimmunization, Manitoba, 1963-75.

The number of Rh-isoimmunized pregnancies in Manitoba has been reduced from 223 and 228 in the years ending Oct. 31, 1963 and 1964 to 60 and 62 in the years ending Oct. 31, 1974 and 1975. The number per 1000 total births in the same years has decreased from 10.0 and 10.6 to 3.4 and 3.5 Perinatal mortality rates in those years decreased from 13.8 amd 15.7% to 0 and 2.2%, respectively. The number of perinatal deaths has been reduced from 55 in the first 2 years reported to 1 in the last 2 years. Among the 121 isoimmunized women pregnant in the 2-year period ending Oct. 31, 1975, isoimmunization was due to failure to give Rh immune globulin after delivery in 33 and failure to give it during pregnancy in 48. Of the remaining 40, 37 were immunized before Rh immune globulin became available. Complete prevention of Rh isoimmunization and therefore of all perinatal deaths from Rh erythroblastosis can only be achieved through universal Rh testing prenatally and immediately after delivery, and institution of an antenatal Rh prophylaxis program.


Isolated thyrotropin deficiency due to a pituitary tumour.

Hypothyroidism due to isolated deficiency of thyrotropin (TSH) associated with an enlarged sella turcica, presumably the result of a nonfunctioning pituitary adenoma, occurred in a 58-year-old man. Low serum concentrations of TSH and thyroid hormones, together with the lack of TSH response to administration of thyroid releasing hormone, indicated a pituitary deficiency of TSH. Serum values of other pituitary hormones were normal.


Intravenous hyperalimentation as an adjunct to colon interposition for carcinoma of the esophagus.

Ten consecutive malnourished patients with undifferentiated squamous cell carcinoma of the esophagus underwent operation for colon interposition. They received intravenous hyperalimentation preoperatively and/or postoperatively for an average time of 21.5 days and had an average weight gain of 6.5 lb. There was no significant gastrointestinal morbidity, but one patient succumbed to aspiration pneumonia. All ten patients had postoperative gastrografin swallow radiography which showed no leaks in either anastomosis. With proper use of intravenous hyperalimentation, morbidity and mortality following colon interposition in the malnourished patient for either palliative or curative bypass can be decreased to acceptable levels.


Stages I--III Hodgkin's disease in children: results of staging and treatment.

Fifty-two children with clinical stages I-III Hodgkin's disease were evaluated for disease extent between April 1969 and March 1975. All underwent laparotomy and splenectomy. Two patients with liver involvement were excluded. Thirty of 31 patients with pathologically staged IA-IIA disease have been continuous complete remission after mantle and para-aortic irradiation. There have been no extensions into the untreated pelvis. Fourteen of 15 patients with pathologic stages IIB and IIIB disease show no evidence of relapse after TNI and MOPP. Three of four patients with stage IIIA disease developed nodal relapse after irradiation; all are alive without evidence of disease after re-irradiation (3) and MOPP (2). Thus 45 of 50 patients (90%) have remained continuously free of disease after completion of the planned treatment, and overall 49 of 50 (98%) are alive, without evidence of disease. Such results justify continuation of our staging and treatment philosophy in children with Hodgkin's disease.


Radiation therapy of carcinoma of the tonsil.

The results of treatment with radiation therapy of 65 cases of epidermoid carcinoma of the palatine tonsil have been reviewed. The major determinant of prognosis is the stage of the primary lesion. Failure usually results from the inability to control local grossly detectable tumor. Thirteen of thirteen T1 lesions but only two of sixteen T3 lesions were locally cured with external beam radiation therapy. No difference in survival was noted between patients with no clinical adenopathy vs those with clinically involved movable ipsilateral adenopathy.


Intravenous hyperalimentation as an adjunct to radiation therapy.

Radiation therapy may induce anorexia with resultant weight loss and inanition that can limit the dose of radiation therapy administered. The purpose of this study was to evaluate 39 nutritionally-depleted patients who had a variety of malignant diseases treated with radiation therapy and concomitant nutritional support with intravenous hyperalimentation (IVH). The average dose of radiation delivered was 3827 rads in an average of 3.5 weeks. Ninety-five percent of the patients completed their planned course of radiation therapy and improved symptomatically. Fifty-four percent of the patients responded with a greater than 50% reduction in tumor size. Responding patients gained an average weight of 13.0 +/- 6.5 lbs. during IVH (av. 36.2 days) and radiation therapy (av. 3832 rads), whereas non-responding patients gained only 4.9 +/- 8.8 lbs. (p less than 0.001) during IVH (av. 42.8 days) and radiation therapy (av. 3819 rads). Serum albumin concentrations rose from 3.12 +/- 0.49 gm/100 ml to 3.51 +/- 0.68 gm/100 ml (p less than 0.05) during treatment in responding patients but did not rise significantly from 3.09 +/- 0.48 gm/100 ml in non-responding patients. In conclusion, IVH allowed a planned course of radiation therapy to be delivered to a group of poor-risk, malnourished cancer patients, and a positive correlation between tumor response and nutritional status was identified. Moreover, IVH was a valuable adjunct in the treatment of six patients who had enteric fistulas that originated from radiated bowel.


Hyperbaric oxygen as a radiotherapeutic adjuvant in advanced cancer of the uterine cervix: preliminary results of a randomized trial.

From September 1968 to March 1974, a randomized clinical trial was carried out, using conventional fractionation, i.e., five treatments per week, in 233 patients with advanced cancers of the uterine cervix--Stages IIB, IIA, IIIB and IVA. The age limit was 70 years and all patients had medical clearance. Lymphangiography and, in some patients, an exploratory laparotomy with selective lymphadenectomy, were done prior to treatment to determine the extent of nodal disease. The staging has not been changed either by lymphangiogram or lymphadenectomy findings. A few patients with bulky Stage I and IIA lesions were entered into the trial because of extensive nodal disease demonstrated either by lymphangiogram and/or lymphadenectomy. First, the patients were grouped according to the clinical stage. The secondary stratification was according to the lymphangiogram and/or selective lymphadenectomy findings. The patients were then randomized to air or hyperbaric oxygen within each group. The patients were pressurized in a Vickers chamber at 3 atmosphere absolute, using a 20-minute soak time prior to the irradition. The size of the external beam portal was determined by the status of the nodes. The difference in absolute NED (no evidence of disease) survival rates for both groups as a whole and by stages is not statistically significant. There is no difference in the incidence of failures in the irradiated area between the HPO and air patients. There is no increase in distant metastases in the HP group. It does not seem that the HPO has had an effect on the major complications. However, there was an increase in the incidence of complications with extended fields. The addition of lymphadenectomy had increased the incidence of fatal complications, even with routine pelvic portals. The negative results of this trial with conventional fractioantion should not lead to the conclusion that HPO could not be useful with schemes using a few high dose fractions.


Effect of thymosin in vitro on T cell levels during radiation therapy: correlations with radiation portal and initial T cell levels.

The effect of thymosin in vitro on percent T cells was determined in 388 blood specimens from patients with head and neck, mediastinal, and pelvic malignancies during radiation therapy, in 94 untreated patients with these malignancies, and 277 normal adults. Changes in percent T cell levels after incubation of lymphocytes with thymosin did not correlate with tumor histology or cumulative radiation dose, but in all groups correlated with radiation portal and initial T cell levels. T cell levels increased by a similar increment in normals and in the untreated patients. During irradiation, the mean levels after incubation with thymosin did not change in patients with head and neck and pelvic malignancies, but in patients with mediastinal malignancies the levels increased significantly more than in normals. For a given T cell level, the increase in patients with mediastinal malignancies was greater than in patients with pelvic malignancies, and as a group was greater than in patients with head and neck malignancies. The results can be explained by an increase in circulating thymosin-responsive lymphocytes during mediastinal irradiation due to suppression of a function of the thymus important for maturation of these cells, and a decrease in these cells during pelvic irradiation due to a deleterious effect on precursors in pelvic bone marrow. The results thus provide a rationale for clinical trials to assess the efficacy of thymosin in averting declines of T cell levels in patients receiving mediastinal irradiation.


