The effect of spermine on transcription of mammalian chromatin by mammalian deoxyribonucleic acid-dependent ribonucleic acid polymerase. Isolated rat liver nuclei demonstrate an increased ability to synthesize RNA in the presence of either spermine or spermidine. Spermidine has more effect on the low-salt alpha-amanitin-insensitive reaction, and spermine has more effect on the high-salt alpha-amanitin-sensitive reaction. Spermine is effective at concentrations of 0.1 mM and 1 muM, showing a biphasic effect. The RNA polymerase activity associated with nuclear chromatin is increased in the presence of spermine only at a concentration of 0.1 mM. Aso the transcription of deproteinized liver DNA by liver form-B polymerase or Escherichia coli enzyme is more efficient in the presence of 0.1 mM-spermine. Only when liver chromatin is transcribed by its homologous enzyme (and not by E. coli enzyme) is spermine active at both 0.1mM and 1 muM as in purified nuclei. The lower concentration of spermine (1 muM) is able to affect chromatin transcription by increasing the affinity of chromatin for the enzyme. Our findings suggest a regulatory role of spermine at the level of genome transcription.


[Clinical-pharmacological and pharmacokinetic studies with naproxen]. d-2-(6'-Methoxy-2'-naphthyl)-propionic acid (naproxen) is bound in blood to a high degree to plasma protein. After oral application naproxen is absorbed rapidly and completely. The mean biological half-life in man is fourteen hours. This gives us the possibility of controlling the symptoms of disease with two daily doses. In man, 99% of naproxen is excreted in the urine, either unchanged or glucuronized and as its 6-des-methyl metabolite. There is a linear increase of plasma levels with daily doses of up to 500 mg. Higher naproxen doses do not produce higher blood levels but lead to a faster excretion. This probably depends in part on a saturation of the binding sites in plasma. Differences in affinity of some compounds for binding sites in plasma are responsible for a potential interaction between naproxen and other agents, such as warfarin, acetyl salicylic acid (ASA) and sulfonylureas. These interactions are discussed. In a double-blind trial the influence of naproxen and ASA on the gastric mucosa of healthy volunteers has been compared. The results of gastroscopic findings as well as laboratory tests are reported.


A Double-blind comparison of naproxen against indometacin in osteoarthrosis. Fifty patients with osteoarthrosis of the knee and hip were entered into a double-blind trial of d-2-(6'-methoxy-2-naphthyl)-propionic acid (naproxen) 500 mg daily versus indometacin 100 mg daily. Two simultaneous trials of identical design were performed, one consisting of patients with unilateral or bilateral knee joint arthrosis only, the other of patients with unilateral or bilateral hip joint disease. Patients were assigned to 4 weeks treatment on naproxen or  indometacin, then crossed over to the other drug. Assessments were made at --2, 0, 2, 4, 6 and 8 weeks, including subjective grading of pain and objective measurement of joint movements, stair climbing time and walking time. In almost all parameters there was significant improvement from baseline on both drugs, the magnitude of improvement being statistically equivalent. Side-effects recorded during the naproxen treatment period were significantly fewer than during indometacin treatment.


[Methods and statistics of multicentral double-blind "cross-over" examination of naproxen compared to indomethacin]. The methodology of documenting clinical findings and statistical analysis of the results of a multicentre double-blind cross-over study, in which the efficacy of d-2-(6'-methoxy-2'-naphthyl)-propionic acid (naproxen) and indometacin in the treatment of rheumatic diseases was compared, is presented. Eight investigators from four clinics conducted this clinical trial according to an identical protocol in a total of 100 patients. 46 patients (5 male and 31 female) with rheumatoid arthritis received either of the two drugs over a period of 26 days. 35 patients (32 male and 3 female) with ankylosing spondylitis and 19 patients (8 male and 11 female) with osteoarthrosis were treated with either of the drugs for 15 days. The following parameters were investigated and documented: pain and its localization, inhibition of joint function, symptoms of inflammation, and the "quasi" irreversible joint changes. These various parameters were for statistical purposes combined as indices for pain, function, and inflammation. This permitted a quantitative evaluation of the analgesic and antiinflammatory efficacy as well as of the improvement of joint function of the two compounds tested. The overall results of the clinical parameters revealed no significant difference in efficacy for the two drugs. When differentiating the patients as to their sex, both drugs showed higher efficacy in male patients, and only slight efficacy in female patients.


[Clinical results of a multicentral double-blind examination of naproxen compared to indomethacin in chronic rheumatoid arthritis, ankylosing spondylitis, and osteoarthrosis]. In a multicenter double-blind cross-over trial the therapeutic effect and the tolerance of d-2-(6'-methoxy-2'-naphthyl)-propionic acid (naproxen) and indometacin were compared including 46 patients with rheumatoid arthritis, 35 patients with ankylosing spondylitis and 19 patients with osteoarthrosis. Duration of treatment with both drugs was two to four weeks each. The daily dose of naproxen was 750 mg, that of indometacin l50 mg. In rheumatoid arthritis the combined clinical effect of indometacin was stronger than that of naproxen while both drugs had the same clinical effectiveness in ankylosing spondylitis and osteoarthrosis. When differentiating the total clinical effect as to indices of pain, inflammation and function, indometacin was shown to be superior in all three diseases with regard to pain index. On the other hand, naproxen showed a better effect in ankylosing spondylitis and osteoarthrosis than indometacin as to inflammation, and as to function in osteoarthrosis.


[A double blind comparison of naproxen and indomethacin on the after-midnight-pain of patients with morbus bechterew]. 27 patients with ascertained ankylosing spondylitis experiencing constant after-midnight-pain received, following three medication-free days, in a double blind, randomized, cross-over fashion indometacin (100 g/day) and d-2-(6'-methoxy-2'-naphthyl)-propionic acid (naproxen) (500 mg/day), as suppositories, for a period of six days each. The intensity of the night-pain was recorded daily. Naproxen was shown to be equally effective as indometacin in alleviating the after-midnight backache of ankylosing spondylitis. Side effects occurred under indometacin in 5 cases, under naproxen in 3 cases.


[Observations of long-term treatment of arthrosis with naproxen]. In a previous open clinical trial with d-2-(6'-methoxy-2'-naphthyl)-propionic acid (naproxen) in 32 outpatients with osteoarthrosis, very good to good results were achieved in 6 patients, satisfactory in 12, fair in 5, and unsatisfactory in 9 patients. The severest side effect observed was a case of gastric bleeding in a female patient who had been previously treated with a drug containing phenylbutazone and prednisone. Since, furthermore, 6 out of 9 such patients who did not respond to therapy had to stop the durg prematurely because of minor side effects and since in another 6 there was a tendency to a reduction of the leucocyte count, we subsequently initiated a long-term efficacy and tolerance study in our department for chronic patients. This series comprised 15 patients, aged 66 to 86 years, with severe, partly immobilizing coxarthroses and gonarthroses. The length of treatment was 2 to 16 months (mean 10.5 months). During the first 3 months ESR determinations and complete blood counts were performed every two weeks, later at four-weekly intervals. The patients were questioned weekly as to the efficacy of the treatment and as to side effects. Neither changes of the laboratory data nor gastro-intestinal side effects were observed. In two patients -- one a female with multiple allergies -- treatment had to be stopped because of itching and exanthema, respectively. The analgesic effect of naproxen was evaluated as very good in 5 cases, good in 5, fair in 2, and unsatisfactory in 3 cases. In addition, we conducted another clinical trial in 15 outpatients with degenerative joint diseases (partly of the spinal column). Their age varied from 16 to 60 years. Naproxen was administered in daily doses from 500 to 750 mg for the duration of 6 to 12 weeks. In no case had treatment to be interrupted, nor was there any gastric intolerance or alteration of the blood count observed in this group. Clinical results were classified as satisfactory to good in 10 patients.


Sinus node function in the denervated human heart. Effect of digitalis. Evaluation of sinus node function was performed in 5 patients with an intact cardiac autonomic nervous system (group I), and in 8 patients with a transplantated, denervated heart (group 2). After baseline data were recorded, the electrophysiological studies were repeated in all group I patients and in 6 of the 8 group 2 patients, 45 to 60 minutes after the administration of digoxin 1.25 mg intravenously. Baseline cycle length, sinus node recovery time, and sinoatrial conduction time were significantly shorter in the transplanted heart than in those with intact autonomic innervation, but correction of the sinus node recovery time and sinoatrial conduction time for heart rate abolished these differences. Digoxin produced a small increase in cycle length, sinus node recovery time, and sinoatrial conduction time which did not reach statistical significance in this small study group of patients with innervated hearts. In the denervated, transplanted patients, no change in cycle length occurred after digoxin in any patient. The sinus node recovery time was unaffected by glycoside administration in 3 of 6 patients, while the sinoatrial conduction time was unchanged in 4 of 6. In one group 2 patient, digoxin produced first degree sinoatrial node exit block, and in a second patient, 2:1 sinoatrial nodal exit block developed. The mechanisms responsible for these effects in the denervated heart are not clear.