Chronic myelogenous leukemia: management of splenectomy in a high-risk population.

Splenectomies have been performed on 58 patients with chronic myelogenous leukemia (CML) during the last 16 years. For the 27 patients operated upon during the first 12 years, the operative mortality was 26%. Four patients also had to be re-explored for bleeding and three required drainage of subphrenic abscesses. There has been no operative mortality in the 31 patients operated using a standardized procedure during the last four years but two had to be re-explored for bleeding and one required drainage of a subphrenic abscess. The operative risks for leukemic patients are infection, perhaps related to granulocyte abnormalities and hemorrhage in patients with thrombocytopenia or qualitative platelet abnormalities. When the splenectomy in patients with CML is timed with the patient's chemotherapy cycle, the use of local antibiotics and platelet concentrates should permit a safe operation. Survival rates were not comprised even when the patients had their splenectomy during CML-blast crisis.


Combined therapy for endometrial carcinoma preoperative intracavitary irradiation followed promptly by hysterectomy.

A group of 99 patients with endometrial cancer clinically confined to the uterus has been treated with preoperative adjunctive intracavitary irradiation followed within 72 hours by total abdominal hysterectomy and bilateral salpingoophorectomy. Those patients with poorly differentiated tumors, deep myometrial invasion, cervical involvement, or pelvic metastasis were then considered for external beam radiotherapy to the whole pelvis because of the increased risk of involvement of pelvic nodes or other pelvic structures. Only one of 16 patients so treated has failed in the pelvis. The overall Berkson-Gage actuarial survival at 3 years, uncorrected for death from intercurrent disease, is 85.8%. No vaginal recurrences have been identified. Although this series has not been randomized, it appears that there has been a reduction in vaginal and pelvic recurrences when compared with patients reported by others who have received no adjuvant radiotherapy. The low recurrent rate and favorable survival in this group of patients was achieved with low morbidity from this treatment technique.


Management and results of endometrial carcinoma treated at Instituto Português de Oncologia de Francisco Gentil.

The experience of 260 patients with endometrial carcinoma was reviewed. The influence of factors such as age, stage of disease, grade and degree of myometrial penetration on the survival was presented, showing that survival decreases in elderly patients, in patients with advanced stage of disease, when the tumor is undifferentiated, and when the tumor deeply penetrates the myometrium. The methods of therapy, fall into three main groups: surgery, radiotherapy, and combined therapy, the latter yielding the best 5-year survival rate, in all stages. The incidence of vaginal recurrences was low, probably due to the fact that 68.8% of the patients were treated by a combined therapeutic modality.


Radiation treatment planning.

Radiation treatment planning has developed into a substantial and effective component of the entire radiation treatment approach. Over the last 2 decades, with the development of high energy electron and x-ray sources, and also with the availability of new radionuclides and techniques for internally applied radiation, all of which permit a high degree of concentration of radiation, treatment planning has been developed to make best use of these modalities. The use of automatic computation has proved necessary in order to handle the large amounts of radiation data involved in treatment dose calculation. Tumor and anatomical localization has been carried out with increasing precision. Several cases are described which illustrate planning and in particular the use of computerized transverse tomography. The use of moving shadow-shields is also described. For internally applied radiation, the features of iodine-125 are illustrated.


Supportive therapy in radiation oncology.

Measures for supportive care of the radiation therapy patient are presented. These include emotional support prior to and during the course of therapy facilitated by a written interview that allows the radiation oncologist to be a supportive communicator of realistic information. A discussion is made of the support of body tissues affected by combination radiation and chemotherapy. These tissues usually include skin, oral, esophageal and intestinal mucosa, and teeth. Means of maintaining nutritional support following weight loss of patients during therapy are described.


High-energy photon and electron beam.

The optimum radiation treatment plan for any given clinical situation can be achieved by combining various irradiation modalities and beam energies. The availability of equipment that provides photon and electron beams of energies from 4 MeV to 25 MeV permits optimal dose distribution throughout the treatment volume. Since no difference in the biological effectiveness of electrons compared with megavoltage photons has been demonstrated in laboratory studies, there is no hesitation in combining electrons with photons. The selection of the various energies, the combination of electrons with photons, and the ratio of the given doses of each beam depend on the location of the tumor and the maximum depth to be treated. With the use of one beam alone, a combination of 25 MeV and 4 to 6 MeV photon beams, or a combination of photons and electrons, the most effective treatment plan with the available beams can be designed for any clinical situation.


Particle radiation therapy: current status and future potential.

Radiation therapy with "heavy" particles offers potential biological and physical advantages compared to irradiation with low LET photons. Clinical studies are in progress with proton beams, which have dosimetric advantages, and fast neutron beams, which have potential biologic advantages. Clinical studies with negative pi mesons and heavy nuclei, which have combined dosimetric and biologic advantages are about to start.


Soft part tumors.

Results of management of rhabdomyosarcoma of childhood have improved in a dramatic manner during the recent 10 years as a consequence of the treatment by three modality approach which relies on an intensive multi-drug multicycle chemotherapy regimen combined with radiation therapy and or surgery. Both local results and total disease-free survival rates are markedly better with this more comprehensive approach. A staging system for sarcoma of soft tissue has just been developed by the A.J.C. Histopathological grade is the important parameter: stage 1,2, and 3 are tumors of histological grades 1, 2, and 3 respectively (an outline of the system is presented). Treatment results of 100 patients with sarcoma of soft tissue (extremities 89, torso 11) treated by radical dose radiation therapy (less than 6300 rad) and limited surgery at M.D. Anderson Hospital are presented. For both local control and disease free survival, results decreased with advancing stage and anatomic site was not a factor per se. Radiation therapy under tourniquet induced hypoxia was not found to be significantly more effective than conventional radical dose therapy. The necessity of planning treatment such that if subsequent surgery is required, the fields will provide the best distribution of unirradiated or low dose tissue for preparation of flaps, etc.


Radiation in bone sarcomas: a re-evaluation in the era of intensive systemic chemotherapy.

We have reviewed the literature and described experience in treating Ewing's sarcoma and osteosarcoma before and during the era of intensive systemic chemotherapy. Local control of Ewing's sarcoma may relate to increasing doses of radiation, especially when intensive chemotherapy is administered also. Problems of radiation enhancement by chemotherapy have caused us to reconsider time-dose and volume parameters in treating these patients. The role of radiation in osteogenic sarcoma is limited to patients with inoperable lesions and metastases.


Radiotherapy in Hodgkin's disease: past achievements and future progress.

Improvements in results of treatment for Hodgkin's disease have been dramatic in the last 20 years. The results of high dose radiation therapy are reviewed in terms of the importance of the extent of the field of irradiation. The importance of adjuvant chemotherapy is also discussed. Problem areas both in staging and in general management of patients with Hodgkin's disease are emphasized. Long-term complications of treatment, both radiotherapeutic and chemotherapeutic are stressed. The excellent results of management for stage I, II (A and B), and IIIA Hodgkin's disease have reached a level at which future studies must aim at reducing the morbidity of treatment.


Radiation oncology: cancer of the prostate.

The role of palliative radiation treatment of prostate cancer is well recognized. Appreciation of the value of definitive radiation therapy in management of locally advanced prostate cancer is increasing. Optimal management requires careful patient selection with multidiscipline evaluation to provide accurate grading and staging, availability of adequate facilities, and careful planning the treatment. Definitive radiation therapy may be used as primary treatment, or in management of endocrine treatment failure, and in postoperative residual or recurrent cancers. Similar techniques may be employed in the management of locally symptomatic Stage D cancer. Definitive radiation therapy is useful management of some Stage B and many Stage C locally advanced and nonresectable cancers of the prostate.


The present status and future potential of radiotherapy in the management of esophageal cancer.