Evaluation of the toxicity of local anaesthetic agents in man. Etidocaine given by intravenous infusion has been compared, using a double-blind technique, with bupivacaine and lignocaine in respect of toxic symptoms and signs. The degree of toxicity is affected considerably by the rate of drug infusion. At 10 mg/min subjects could tolerate twice the dose of etidocaine as bupivacaine. Tolerance to etidocaine was less at 20 mg/min but still compared favourably with bupivacaine at 10 mg/min. Considering the difference in potency of the two agents it was considered that the therapeutic ratios would not be substantially different. Lignocaine at 20 mg/min was better tolerated than etidocaine. Venous plasma concentration during these experiments showed a more rapid decrease in the case of etidocaine compared with bupivacaine, but the concentrations were unhelpful in predicting the toxic effects. Similarly electroencephalography revealed no abnormalities in spite of marked subjective and objective signs of toxicity.


Change in end-tidal position in children after suxamethonium. The change in end-tidal position (ETP) after suxamethonium-induced paralysis was measured in 15 children during routine general anaesthesia. In all patients the onset of muscle paralysis was associated with an increase in lung volume from ETP. This increase may be the result of paralysis of the expiratory muscles which are active during anaesthesia.


Effect of CO2 on myocardial contractility and aortic input impedance during anaesthesia. The haemodynamic responses to hypocapnia and hypercapnia have been studied in the dog during intermittent positive pressure ventilation under halothane anaesthesia (1% halothane in oxygen) and under nitrous oxide anaesthesia (30% oxygen in nitrous oxide). In the absence of significant variations of either myocardial contractility or left ventricular end-diastolic pressure, the changes of stroke volume and cardiac output (diminution because of hypocapnia, augmentation because of hypercapnia) were determined by alterations of systemic vascular resistance (augmentation because of hypocapnia, diminution because of hypercapnia).


Mechanics of breathing, gas distribution and functional residual capacity at different frequencies of respiration during spontaneous and artificial ventilation. Nine healthy volunteers were investigated, both while awake and breathing spontaneously, and while anaesthetized with IPPV, in all cases at rates of both 12 and 24 b.p.m. Gas flow and volume were measured with a pneumotachography. The transpulmonary pressure (the pressure difference between the trachea or the buccal cavity and the oesophagus) was also recorded. The distribution of gas was analysed by means of nitrogen washout curves, which also permitted the determination of functional residual capacity (FRC). Lung compliance during IPPV was approximately half that during spontaneous breathing. During IPPV the compliance was dependent on the frequency of ventilation, being lower with the greater frequency. Pulmonary resistance was approximately twice as great with artificial ventilation, but no significant relationship to frequency was demonstrated. Gas distribution was within normal limits and in this respect there was no difference between low and high rates of spontaneous breathing. With IPPV at the higher rate, gas distribution was significantly less even, but still within normal limits. Dfferences in FRC under the different conditions during the experiments were not significant, but the values obtained were lower with artificial ventilation. Neither the reduction in dynamic lung compliance induced by anaesthesia and artificial ventilation, nor its dependence on the frequency of such ventilation, can be explained with certainty by changes in gas distribution.


An autoclavable ventilation failure alarm. A small ventilation failure alarm which can be used with all types of lung ventilator is described. The entire patient circuit can be removed and autoclaved easily. Activation of the unit is by a removable key, a mains failure warning is provided, and alarm may be muted during aspiration of secretions from the tracheobronchial tree.


Mechanism of action of extradural analgesia. The factors governing the spread and site of action of local anaesthetics in the extradural space are discussed. Local anaesthetic drugs injected into the extradural space may cause nerve blocks at three possible sites: (a) On the spinal nerves in the paravertebral space. This probably occurs only in young subjects. (b) On the spinal nerves intradurally. This is probably the essential site of blockade. (c) On the spinal cord.


A review of the metabolism of amide local anaesthetic agents. (1) Amide local anaesthetics are almost completely metabolized before excretion in both animals and man. (2) Considerable interspecies variability occurs in the quantitative excretion of local anaesthetic metabolites; however, qualitative similarities often exist. (3) The secondary amine metabolite of lignocaine, MEGX, is capable of reacting in vivo with acetaldehyde, formed as a metabolite of ethyl alcohol, to produce a cyclic condensation product. (4) Although hydrolysis in man of the secondary amine metabolite of lignocaine, MEGX, appears extensive, the same is not true for the metabolic analogue produced from mepivacaine, PPX. (5) Metabolism of mepivacaine by the neonate is impaired, but the excretion process in the newborn is capable of eliminating the drug within 24 hr after birth. (6) Metabolic data on bupivacaine and etidocaine are incomplete. The latter compound appears to have a more rapid plasma clearance.


Cardiovascular pharmacology of local anaesthetics. Although most local anaesthetic drugs are generally regarded as vasodilators at their site of injection, they produce systemic effects characterized by increases in peripheral resistance, cardiac output, arterial pressure heart rate. The direct depressive effects of the drugs are counterbalanced by increased sympathetic activity. There is evidence that many local anaesthetic agents increase the myogenic tone of certain vascular beds. A proper understanding of these phenomena could lead to improvements in the development of new local anaesthetic agents and appropriate vasoconstrictors.


Cardiovascular effects of extradural anaesthesia. The changes that occur in the circulation following extradural block are described and compared with spinal anaesthesia. The contribution of height of blockade, the effects of the local anaesthesic agent after absorption and the modification in the haemodynamic changes caused by vasopressors added to the local anaesthetic agent, are discussed.


Intercostal nerve blockade for evaluation of local anaesthetic agents. Bilateral intercostal nerve block provides the opportunity to subject as many as 16 separate peripheral nerves in a single subject to known or unknown local anaesthetic agents in a variety of concentrations, volumes, and additives. It permits the observation of local (e.g., neuritis), clinical (e.g., onset and duration), and systemic (e.g., toxicity and blood concentration) effects of these variables. In double-blind studies, bilateral intercostal nerve block allows the use of each side of the trunk for comparison of two experimental drugs, a new drug against a standard, or two new drugs. Subtle differences in clinical properties as well as simultaneous blood concentrations may be detected in these studies. The advantages of this technique in evaluating local anaesthetic agents are primarily the use of a single subject as his own control while studying may separate peripheral nerves. This aids appreciably in limiting the variable of age, temperature, and perfusion, as well as techniques of administration and evaluation. The constancy of the anatomy of the intercostal nerve provides a highly reliable and reproducible block technique.


Double-blind controlled crossover high-dose study of Azaribine in psoriasis. Azaribine used in high doses of 200 mg/kg a day is an effective agent in inducing temporary remissions in patients with severe psoriasis but potentially serious neurotoxicity may occur. Therapy should be initiated at lower doses of 125 mg/kg a day and advanced carefully only if clinical response does not ensue at the lower level.


Dermal-epidermal deposition of complement components and properdin in systemic lupus erythematosus. Skin lesions and clinically normal skin of thirteen patients with systemic lupus erythematosus (SLE) were examined, by the use of immunofluorescent techniques, to determine the presence of CIq, C3, C3 proactivator (C3PA), properdin, immunoglobulins (IgG, IgA, and IgM), and fibrin. IgM deposition was present in all thirteen skin lesions and in eleven normal areas tested, whereas IgG deposition occurred in eleven of the lesions but in only six normal areas. IgA was the least frequently encountered immunoglobulin. CIq deposition occurred in all thirteen skin lesions, and C3 deposition was present in twelve. Seven of nine and eight of eleven clinically normal areas demonstrated CIq and C3 deposition, respectively. Although less intense that CIq and C3 deposition, properdin deposition occurred in eight of the thirteen skin lesions tested but in only two of nine normal areas. C3PA deposition was of greater intensity than was properdin, but was seen in only five lesions and three clinically normal areas. Seven skin lesions and two normal areas also demonstrated fibrin deposition. Although alternate pathway activation is apparent, our findings suggest that the classical pathway (CI to C9) is the primary complement pathway involved in SLE skin.


Growth inhibition in normal mammalian melanocytes in vitro. Studies on primary cultures of normal adult melanocytes from the ear skin of a black strain of guinea-pigs are reported. Melanocytes have a low proliferation rate and tend to form cohesive groups on a glass surface. The cells undergo division while maintaining close contacts with each other whereas DNA synthesis is rare in melanocytes with multiple contacts.


The junctional zone beneath migrating epidermis. The changes that take palce in skin explants in vitro at the interface between migrating epidermal cells and dermis have been examined by light and electron microscopy and by immunofluorescent antibody techniques. In the first 24 hr epidermal cell migration occurred in the absence of any of the usual components of this junctional zone. After 48 hr of incubation antigenic determinants of the basal zone were detected and basal lamina was demonstrated ultrastructurally. It is proposed that migrating epidermal cells do not need a complex and specialized junctional zone and that these cells are themselves responsible for the elaboration of new basal lamina.


High dose ascorbic acid in the management of thalassaemia leg ulcers--a pilot study. Eight patients with beta thalassaemia major suffering from leg ulcers, were treated over an 8-week period with 3 g ascorbic acid daily in a controlled double-blind crossover study. The ulcers of all the patients showed a high rate of either complete or partial healing.


Oral retinoic acid as therapy for congenital ichthyosiform erythroderma. Oral retinoic acid has been therapeutically beneficial for three patients with congenital ichthyosiform erythroderma and one with congenital bullous ichthyosifrom erythroderma. Response to therapy was slow but definite with suppression of scale formation, marked reduction of painful cracking of the underlying epidermis and disappearance of skin irritation. Side effects have not occurred. Therapy had to be maintained to prevent recurrence of symptoms.