The management of 2032 patients with squamous esophageal cancer seen in Edinburgh from 1931 to 1969 was studied. Since 1949 more radical surgical treatment has been offered and since 1956 more radical radiotherapy. From 1949 to 1969 there has been a  swing over from mainly surgical to mainly radiation treatment associated with a rise in the overall one and five-year survival rates to 32% and 9% respectively. The main reason for failure is the prevalence of disseminated disease. The significant mortality of surgical treatment limits its usefulness. Too often, radiotherapy fails because of recurrence of tumor within the irradiated volume. In the upper 2/3 of the esophagus irradiation is preferable to surgery because of better sirvoval. In the lower 1/3 the two methods achieve similar long term survival but following irradiation the short term survival is better and the patient retains a normal stomach. Improvement could be achieved by prevention, earlier diagnosis, improved tumor delineation and a wide range of measures to improve the local effectiveness of irradiation.


Radiation therapy as initial treatment for early stage cancer of the breast without mastectomy.

This report describes 150 patients with clinical stage I and II carcinoma of the breast treated at four institutions--Yale University School of Medicine, Harvard Medical School-Joint Center for Radiation Therapy, Hahnemann Medical College, Jefferson Medical College--with radiotherapy only following excisional biopsy. Closely similar treatment policies were followed at all four centers, 4500-5000 rads minimum tumor dose being delivered to the entire breast and axillary, supraclavicular and internal mammary nodes. Forty-six of 49 stage I patients treated are alive without disease, the actuarial relapse-free survival being 91% at 5 years. Of the 101 stage II patients, 75 are alive without disease with a relapse-free actuarial survival of 60% at 5 years. Local failure has occurred in 10 patients (9 stage II and 1 stage I, 6.6%) 5 of whom are disease-free following mastectomy. The results obtained in this study are comparable to those of conventional surgery. It is our conclusion that mastectomy is not a necessary part of the treatment of small breast cancers, that radiation without mastectomy is an acceptable alternative with far superior cosmetic and functional results. Adjuvant chemotherapy should be considered particularly in stage II patients in view of their 40% relapse rate.


Radiotherapy in the postoperative treatment of operable cancer of the breast. Part I. Critique of the clinical and biometric aspects of the trials.

A statistical assessment of several "randomized" clinical trials was carried out and the following conclusions were noted. 1. There is no statistically significant evidence that radiation to the peripheral lymphatics and/or chest wall following radical mastectomy affects survival. 2. There is significant evidence that postoperative irradiation to the peripheral lymphatics and/or chest wall does decrease local recurrence rate. 3. There is reliable evidence that the treatment of local recurrence, once it develops, is successful in only 50% of the cases. 4. There is evidence that radiation plus simple mastectomy is as effective in the treatment of breast cancer as radical mastectomy alone. It is our considered opinion that radiation remains a useful adjuvant and complementary agent in the treatment of breast cancer, is not harmful vis a vis survival and should be used when and where indicated.


Carcinoma of the cervix: present status and future.

The incidence of invasive carcinoma of the cervix is decreasing in Puerto Rico and Continental U.S.A. This appears to be related to improvements in socioeconomic level and adequate utilization of the Pap smear in the population. Mortality from carcinoma of the cervix has shown a 60% reduction in the last 20 years. Most likely this is related to the observed reduction in the incidence  of the invasive forms of the disease, earlier diagnosis, and improvements in therapy. Optimal therapy of the clinically evident invasive forms of the disease is a combination of external irradiation and intracavitary brachytherapy. Surgery would be of value for specific clinical situations as an adjuvant to irradiation (barrel-shaped types), in post-irradiation recurrences, inadequate brachytherapy, etc. The yield in terms of survival and disease-free status in the pelvis is high for the early stages of the disease (approximately 90% 5-year survival and 97% control of pelvic tumor for stage I), but stage IIIB and IV cases show a failure rate of close to 50% or more in the irradiated volume and a high incidence of metastases to the para-aortic nodes and elsewhere.


Carcinoma of the testis.

Patients with Stage I or II malignant testis germ cell tumors underwent a randomized prospective study at Walter Reed Hospital, Washington, D.C., from 1968 to 1973. Pure cell lines of seminoma or choriocarcinoma were excluded. Forty patients had inguinal orchiectomy followed by irradiation to the inguinal, iliac, lumbar para-aortic, mediastinal and supraclavicular lymph nodes. Thirty-four patients (85%) in this group are alive and free of tumor a minimum of 3 years. Fifty-one patients received pre- and postoperative irradiation to primary lymphatic pathways in association with bilateral retroperitoneal lymphadenectomy as well as elective irradiation to the mediastinum and supraclavicular regions. Forty-six patients (90%) in this second group are alive and free of tumor a minimum of three years. Both treatment methods represent marked improvement in cure rates compared to lymphadenectomy and postoperative irradiation utilized prior to 1968, but no statistically significant difference from each other.


Hyperplastic alveolar nodules of the rat mammary gland: tumor-producing capability in vivo and in vitro.

Carcinogen-induced hyperplastic alveolar nodules failed to form tumors after transplantation into isologous female rats; instead they developed ductal or ductal-alveolar outgrowths. Mammary glands from mid-pregnant female rats showed similar outgrowths following transplantation to the isologous hosts. In vitro and in vivo studies failed to demonstrate that subcarcinogenic doses of 7,12-dimethylbenz(a)anthracene can induce mammary tumorigenesis in the hyperplastic alveolar nodules. Our observations suggest that carcinogen-induced hyperplastic alveolar nodules are not 'preneoplastic' lesions in mammary carcinogenesis in the rat.


Molecular structure of the K-region cis-dihydrodiol of 7,12-dimethylbenz[a]anthracene.

The molecular structure and conformation of the cis-5,6-dihydrodiol of 7,12-dimethylbenz[a]anthracene has been determined by an X-ray crystallographic analysis. The compound crystallizes in the space group P21/a with cell dimensions a equals 17.799(6), b equals 33.211(8), c equals 5.241(1) A, beta equals 91.88(2)degrees. There are two molecules, designated A and B in the asymmetrical unit, that are not related to each other by crystallographic symmetry. Their conformations are almost identical, and there are no significant differences in their bond lengths or angles. In both molecules the 5-hydroxyl group is equatorial while the 6-hydroxyl group is axial. This conformation is probably forced by steric hindrance between the hydroxyl group, 0-6, and the hydrogen atoms of the 7-methyl group. The molecules pack in the crystal by forming hydrogen bonds between the hydroxyl groups of adjacent molecules, A with A, B, with B, and A with B. The ring system of the cis-5,6-dihydrodiol is much more buckled than is that in 7,12-dimethylbenz[a]anthracene itself. The angle between the two outermost rings is 36 degrees, the deviation from planarity being primarily a consequence of the partial saturation in the ring containing the two hydroxyl groups. Extrapolation of these results to other dihydrodiol derivatives of carcinogenic hydrocarbons permits some predictions of preferred molecular geometry. Thus, the 8,9-dihydrodiol-10,11-epoxide of 7,12-dimethylbenz]a[anthracene, analogous to the biologically active 7,8-dihydrodiol-9,10-epoxide of benzo]a[pyrene, a mutagen that is believed to be an active intermediate in carcinogenesis by benzo]a[pyrene, should probably exist preferentially in a conformation bearing the8-hydroxyl group in the axial orientation.


Isolation and characterization of the glycoprotein (JBB5) in the urine of a patient with carcinoma of the colon.

Patient J. B. with metastatic carcinoma of the colon excreted 0.5 to 1.0 g protein daily, about one-third of which was in Molecular Weight Class 30,000 to 60,000. The major component of this class was isolated by gel filtration and ion-exchange chromatography. The purified protein, labeled JBB5, contained about 11% sialic acid, 8% hexose, and 4% hexosamine. Its molecular weight was between 51,000 and 59,000. It did not react detectably with antisera to any of the recognized normal human plasma proteins. A specific antiserum to JBB5 was raised in the rabbit. Urine from 4% of subjects with nonneoplastic illnesses reacted in double immunodiffusion with anti-JBB5. Thirty-three % positive reactions were obtained with urines from patients with advanced neoplastic disease, the percentage varying from 64% in metastatic cancer of the pancreas to 15% in chronic lymphocytic leukemia.


Immuno-cytochemical demonstration, in the external region of the amphibian median eminence, of separate vasotocinergic and mesotocinergic nerve fibres.

By means of single and double immuno-enzyme cytochemical staining techniques, it was shown that the external region of the amphibian median eminence contains separate vasotocinergic and mesotocinergic nerve fibres. Moreover, it was demonstrated that the vasotocinergic fibres also contain neurophysin. In animals in which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive vasotocinergic and mesotocinergic fibres of the median eminence had disappeared. From this result, it is concluded that, at least the great majority of the vasotocinergic and mesotocinergic fibres of the external region of the amphibian median eminence are processes of neurosecretory perikarya located in the hypothalamic magnocellular preoptic nuclei. On the other hand, our results do not exclude the possibility that a minority of these neurosecretory fibres originate from small immuno-reactive perikarya which were found in the tuber cinereum. The observation that both kinds of processes accumulate around blood capillaries of the hypophysial portal system strongly suggests that they play a role in the control of the activity of the pars distalis of the hypophysis.


Studies on the juxtaglomerular apparatus. V. The juxtaglomerular apparatus in Tupaia with special reference to intercellular contacts.

The vascular pole of the juxtaglomerular apparatus in Tupaia belangeri was studied with special reference to the intercellular contacts of the periendothelial cells and the endothelium of the vas afferens. The periendothelial cells of the vascular pole of the glomerulum are connected by numerous gap junctions; and the granulated epithelial cells are suggested to form a functional unit. Probably there is a continuity of this system throughout the entire vascular pole including (1) all granulated cells, (2) all lacis cells, (3) the mesangium cells and (4) the adjacent smooth muscle cells of the vas afferens and vas efferens. Analysis of the endothelial junctions shows a zonular arrangement of tight junctions indicating a rather tight blood-tissue barrier next to the glomerular vascular pole; The ultrastructure of the different cell types of the vas afferens is also described, emphasizing the granulated epithelial cells and their innervation.


The third ventricle of monkeys; Scanning electron microscopy of surface features in mature males and females.

Surface features of the ependymal lining of the third ventricle in mature male and female monkeys have been investigated with scanning electron microscopy (SEM); Broad aspects of third ventricular morphology from three species of monkey are similar regardless of sex. The lateral walls are heavily ciliated whereas the ventral floor and most ventral parts of the lateral walls are not. Clumps of cilia on the lateral walls are so dense that underlying surface details are usually obscured. There is a transition zone between the ciliated lateral wall and nonciliated ventral floor. The floor and lower part of the lateral walls of the third ventricule exhibit a characteristic polygonal pattern upon which surface specializations such as microvilli, blebs and polymorphous membrane protrusions are superimposed. Ependyma of the choroid plexus of the third ventricle also display membrane specializations. Supraependymal cells are more visible in nonciliated regions.


Immunohistochemical localization of thyrotropin-releasing factor in the rat median eminence.

Antibodies raised in rabbits against a synthetic preparation of thyrotropin-releasing factor (TRF) were used in association with the immunoperoxidase histochemical technique to localize TRF in the rat median eminence. Using coronal sections cut through the mid-arcuate region, specific immuno-reactive material was observed in the medial and lateral regions of the superficial layer of the external median eminence.


Taste areas of the plica sublingualis of Alouatta and Aotus (Primates, Platyrrhini).

A conspicuous accumulation of taste buds occurs in the rostral part of the plica sublingualis ("frenal lamella") of Alouatta and Aotus forming taste areas (area gustatoria) superficially situated in the oral mucous membrane. They are found in close vicinity to the orifices of the sublingual salivary glands, but are lacking in the aboral part of the plica sublingualis. They do not occur in all primate species studied. A taste area does not projects above the surface of the surrounding tissue like a papilla. The taste buds open not in crypts of furrows of the oral mucosa, but directly into the spatium sublinguale of the oral cavity proper. In the anterior part of the cavum oris proprium different kinds and very differentiated qualities of sensoral information are perceived (touch, olfaction, temperature). It is conceivable that the taste areas play an important role in perceiving fresh saliva, together with the other sensorial structures in this part of the mouth. This problem can be solved experimentally and by behavioral studies, In addition to its topographical relation to the tongue, the organon sublinguale of Callicebus is structurally very similar to the plica sublingualis of Aotus and Alouatta. Since a sublingua does not occur in New World monkeys, it can be concluded that this organ represents a plica sublingualis which became adherent to the undersurface of the tongue.


Ultrastructural and immunohistochemical demonstration of pancreatic polypeptide-containing F-cells in the stomach and pancreas of Tupaia belangeri.

The gastro-entero-pancreatic (GEP) system in Tupaia belangeri contains a specific cell which reacts with antisera to pancreatic polypeptide (PP). The cells are scattered between exocrine pancreatic cells and also found in the pancreatic islets. Furthermore, they are also located in the fundic glands and to a very small extent in the corpus mucosa and in the glands of the upper duodenum. The cell reacting with antisera against PP is identified as the F-cell which has a specific ultrastructure especially with respect to its secretion. The present identification of the F-cell as the PP-cell in pancreas and stomach is discussed with respect to its possible functional implications.


Differences in pulmonary microvascular anatomy between Bufo marinus and Xenopus laevis.

1. Histological and corrosion-casting techniques have been used to provide a comparison of lung structure between a terrestrial load (Bufo marinus) and an aquatic one (Xenopus laevis). The pulmonary microvascular anatomy is distinctly different in these two species and both differ from classical descriptions of other anuran lungs. 2. In X. laevis large peribronchial vessels form a number of direct connections between the pulmonary artery and vein at the base of the lung. As a result of their location these vessels could easily function as respiratory bypasses or shunts. No similar connections were found in B. marinus lungs, nor have they been previously described for any other anuran amphibians. Possible respiratory implications of their presence in X. laevis are discussed. 3. The patterns of major vessel distribution are markedly different in the two species. Despite these differences the appearance of the respiratory capillary beds and their patterns of supply and drainage are surprisingly similar, suggesting that common design problems have been encountered in the evolution of both lung types. 4. The lungs of X. laevis have considerably less septal and lung wall smooth muscle than is the case in B. marinus. However, Xenopus lungs are generally thicker-walled and of more solid construction. The differences are discussed in terms of the widely different habitats of the two species.


Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture.

A method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture. In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or "dedifferentiated" after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification of "dedifferentiation" process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium. Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of "dedifferentiated" cells at all times. The advantages of differentiated rather than "dedifferentiated" smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.


Frequency of mast cells in the olfactory mucosa of rhesus monkeys.

During studies of the olfactory mucosa and its response to the different levels of circulating sex hormones, considerable numbers of mast cells have been observed in its epithelia and subepithelial regions. The number of these cells in the olfactory mucosa of male monkeys differs greatly from that found in females. The frequency of these cells in the olfactory mucosa of females fluctuates significantly during the menstrual cycle. These fluctations stimultaneously correspond to the well known changes in olfactory sensitivity: around ovulation, when the olfactory sensitivity for certain odorants is high, the number of mast cells in the olfactory mucosa also increases.


Myocardial bridges in man: clinical correlations and angiographic accentuation with nitroglycerin.

Little is known of the clinical significance of myocardial bridges, which may be recognized angiographically as systolic coronary artery narrowing (SCAN). A retrospective review of a 1 year's experience (313 consecutive coronary arteriograms) revealed 5 patients with SCAN, an incidence of 1.6%. SCAN involved the proximal and/or middle segments of the left anterior descending coronary artery in all patients. It is of particular note that the administration of nitroglycerin noticeably accentuated the SCAN phenomenon in each of 3 patients to whom it was administered. Four of the 5 patients had left ventricular hypertrophy due to hypertrophic cardiomyopathy (2), aortic stenosis (1), and hypertension (1). All 5 patients with the SCAN phenomenon had anginal chest pains, and critical obstructive coronary atherosclerosis was observed in only 2 cases. The other 3 patients showed, otherwise normal coronary arteriograms. Thus, myocardial bridges appear to be angiographically manifest predominantly in patients with cardiac hypertrophy. Nitroglycerin, which accentuates SCAN, might be useful as a provocative test to enhance the angiographic recognition of this phenomenon. The possible role of myocardial bridges in the production of myocardial ischemia warrants further investigation.