The effect of a low calorie diet or a thiazide diuretic on the incidence of pre-eclampsia and on birth weight. A 1200 calorie diet or cyclopenthiazide with potassium was given to two groups of 51 high weight gain primigravidae and baby weight and the incidence of pre-eclampsia were compared with those in a matched control group.  There was no difference in the development of pre-eclampsia, but the weight of babies in the control group was higher than in the treated groups.  The body fat was reduced in the diet group and the total body water in the diuretic group.


Oral malignant melanoma.

Oral malignant melanoma. Sixty-five cases of oral malignant melanoma are reviewed, comprising 53 cases taken from the literature and 12 unreported cases.  The classification of tumours on clinical and histopathological grounds is suggested to be comparable to that for cutaneous melanoma.


Sequence homology of the mitochondrial leucyl-tRNA cistron in different organisms. Sequence divergence of the mitochondrial leucytl-tRNA cistron in several eukaryotes has been examined by RNA-DNA hybridized. Rat mitochondria Leucyl-tRNA was hybridized with rat, mouse, guinea pig, monkey, chicken, and yeast mitochondrial DNAs (mtDNA) immobilized onfilters. Hybridization was carried out in 50% formamide (Tm -12degrees) or in 20% fromamide (Tm -21degrees). melting profiles of the hybrids were obtained for evaluation of the extent of base sequence micmatching. Under the more stringent hybridization conditions (50% formamide, Tm -12degrees), only mouse and quines pig mtDNAs hybridized with rat mitochondrial leucyl-tRNA. The Tm's of the heterohybrids were depressed by 2 and 9 degrees, respectively. Under less stringent hybridization conditions (Tm-21 degrees), monkey mtDNA also hybridized, and the Tm was depressed by about 15 degrees. Chicken and yeast mtDNAs did not form specific hybrids with rat mibochondrial leucyl-tRNA under these hybridization conditions. Mitochondrial leucyl-tRNA sequences in different eukaryotes appear to be conserved to a less extent than cytoplasmic rRNA, 5S RNA, or hemoglobin mRNA sequences.


The interaction of heavy meromyosin and subfragment 1 with actin. Physical measurements in the presence and absence of adenosine triphosphate. Viscosity, turbidity, and laser-light fluctuation autocorrelations of acto-heavy merymyosin (HMM) and acto-subfragment 1 (S-1) solutions were measured under conditions where the actin-activated ATPase is close to its maximal value. The results were compared to similar data obtained in the absence of ATP where the actin and myosin fragments were completely domplexed, and in the presence of ATP but at 0.1 M KLC where the actin and HMM or S-1 were almost completely dissociated. It was found that at maximal actin activation, the viscosity, turbidity, and autocorrelation data were all much closer to the values for the completely dissociated systems than to the values for the completely complexed systems. Assuming that viscosity, turbidity, and autocorrelation measurements approximate a linear measure of binding between actin and HMM or S-1, the results suggest that at maximal actin activation less than 10% of the HMM or S-1 are bound to the actin. Therefore as was suggested previously by ultracentrifuge and kinetics studies, it appears that under conditions of maximal actin activation, most of the HMM and S-1 occur in a refractory state unable to bind to actin.


Identification of a unique ethidium bromide binding site on yeast tRNAPhe by high resolution (300 MHz) nuclear magnetic resonance. The binding of ethidium bromide to yeast tRNAPhe has been studied by high resolution (300 MHz) proton nuclear magnetic resonance. Under appropriate experimental conditions one ethidium bromide is bound to each tRNA and two resonances from ring NH protons are shifted upfield. These observations taken in conjunction with the low-field spectrum of yeast tRNAPhe show that the unique ethidium bromide binding site is located between base pairs AU6 and AU7 of the amino acid acceptor stem. This information should be of value in understanding the way in which ethidium bromide binding alters the biochemical properties of the tRNA molecules.


Kinetic evidence for the obligatory formation of a 30S initiation complex in polyphenylalanine synthesis initiated with N-acetylphenylalanyl-5RNA. The problem of whether the initiation of bacterial protein synthesis involves the obligatory formation of a 30S initiation complex intermediate was examined in a model system with N-acetylphenylalanyl-tRNA as initiator 5RNA nad poly(uridylic acid) as mRNA. The time courses of the formation of the 30S and 70S initiation complex with Escherichia coli ribosomes were measured simultaneously by stopping the reaction with dextran sulfate and differentiating the N-acetylphenylalanyl-tRNA bound to 30S ribosomal subunits from that bound to 70S ribosomes with RNase I, which hydrolyzes N-acetylphenylalanyl-tRNA bound to 30S subunits but not that bound to 70S ribosomes. A maximum in the 30S complex concentration was observed within the first 10-15 sec of the reaction, whereas 70S complex formed formed more slowly with a slight initial time lag. When an analog computer was programmed with rate constants determined separately for the formation of the 30S initiation complex from preformed 30S complex, kinetic curves very similar to the empirical curves were obtained for the entire time course of the reaction. The results show clearly that formation of the 70S complex obeys the kinetic laws for consecutive reactions, and the 30S complex is, therefore, an obligatory intermediate in the initiation of polyphenylalanine synthesis in the model system.