A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA.

The sequences corresponding to the 18S and 28S rRNAs have been mapped within a cloned 17 kilobase (kb) fragment formed by Eco R1 cleavage of Drosophila melanogaster rDNA. This fragment, Dm103, represents the longer of two major types of repeating units that are present in the rDNA of this fly, and was cloned as a hybrid plasmid, pDm103, consisting of Dm103 inserted at the Eco R1 site of the pSC101 vector (Glover et al., 1975). Mapping of the 18S and 28S rDNA in Dm103 was accomplished by quantitative determination of the amount of these rDNAs in each member of an ordered set of restriction fragments obtained by Hind III and Eco R1 ccleavage of pDm103. The amounts of 18S and 28S rDNAs were determined by hybridization of the rRNAs to fragments that were purified by cloning, and an unambiguous order of the fragments within pDm103 was established by heteroduplex mapping and from the stoichiometry of the fragment lengths. The resulting map revealed that the 4 kb of 28S rDNA within the long repeating unit represented by Dm103 is divided into two blocks that are separated by 5.4 kb of DNA of unknown function. It is this unusual arrangement of the 28S rDNA that distinguishes the long repeating units (17 kb) from the short units (11.5) kb), whose 4 kb of 28S rDna is confined to a single block, as is shown in the accompanying paper (White and Hogness, 1977). The remainder of the DNA in this long unit appears to be typically arranged, with the 2 kb of 18S rDNA confined to a single block that is separated by about 1 kb from the closest block of 28S rDNA.


R loop mapping of the 18S and 28S sequences in the long and short repeating units of Drosophila melanogaster rDNA.

Cleavage of D. melanogaster rDNA with the Eco R1 restriction endonuclease reveals two major classes of repeating units: a long class of 17 kilobases (kb) and a short class of 11.4 kb. R loop mapping has been used to determine the topography of the sequences corresponding to the 18S and 28S rRNAs in both classes, including a cloned member (Dm103) of the long class. This mapping procedure derives from a novel reaction that we discovered between single-stranded RNA and homologous regions in duplex DNA molecules. In high formamide and at an elevated temperature, the RNA pairs with one of the two DNA strands in the region of homology to form an R loop in which one element is an RNA/DNA duplex and the other is single-stranded DNA (Figure 1). Mapping is accomplished by visualization of R loops in the electron microscope. The R loop map of Dm103 parallels that determined independently by Glover and Hogness (1977) from an analysis of its restriction fragments. Both maps indicate that the 28S rDNA in this cloned unit is divided into two blocks by a 5 kb insertion segment. R loop mapping of a population of long units obtained directly from the rDNA (that is, without cloning) has demonstrated that this interruption of the sequence coding for the 28S rRNA is a characteristic of the long class. By contrast, the 28S rDNA in the short units that we examined is not interrupted by an insertion segment. Otherwise, the R loop maps of the long and short units do not differ significantly. The two classes therefore correspond to repeating units that do (IN+) or do not (IN-) contain an insertion segment. Models for the transcription and function of these two classes of repeating units are discussed.


The structural organization of ribosomal DNA in Drosophila melanogaster.

The genes coding for the large rRNAs (rDNA) were purified from Drosophila melanogaster embryos by two different methods. Purified rDNA was analyzed by gel electrophoresis after digestion with restriction endonucleases and by electron microscopy. Each repeating unit of rDNA consists of a gene region that codes for a transcript which includes the 18S and 28S rRNA sequences plus a nontrascribed spacer. Analysis of rRNA/rDNA hybrids in the electron microscope reveals that two types of repeating units exist in the rDNA from this fly. One of these is characterized by insertions of various lengths within the 28S gene. Insertions range in size from about 0.5 kilo base pairs (kb) to 6.0 kb and occur in distinct size classes which are multiples of 0.5 kb. Figure 10 summarizes the structure of the rDNA repeating unit. Repeats with insertions constitute about two thirds, and repeats without insertions about one third of all repeating units in the sample studied where 86% of the rDNA is derived from the X chromosome nucleolus organizer. Statistical analysis of pairs of of nearest-neighbor repeats demonstrates that repeats with and without insertions are interspersed in the X chromosome nucleolus organizer, and that scrambling is close to that expected for random arrangement. Insertions are the major source of length heterogeneity in this rDNA. A minor length heterogeneity also exists in thie nontranscribed spacer of Drosophila rDNA as demonstrated by heteroduplex mapping of restriction fragments in the electron microscope.


Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.


[Isolation of a pathogenic rhabdovirus of brown trout (Salmo trutta L., 1766)].

A Rhabdovirus pathogenic both for Rainbow and Brown Trout has been isolated from Brown Trout suffering heavy mortalities associated with muscular haemorrages and necrotic lesions of kidney and spleen. According to seroneutralization tests and host range, this virus appears to be different from the other known salmonids rhabdoviruses. Upon the basis of its in vitro gross characteristics, it is identical to Viral Haemorragic Septicemia Virus (VHS), F1 serotype, but differs from Infectious Hematopoïetic Necrosis and VHS type II viruses.


[Immunological demonstration of large quantities of glycogen or of a glycogen-like substance in human embryonic colon cells and in colon carcinomas by means of rabbit antisera raised against a strain of Escherichia coli 013].

Rabbit antisera raised against a strain of E. coli 013, with a strong antiglycogen activity, were tested on human fetal and normal adult colons, on colon carcinomas, and on colon tumor cells in culture (HT29). Only very rare granules were present in adult normal colons when tested with the immunofluorescence method. In faetal colons, in 12 out of 14 carcinomas, and on HT29 cells, the immunofluorescent reactions were similar to those observed in normal liver. The reactions were negative after previous treatment with alpha-amylase. They were inhibited with glycogen, with phenol-alcohol, perchloric, and trichloroacetic extracts from faetal colons, and with a tumor trichloroacetic extract. The extracts precipitated with anti-E. coli 013 antisera. They had a strong inhibiting activity in a radioimmunoassay test with labeled glycogen. The extracts from normal adult colons did not precipitate with the antisera and they had no inhibiting activity in either immunofluorescence and radioimmunoassay tests.


[Protein synthesis in vivo in muscles of the growing lamb].

The relationship between incorporation of intravenously injected 14C lysine and specific radio-activity of precursor was used to estimate protein synthesis in muscle of growing lambs. The rate of protein synthesis per unit of muscle weight in Supraspinatus and Extensor digitorum longus decreased strongly from one week of age to puberty (10 weeks); afterwards it decreased in supraspinatus and increased slightly in Extensor digitorum longus. The rate of protein synthesis increase in muscle protein weight was constant during the whole experiment (1 week-16 weeks). In preruminant Lambs )1 week-5 weeks) the rate of protein synthesis per unit of muscle weight decreased; however, due to the increase in muscle weight, the rate of protein synthesis in whole muscle remained relatively constant. In order Lambs the rate of protein synthesis in whole muscle decreased. The turnover time of protein increased with age. These results give some explanation on muscular development of Lambs.


[Demonstration of exocytosis in the sinus gland of Astacus Leptodactylus (Nordmann) under the influence of serotonin injections].

Following a serotonin injection, a rise of exocytosis in the sinus gland of the crayfish Astacus leptodactylus is observed together with an increase of the glucose level in the hemolymph. The phenomenon of exocytosis reaches a peak between 2 and 4 h after injection, wheras the highest glucose level in the hemolymph appears between 4 and 6 h after the same injection.


[Effect of light on variations in blood testosterone in the ram: demonstration of a photosensitive phase in diurnal rhythm].

6 groups each of 5 adult rams were subjected in light controlled pens to a pretreatment of normal variations in daylength during May and June. Then one group followed the normal daylength and the other five different "skeleton photoperiods" of 8 hrs of light per day consisting of: 7 hrs continuous light (7 L) + 1 hr light (1 L) situated at different times of the dark period (D) according to groups. Blood samples were taken once each week and hourly during one 24 hr period in June, July and September. Peripheral plasma testosterone measured by radio-immunoassay was used as an index of testicular activity. There was increase in the level of testosterone resuting from a rise in the number of peak release during the 24 hrs. In the different light treatments tested, only the treatment (7L+9D+1L)+7D) stimulated testosterone secretion with the pattern of testosterone of this group being similar to that of the control group. This indicates that there exists a photosensitive phase at 16 to 17 hrs after the beginning of the principal light (the subjective dawn).


[Respective effects on locomotor activity of injections of 5, 7-DHT in median raphe nucleus and of 6-OHDA in the mesolimbic dopaminergic group area in the rat].

This experiment was performed in order to demonstrate that the locomotor hyperactivity provoked by a radiofrequency lesion of the ventral mesencephalic tegmentum-A10 DA group area was not due to a 5-HT fiber damage. Four groups of Rats were used. First groups II and IV received a 5, 7-DHT injection in the median raphe; groups I and III received the vehicle. Locomotor activity was measured in a circular corridor 10 and 30 days; no hyperactivity was obtained. Then the same groups received a 6-OHDA injection, bilaterally in the A 10 area (groups III and IV) or the vehicle (groups I and II); the activity was measured 10 days later: significant hyperactivity was obtained with groups III and IV, without statistical differences between these two groups. In conclusion (i) 5-HT neurons are not directly involved in the VMT-hyperactivity, (ii) the DA A 10 neurons seem to be a critical anatomical target for this symptom.


[Hemolymph coagulation in Macropipus puber (L.) Crustacea Decapoda. Respective roles of hemolymph and plasma].

The process of coagulation of the hemolymph in Macropipus puber (L.) Crustacea Decapod depends on one or several plasmatic factors and on a thermo-labile cellular factor which does not seem to be specific. The existence of these factors has been demonstrated.


[Binding sites for 3H-Leu-enkephalin in rat straitum].

The binding of 3H-Leu-Enkephalin to a particulate fraction from Rat striatum has been investigated in the presence of 20 mum bacitracin which prevents its hydrolysis. After deduction of a low-affinity, "non-saturable" component, the saturation kinetics at equilibrium provide evidence for two distinct saturable sites. The first exhibits Michaelis kinetics with a Kd value of 2.7 +/- 0.1 nM; both morphine and naloxone compete with the binding of 3H-Leu-Enkephalin on this site but not on the second one. The marked decrease in binding of 3H-Leu-Enkephalin observed in the presence of high concentration of sodium ions indicates that these two pentapeptides have the same properties as morphinomimetic analgesics. Taken together, these data indicate that the first site is identical to the "opiate receptor", while the nature of the second saturable site remains to be established.


[DNA-polyintercalation compounds. Synthesis of ellipticine dimers].

Many symmetrical bis [9-methoxy 2-ellipticinium] dioxodiazaalkanes salts were synthesized from 9-methoxy ellipticine. An analog derivative of 9-hydroxy ellipticine was also prepared. Some of these dimers bind to DNA by a bifunctionnal mode of intercalation.


[Activity of the principal alkaloids of Polyalthia oliveri Engler (Annonaceac) on the central nervous system and the cardiovascular system].

Polyalthia oliveri leaf alkaloid, oliveroline, shows in Mice antiparkinsonian properties which decrease or disappear with the alkaloïds extracted from the bark of the trunk, oliveridine and oliverine. The latter alkaloid has an antihypertensive effect which is not found in oliveridine, only hypotensive by a relaxant close to papaverine action on vascular smooth muscles.


[Ultrastructure of neurosecretory granules in the sinus gland of Palaemon serratus Pennant].

Five types secretory granules are distinguished in the sinus gland of Palaemon serratus on the basis of differences in their diameter and their electron density. Variations in the density of the acoplasm and of the granules are observed for three of these granule types.


[Ultrastructure of epiphyseal cartilage after extraction on thin sections of "Epon" of proteoglycans by calcium chloride].

Dissociative methods are commonly used to extract proteoglycans. With a 2 M CaCl2 solution these components can be also extracted from thin sections of fixed, "Epon" included material. Secretory granules of the chondrocytes, granular components of extracellular matrix as matrix vesicles lost their electron density. Glycoproteins of the cell coat as non collagenous glycoproteins disappear. The method seems to be valuable for extracting proteoglycans from thin sections prepared for electron microscope investigations.


[Action of hyperoxia on hemolytic activity of the complement system].

Normobarie oxygen exposures momentarily raise by about 20% the haemolytic activity of the guinea pig complement likely by a rise of protein synthesis. About the end of this treatment the complement activity quickly decreases back to its initial value. Hyperbaric oxygen exposure immediately decreases the haemolytic activity. This shift might be result of a release in the serum of cytoplasmic elements bearing an anticomplementary activity or of an inactivation of the complement components involved in the stress reaction appearing in the treated animals. 2 days after the first exposure, during the resting phase, the complement rate increases by about 25% then decreases slowly back to normal.


[Characterization of the "4 S-trypsin" form of the cytosoluble estradiol receptor of calf uterus in a nondenaturing system].

The characterisation of the "4 S-trypsin" form of the estradiol-receptor from calf uterus cytosol was carried out by polyacrylamide gel electrophoresis under non-denaturing conditions. In a multiphasic buffer system (upper buffer Tris-glycine pH25degrees C = 8,6, lower buffer Tris HCl pH25degrees C = 7,4), three radioactive estradiol peaks were observed. The first was free estradiol, the second estradiol initially complexed with a macromolecule and dissociated during electrophoresis, and the third the hormone-receptor complex. The sedimentation coefficient (4 S) of this complex was the same before and after electrophoresis. Its mean geometric radius is R = 2.53 + 0.08 nm and its molecular weight was estimated to be 55,000.


[Overlapping genes: a priority].

Three British biologists have discovered that in the "bacteriophage phi X 174 genes D and E are translated from the same DNA sequence but in different reading frames." In 1973, the present author, on the basis of biological data and theoretical considerations, had foreseen existence of overlapping genes. The result of the British biologists confirms the hypothesis of P.-P. Grassé.


[Facilitation of a stereotyped motor behavior (climbing behavior) by previous stimulation of dopaminergic receptors: hyposensitivity of autoreceptors?].

The sensitivity of dopamine receptors in Mouse striatum has been evaluated both behaviourally (responsiveness to apomorphine as regarviour) and biochemically (striatal level of homovanillic acid and its decrease induced by apomorphine) After a single administration of apomorphine (0.25 mg.kg-1 or 5 mg.kg-1) or piribedil, another dopamine agonist, a state of "behavioural facilitation" develops which differs from the state of hypersensitivity following blockade. This state of facilitation is characterized by a lower threshold dose of apomorphine eliciting the stereotyped behaviour, without modification of the response to higher doses. In contrast with the state of hypersensitivity, the level of homovanillic acid is not modified and the decrease of this level by a low dose of apomorphine is less important. The hypothesis is put forward that "behavioural facilitation" results from the hyposensitivity of a class of dopamine receptors, possibly autoreceptors, mediating an impaired activity of dopaminergic neurons and, consequently, inhibitory behavioural effects.


Extraction and measurement of circulating angiotensins I and II.

We describe a method for simultaneous extraction of angiotensins I and II from human blood. Blood was diluted with 10 volumes of isotonic saline containing ammonium ethylenediaminetetraacetate, and the angiotensin peptides were extracted by cation-exchange chromatography. The dilution effectively inhibits in vitro production of angiotensin I, conversion of angiotensin I to angiotensin II, and degradation of the peptides. Analytical recovery of exogenous angiotensin I or II added to whole blood in physiological concentrations was essentially complete. The extraction procedure removes blood and plasma components that interfere with radioimmunoassay of angiotensin, thus eliminating the need to correct for nonspecific interference. Circulating angiotensins I and II, measured in patients with essential hypertension before and after a week of diuretic administration, increased in parallel with plasma renin activity.


Evaluation of serum triiodothyronine and adjusted triiodothyronine (free triiodothyronine index) in pregnancy.