The isolation and characterization of nuclear ghosts from cultured HeLa cells. Macromolecular complexes, which appear as ghosts when viewed by phase contrast microscopy, have been isolated from the nuclei of HeLa cells grown in culture. The preparation of these ghosts involves a detergent wash which removes the unit membranes of the nuclear envelop structure but leaves intact both the nuclear pores and the dense structure conferring nuclear margins (possibly the dense lamella). Detergent-washed nuclei are subsequently treated with 0.5 M MgCl2 and fractionated on continuous sucrose gradients containing 0.5 M MgCl2. The ghosts are recovered as a sharp band at an apparent sucrose density of 47-52% and consist of 72% protein, 10% phospholipid, 14% DNA, And 4% RNA. The release of the majority of intranuclear components is indicated by the large loss of nuclear DNA (95%), RNA (71%), and protein (87%) contrasted to the small loss of phospholipid (27%) druing the conversion of detergent washed nuclei to isolated ghosts. Sodium dodecyl sulfate-polyacrylamide gel patterns of the ghost proteins consist of two major bands with approximate molecular weights of 20,000 and 35,000. The isolation of ghosts with a similar density and protein composition from nondetergent-washed nuclei indicates that the ghost is not an artifact induced by the detergent treatment. The absence of cytoplasmic contamination in the preparations of detergent washed nuclei and nuclear ghosts was demonstrated by chemical, enzymatic, and electron microscope studies. We suggest that the isolated ghosts represent a structural macromolecular complex which underlies and is probably attached to the inner nuclear membrane of intact nuclei. The possible additional presence of intranuclear network proteins has not been excluded.


Incorporation of fluorotryptophans into proteins of escherichia coli. A tryptophan-requiring strain of Escherichia coli can go through two doublings of optical density after L-tryptophan is replaced in the medium by 4-fluorotryptophan, during which the fluoro analog displaces approximately 75% of the L-tryptophan in cell protein. One doubling occurs in the presence of 5- or 6-fluorotryptophan, with 50-60% replacement of L-tryptophan by analog. When beta-galactosidase is induced at the time of addition of analog, it reaches 60% of the control specific activity in the presence of 4-fluorotryptophan, 10% of normal in the presence of 5- or 6-fluorotryptophan. Lactose permease activity is 35% of the control in the presence of 4- and 6-fluorotryptophan, less than 10% in the presence of 5-fluorotryptophan. D-Lactate dehydrogenase shows a specific activity twice that of the control in the presence of 4-fluorotryptophan, one-half with 5- or 6-fluorotryptophan. Thus fluorotryptophan can be incorporated into proteins and affect their activities, although the nature and magnitude of the effect cannot be predicted for any given enzyme. Such substituted proteins should be useful for the study of protein structure and function by 19F nuclear magnetic resonance and other techniques.


Cooperative interactions in hybrids of aspartate transcarbamylase containing succinylated regulatory polypeptide chains. Succinylated derivatives of the regulatory subunit of aspartate transcarbamylase of Escherichia coli were prepared by treating the intact enzyme with succinic anhydride followed by dissociation of the modified protein into catalytic and regulatory subunits which were separated by ion exchange chromatography. The succinylated regulatory subunits were used in hybridization experiments with native subunits to study the organization of the six regulatory polypeptide chains in the intact enzyme. Rapid mixing of succinylated and native regulatory subunits with native catalytic subunits yielded a four-membered hybrid set of reconstituted enzyme-like molecules; hence, the assembly process involves three regulatory combining units and the six regulatory polypeptide chains in the intact enzyme must be arranged as three dimeric subunits. When the modified and native regulatory subunits were incubated together for only brief periods (less than 1 min) followed by the addition of catalytic subunits, the resulting hybrid set was complex with no resolution of discrete species. Apparently the isolated regulatory dimers dissociate readily and reversibly into single polypeptide chains due to relatively weak intra-subunit bonding domains. In contrast, after reconstitution of enzyme-like molecules, the incorporated succinylated regulatory subunits did not exchange with free subunits. Enzyme-like molecules containing three extensively succinylated regulatory subunits show reduced binding of the inhibitor, CTP, and lack both the homotropic and heterotropic effects characteristic of native aspartate transcarbamylase. Preparations containing only slightly succinylated regulatory subunits showed only little inhibition by CTP and considerable cooperativity. The decrease in homotropic effects in these reconstituted molecules correlated with the reduction in the succinate-promoted change in the sedimentation coefficient. Reconstituted enzyme-like molecules containing regulatory subunits which had been extensively succinylated in the presence of CTP retained their binding capacity even though they were only slightly inhibited by CTP and exhibited reduced cooperativity. Hybrid molecules containing both native and succinylated regulatory subunits also possessed reduced allosteric behavior.