We measured serum thyroxine (free and total), triiodothyronine (free and total), thyroxine-binding globulin, and triiodothyronine uptake by talc in 97 normal men and 50 pregnant women. Mean serum thyroxine and triiodothyronine concentrations were higher in the pregnant subjects (104 vs. 78 mug/liter and 1.69 vs. 1.30 mug/liter) because of a higher mean thyroxine-binding globulin concentration (70 vs. 38 mg/liter). Mean triiodothyronine uptake by talc was lower in the pregnant subjects (0.82 vs. 1.03). Mean free thyroxine concentrations were similar in the two groups, but mean free triiodothyronine concentrations were 10% lower in the pregnant subjects. Triiodothyronine uptake by talc and the diayzable thyroxine and triiodothyronine fractions were highly correlated (r = 0.85 and r = 0.82, P less than 0.001). Calculated free thyroxine index and free triiodothyronine index values (hyroxine and triiodothyronine indirectly adjusted, using triiodothyronine talc uptake to compensate for differences in thyroxine-binding globulin concentration), were statistically similar (84 vs. 82 and 1.38 vs. 1.34) in pregnant and male subjects. The results indicate that the total triiodothyronine concentration can be normalized on the basis of the triiodothyronine uptake by talc to correct for variations in thyroxine-binding globulin concentration.


Intra-individual variation in the concentrations of IgA, IgG, IgM, and complement component C3 in serum of a normal adult population.

A recent study [Clin. Chem. 22, 1635 (1976)] reported intra-individual variation in 10 serum proteins to be much smaller than interindividual variation. We report results of a similar study involving about 700 apparently healthy adults in whom we estimated the relative magnitude of the intra- and interindividual variation in serum IgA, IgG, IgM and complement component C3. Specimens were collected from each subject weekly for as long as 10 weeks (average, four weeks). The four serum proteins were quantitated by radial immunodiffusion by the maximal-diffusion technique. Traditional 95% reference intervals were computed relative to WHO reference preparations for the immunoglobulins. For C3, the reference interval was computed relative to a commercial reference preparation. We, too, found the ratios of intra-individual to interindividual variation for adults to be so small that the traditional reference intervals do not have the assumed diagnostic sensitivities. Furthermore, these ratios did not change after dividing the study population into subgroups according to sex and age; evidently such subgrouping do not improve the diagnostic sensitivity. The relatively small intra-individual variations were also observed at the extremes of protein concentration ranges.


Evaluation of sodium etidronate in the treatment of Paget's disease of bone. Osteitis deformans.

Thirteen cases of advanced Paget's Disease of bone were treated with Sodium Etidronate (EHDP) at 20 mg/kg/day for 6 months and followed at 2 to 3-month intervals for 20 months with serum alkaline phosphatase, 24-hour urinary hydroxyproline, radiographic skeletal survey, whole-body scanning with Tc-99m-Sn-EHDP and F-18, external body counting with the same radiopharmaceuticals over preselected areas, skin temperature, densitometry of normal phalanges and bone biopsies. Sodium etidronate had a marked effect on Pagetoid bone in all cases with reduction of bone turnover demonstrated by the chemistries, scanning, external counting, skin temperature and X-ray diffraction studies of the bone biopsies. Normal bone did not appear to be materially affected by the drug. Complications drug dose-related included new pain in 6 cases, two fractures in Pagetoid areas and one case of severe demineralization. There was one case of spinal cord compression unlikely to be drug related. All complications cleared or were successfully treated by the end of the study. Some patients continued to show reduction in bone turnover to the end of the study, as long as 14 months after stopping EHDP. Long-term follow-up is needed for final evaluation of the efficacy of the drug. Sodium etidronate shows promise as an agent in the treatment of Paget's Disease. Smaller doses or shorter courses of therapy or combination of EHDP and calcitonin may be just as efficacious and may avoid complications.


Trimalleolar fractures of the ankle. An end result study.

Fifty-one trimalleolar ankle fractures in 50 patients were evaluated subjectively, objectively, and roentgenographically at an average of 42 months following injury. Anatomic reduction of the posterior marginal fragment was achieved in 6 of 7 ankles treated by open reduction and internal fixation of the fragment. Anatomic reduction of the posterior marginal fragment was achieved in three of 44 ankles treated by closed reduction and in none of the eight with large fragments. End results rated as fair or poor occurred more frequently among those patients with large posterior lip fragments and those with residual subluxation of the talus. The incidence of moderate or marked post-traumatic arthritis was greatly increased in those patients with residual subluxation of the talus. Post-traumatic arthritis was present in 11 of 12 patients with moderate or severe pain and in 11 of 17 with moderate or marked restriction of motion. Failure to reduce a posterior lip fracture involving less than 25 per cent of the tibial articular surface did not affect the overall end result significantly. In ankles with a posterior marginal fragment involving 25 per cent or more of the articular surface, open treatment was associated with better subjective, objective, and roentgenographic end results than closed treatment.


Acute renal failure in the newborn.

Recent advances in prenatal and neonatal care have increased the number of live births and extended the life expectancy of critically ill premature infants. These infants represent a formidable therapeutic challenge in that multisystem involvement and previously uncommon conditions, such as intravascular coagulation, acute tubular necrosis, and acute cortical necrosis are now seen with increased frequency. This review begins with a discussion of the development of renal function in the neonate followed by a description of the more common causes of acute renal failure (ARF) in this age group. Finally, the pathophysiology, diagnosis, and management of this condition are discussed.


Fucosidosis.

Disseminated angiokeratomas can be seen in patients with an inherited deficiency of alpha-l-fucosidase as well as in patients with classic Fabry's disease. Patients with deficiency of this lysosomal enzyme, or fucosidosis, have spasticity, mental retardation, and retardation of growth in addition to angiokeratomas. We herein report three new cases of this rare syndrome.


Kinetics of reduction in viability of cultured leukemia L1210 cells exposed to purine analogs.

Rates of reduction in viability and degree of cell killing were relatively independent of concentrations when replicating cultured L1210 cells were exposed to increasing concentrations of 6-thioguanine, 6-methylthiopurine ribonucleoside, 9-beta-D-arabinofuranosyl-9H-purine-6-thiol, or 9-ethyl-6-thiopurine. The relative lack of dependence of cell killing rate on cytotoxic concentrations suggest (a) that these agents may be only effective aganinst proliferating cells, and (b) that only limited therapeutic advantage can be gainged by increasing their concentration beyond a minimum effective level. In contrast, the rate and degree of cell killing were dependent upon concentrations when cells were exposed to increasing amounts of 4-aminopyrrolo(2,3-d)pyrimidine-beta-D-ribofuranoside or to 2-fluoroadenosine, indicating that these analogs are not cell-cycle-stage-specific and that nonreplicating cell populations may be sensitive to them.


[Effect of nitroglycerin sublingually in the emergency management of "classical" pulmonary oedema (author's transl)].

Of 22 patients with the classical clinical signs of pulmonary oedema (orthopnoe, cyanosis, sweating and rales heard at a distance) 15 (Group A) were observed clinically, while seven (Group B) underwent haemodynamic studies. Those in Group A were given 0.8-2.4 mg nitroglycerin sublingually one to six times at 5--10 minute intervals. Within five minutes of nitroglycerin administration 7 of the 15 had their first signs of clinical improvement. In 11 patients the rales had disappeared after 15--20 minutes or had regressed. In the remainder the dyspnoea had decreased so that at this point in 14 of the 15 patients various degrees of improvement had occurred. Four patients were completely without clinical signs after 30 minutes. The increased arterial blood pressure and heart rate had fallen markedly. In the seven patients of Group B mean left ventricular filling pressure fell within ten minutes of nitroglycerin administration (1.6 mg) from 33 +/- 10 to 24 +/- 8 mmHg, cardiac output rising sifnificantly from 3.3 +/- 0.8 to 3.7 +/- 0.8 1/min. Such favourable results with nitroglycerin are to be expected only if the pulmonary oedema is of cardiac origin.


Ontogeny of the subcellular compartmentalization of thyrotropin releasing hormone and luteinizing hormone releasing hormone in the rat hypothalamus.

The 900 x g supernatant fluid prepared from hypothalamic homogenates from male and female rats (ranging in age from -1 to 120 days) was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone (TRH) and LH releasing hormone (LHRH) in the gradient fractions were quantified by radioimmunoassay. In adult hypothalamic homogenates, TRH and LHRH were associated with two populations of particles distinguishable by their sedimentation properties. Each peptide was in turn distributed in two subpopulations of parties differing in size but similar in density. The distribution of each peptide within its subpopulations of particles was found to be a function of age. In hypothalami of 22-day-old fetuses, TRH was associated almost entirely with the subpopulation of small particles. However, in the neonates, an age-dependent increase in the fractional amount of the TRH confined to the subpopulation of large particles was observed. By the 7th day of age, the peptide was equally distributed in the two subpopulations. The buoyant density of the 1-day-old neonatal particles and that of the adult small and large particles containing TRH was similar. The ontogeny of the subcellular compartmentalization of LHRH differed appreciably from that of TRH. LHRH was barely detectable in hypothalami of 22-day-old neonates. Nevertheless, at this age, the peptide was confined primarily to the subpopulation of large particles, and a similar compartmentalization was noted in hypothalami of 5- and 7-day-old neonates. However, in hypothalami of 14-day-old males and 21-day-old females, association of LHRH with the subpopulation of small particles was evident. It is concluded that 1) the nature of the hypothalamic subcellular compartmentalization of TRH and LHRH is age dependent, 2) the compartmentalization of each peptide in neonatal hypothalami differs from that in the adults, and 3) the development of the mature profile of subcellular compartmentalization of TRH and LHRH proceeds asynchronously.


Sodium valproate: a review of its pharmacological properties and therapeutic efficacy in epilepsy.

Sodium valproate has a broad spectrum of anticonvulsant activity, but is structurally unrelated to conventional antiepileptic drugs. Its proposed mode of action is mediated through effects on the function of brain gamma-aminobutyric acid (GABA). However, the elevations in brain and cerebellar GABA, and the concomitant reductions in levels of cyclic guanosine monophosphate, occur in animals at dose levels which are unlikely to be achieved during treatment of epileptic patients.


The role of sulfhydryl groups in the binding of glucocorticoids by cytoplasmic receptors of lung and other mammalian tissues.

We examined the levels and stability of glucocorticoid receptor activity in cytosol preparations of rat lung and other tissues. [3H]Dexamethasome binding capacity at 2 C in lung cytosol decreases with a t1/2 of 40 min in the absence of steroid or a sulfhydryl compound. This rapid inactivation of unbound receptor is prevented and reversed by addition of a sulfhydryl compound (t1/2 = 22h); maximal binding occurs with 2 mM dithiothreitol (DTT), or with 20-25 mM mercaptoethanol, thioglycerol or glutathione. Binding activity is also stabilized by formation of the receptor-steroid complex; dissociation of the complex occurs at the same rate (t1/2 = 18.6h) in both the presence and absence of DTT. In the presence of DTT, cytosol of adult rat lung specifically binds 0.82 pmol of [3H]dexamethasone per mg protein (average 19,000 sites per cell) with an equilibrium dissociation contant of 2.5 nM at 2 C. At 37 C, dispersed lung cells show specific nuclear binding of hormone. DTT also increased dexamethasone binding activity in cytosol prepared from lung of adult hamster, adult mouse, fetal monkey and 11 of 15 tissues of adult rat. There was a minimal effect of DTT in reactions using cytosol of fetal rat lung, fetal and newborn human lung, rabbit lung, and liver, kidney, heart and testis of adult rat. Liver contains a heat-stable factor which mimics that stabilizing effect of DTT. The variable requirement for DTT may reflect in part tissue concentrations of endogenous sulfhydryl compounds. Our findings indicate that glucocorticoid binding activity of lung and many other tissues decays rapidly in vitro due to oxidation of receptor sulfhydryl groups. Maintenance of these groups in the reduced form by endogenous tissue factors, addition of sulfhydryl compounds, or binding of glucocorticoid stabilizes receptor and allows its detection in lung and other tissues previously found to contain little if any activity.


The effect of basal hypothalamic isolation on pituitary-thyroid activity and the response to propylthiouracil.

Basal hypothalamic deafferentation extending from the posterior border of the optic chiasm to the mid-mammillary bodies resulted in depression of plasma TSH, thyroxine (T4), and triiodothyronine (T3) concentration to 50% of normal controls within 7 days. Administration of 0.15% propylthiouracil (PTU) in the diet form postoperative day 26 caused a pronounced drop in the plasma T3 level and a rise in plasma TSH level within two days in the control animals, but had little effect during this interval in the deafferented animals. After 12 days of PTU, plasma T3 and T4 concentrations had dropped to undetectable concentrations in the control animals but both were still detectable in the deafferented animals. After 25 days of PTU, plasms T4 and T3 levels were undetectable and plasma TSH levels were significantly elevated above normal in all animals. Thyroid hypertrophy at that time was as great in the deafferented as in the control rats, although plasma TSH concentration was 50% lower in the former. Administration of 0.1 mug/100 g BW TRH iv on postoperative day 37, when plasma T4 and T3 were undetectable in the controls but still present in the deafferented animals, produced an equally high concentration of plasma TSH in all animals. We interpret these data to support the concepts that: 1) a major source of neural drive of that TRH which stimulates the secretion of TSH by the adenohypophysis lies outside the medial basal hypothalamus, 2) a decrease in TRH reaching the adenohypophysis causes a lower setting of the "thyrostat" sensitive to the concentration of circulating thyroid hormone, and 3) increased TSH secretion and resultant goitrogenesis is delayed in animals with impaired TRH secretion because of the slower rate of secretion of thyroid hormone than in intact controls and the longer time thus required to markedly reduce the concentration of circulating thyroid hormone.


Insensitivity of bonnet monkeys to (D-Ala6, Des-Gly10) LHRH ethylamide, a potent new luteinizing hormone releasing hormone analogue in rats and mice.

The Luteinizing Hormone Releasing Hormone (LHRH) activity of (D-Ala6, Des-Gly10) LHRH ethylamide was compared with that of LHRH in oöphorectomized bonnet monkeys by determining serum LH and FSH concentrations at various time intervals after a sc injection of 100 mug of LHRH and either 100 mug or 1 mg of the analogue. Following administration of synthetic LHRH, a significant rise in both serum LH and FSH was observed. In contrast, no discernible change in serum gonadotropin concentrations was noted following injection of the analogue (D-Ala6, Des-Gly10) LHRH ethylamide, previously reported to have greatly increased potency in rats and mice.


Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.


Membrane fractions from the outer layers of spores of Bacillus thuringiensis with toxicity to lepidopterous larvae.

Two membrane fractions, F1 and F2, have been purified from the outer layers of spores of Bacillus thuringiensis. Both fractions contain 6-7% cysteine and appear to be similar in composition. Amino acids account for about 75% of the dry weight, carbohydrate for about 2% and lipids for about 25%. The fractions are both toxic to Pieris brassicae and the toxicity is inactivated by antiserum to the toxic crystal of Bacillus thuringiensis. The fractions can be distinguished by examination under the electron microscope; both fractions show similar hexagonal patterns but with different spacings. The same fractions from an acrystaliferous mutant (cr) were prepared. These were identical in density and in appearance under the electron microscope; the amino acid analysis of fraction F2 from both strains was identical. However, the spores and fractions F1 and F2 from this strain lacked toxicity. Fraction F2 from the cr strain was used to prepare antiserum specific to fraction F2. Using this anti-serum and anticrystal serum, crystal and F2 antigens were shown to appear simultaneously in sporulating cultures. Crystal and F2 antigens appeared some time before the maximum rate of uptake of [35s]cysteine. It is concluded that fraction F2 is derived from the exosporium and that fraction F1 probably originates from the spore coat. The exosporium in Bacillus thuringiensis appears to be synthesised during stages II and III of sporulation although uptake of [35S]cysteine occurs much later.


The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.


Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobacter cloacae (Clo DF13). Dissociation and reconstitution of the complex.

Cell of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmid-specific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.