Proton magnetic resonance studies on the conformation of the hexanucleotide, GmpApApYpApsiP, and Related fragments from the anticodong loop of baker's yeast phenylalanine transfer ribonucleic acid. A hexanucleotide, GmpApApYpAppsiP, and 11 related compounds from the anticodon loop of Baker's yeast tRNA-Phe were studied by proton magnetic resonance from 100 to 250 MHz. Totally 19 resonance lines from all the base protons (H8, H6, and H2), H1', and methyl proton resonances of the hexamer have been assigned by a systematic "incremental procedure" in comparing all the related shorter fragments. Emphasis is given to the Y base and its stacking conformation with respect to its nearest neighboring bases. The results reveal a strong tendency of the purine bases to have a maximal extent of base-base overlap with their neighbors in the sequence. This tendency is manifested in a zigzag (or balcony-like) mode of base-stacking pattern of the -ApYpA-sequence in the hexamer in which the -pA-residue tends to stack toward the adduct ring (C10, C11, and N12) of Y. This tendency is also shown in the formation of a stack of GmAAA closing the gap left behind by the excision of Y in the hexamer GmpApAp--pApp. The implication of these findings to the structure and function of tRNA is discussed.


The binding of complementary oligoribonucleotides to yeast initiator Transfer RNA. Oligoribonucleotide binding to baker's yeast initiator tRNA was measured by equilibrium dialysis in order to determine which regions of the tRNA were free to bind complementary oligomers and which were involved in secondary and tertiary structure. Association constants of trinucleoside diphosphates and tetranucleoside triphophates complementary to the single-stranded regions of the cloverleaf structure of yeast tRNAfMet were measured at o degrees in 1.0 M NaCl, and 0.01 M MgCl2. The only regions of the tRNA whose complementary oligomers bound to the tRNA were the amino acid acceptor end and the five nucleotides at the 5' end of the anticodon loop. These results differ from those for the other tRNAs studied by this technique; usually oligomers complementary to the dihydrouracil loop bind to the tRNA. The sequence of yeast tRNAfMet and other eucaryotic initiators is unusual. The "TpsiC loop" contains the sequence A-U-C instead of T-psi-C, yet the binding pattern to the THE TpsiC LOOP IS LIKE THAT FOR OTHER TRNAs; no oligomers bind.


Tyrosyl-tRNA synthetase from Escherichia coli. Stoichiometry of ligand binding and half-of-the-sites reactivity in aminoacylation. The tyrosyl-tRNA synthetase from Escherichia coli binds only 1 mol of tRNA, tyrosine, and tyrosyl adenylate per mol of enzyme dimer. However, like the enzyme from Bacillus stearothermophilus, once one active site is occupied by tyrosyl adenylate the other becomes accessible to bind a further molecule each of tyrosine and ATP. Both bacterial enzymes show biphasic kinetics with respect to tyrosine in the aminoacylation of tRNA. Equilibrium dialysis experiments show that this is due to 2 mol of tyrosine binding in the presence of ATP and tRNA. A method is given for a correction for the effects of hydrolysis of the charged tRNA on the aminoacylation kinetics.


Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique. A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus. This has been used to measure the initial rate of acylation of tRNATyr by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme, and also the rate constants of the partial reactions of amino acid activation and transfer to the tRNA. It is shown that at saturating conenctration of tRNA the reaction proceeds by a ternary complex mechanism. The rate-determining step is either the aminoacyltion process or a step preceding it. At low concentrations of tRNA the reaction proceeds by the stepwise process of formation of tyrosyl adenylate followed by acylation of the tRNA. The rate constants for these partial reactions are faster than that for the ternary complex reaction. But the prior binding of tRNA greatly decreases the rate of tyrosyl adenylate formation. Both pathways are probably important at physiological concentrations. 88% of the tyrosine from the tyrosyl adenylate complex is transferred to tRNA. The presence of added tyrosine and ATP reduces this to 78%. However, the addition of aliquots of ATP to a mixture of enzyme, tyrosine, and a saturating concentration of tRNA (i.e., ternary complex conditions) leads to at least 0.97 mol of tRNA being acylated/mol of ATP hydrolyzed. Trapping experiments show that the 12% of adenylated that is not transferred to tRNA is hydrolyzed on the enzyme rather than expelled into solution.


Fragments of bovine serum albumin produced by limited proteolysis. Isolation and characterization of tryptic fragments. Several fragments of bovine serum albumin have been isolated following limited tryptic hydrolysis and their positions then determined in the bovine serum albumin sequence published by J. R. Brown ((1975), Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, 591). When bovine serum albumin was coupled to palmityl-aminoethylamino-agarose and digested with trypsin, two fragments were obtained: (a) peptide 115-184, containing the highly aromatic disulfide loop 3 of Brown's model, and (b) a larger fragment, residues 377-581, containing disulfide loops 7-9. This fragment constitutes the third of the three domains of the albumin molecule. From bovine serum albumin digested in solution, peptide 115-184 was again obtained, as well as (c) a 39,000-dalton fragment identified as residues 198-581, loops 4-9 of the second and third domains, but with a long, tryptophan-containing segment 204-238 missing from loop 4. The ability to isolate these fragments without cleaving disulfide bridges is partial confirmation of the proposed model of bovine serum albumin as a series of nine independent loops.


Difference in pyruvate kinase regulation among three groups of yeasts. Yeast pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase EC 2.7.1.40) was classified into three groups based on the interaction with fructose-1,6-bisphosphate. The pyruvate kinases of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were activated by fructose 1,6-bisphosphate in the concentration range tested (up to 10 mM) of the substrate, phosphoenolpyruvate; the enzymes of "fermentative Candida" (Candida tropicalis and Candida utilis) were affected by fructose 1,6-biphosphate only when the substrate concentration was below 2 mM. Although the pyruvate kinase of Candida lipolytica (a yeast belonging to "oxidative Candida") was also affected by fructose 1,6-bisphosphate, the degree of the activation was extremely small as compared with the above four yeasts. The pyruvate kinase of C. tropicalis was inhibited by ATP more strongly in the absence of fructose 1,6-bisphosphate than its presence. In the case of the C. lipolytica enzyme, however, the enzyme was inhibited to a lesser extent by ATP, and fructose 1,6-bisphosphate did not reverse the inhibitory effect of ATP. Time course changes of the enzyme levels in the yeasts grown on glucose and on ethanol indicate that the pyruvate kinases of S. cerevisiae and C. tropicalis can be controlled both by an allosteric mechanism and by changes in the enzyme concentration, although a marked difference was observed in the susceptibility to the allosteric effect by fructose 1,6-biphosphate  between these fermentative yeasts. On the other hand, that of C. lipolytica would be controlled only by the latter mechanism.


Isolation, chemical and immunological characterization of two strongly basic nuclear proteins from human spermatozoa. Human spermatozoa were solubilized by treatment with urea/guanidine and fractionated by ion-exchange chromatography with Bio-Rex 70 resin. Four fractions were obtained. The acidic proteins pass unretarded, whereas the moderately basic and two strongly basic protein fractions are eluted by means of guanidine gradients. The main protein components of the strongly basic protein fractions have been isolated by gel filtration on Sephadex G-50. The purified proteins have been named human sperm protamine 1 and 2. They contain 47 and 51 aminoacid residues (mol. wt 6280 and 6840), of which 22 and 24 are arginine and 5 and 4 are half cystine residues, respectively. The electrophoretic mobility in urea/polyacrylamide gels is between that of calf thymus histone and salmon protamine. Human protamine 1 and 2 are both auto-antigens as has been detected by a quantitative immunofluorescence inhibition test.


Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase. The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA.


Analysis of the breaking sites in the physical degradation of DNA. The 3'-termini of procaryotic and eucaryotic DNA fragments obtained by shearing, sonication and irradiation have been analyzed. Analysis of the 3'-ends of DNA fragments was done by 32-P-labeling with terminal transferase, hydrolysis of products by spleen DNAase and spleen exonuclease and separation of labeled 3'-terminal nucleotides on polyethyleneimine plates. In the case of sonication the deviation from DNA base composition of 3'-terminal nucleotides is very close to random for Escherichia coli and Haemophilius influenzae DNA, whereas for calf thymus, mouse and yeast mitochondrial DNAs a significant increase in dA and decrease in dC was observed. Shearing at high salt released random 3'-termini in E. coli DNA and 3'-termini with 4 to 6% deviation from base composition in calf thymus DNA. In the case of gamma-irradiation no real differences have been found between E. coli and calf thymus DNA and 3'-terminal nucleotide composition is very close to random. Mechanical breakage of eucaryotic DNA seems to release 3'-termini having a composition which differs slightly but significantly from the average base composition of the DNA.


Physiological transition of a coliphage lambda DNA replication. The "rolling-circle" replicative intermediate (sigma-type molecules) which is normally produced in the late stage of coliphage lambda DNA replication can be found during the first round of lambda DNA replication if cells infected with lambda replication mutant Ots28 are incubated at the nonpermissive temperature until the late stage of the latent period of lambda infection. After shifting to the permissive temperature, the vast majority of replicating forms are sigma-type rolling circle even during the first round of DNA replication. Concatemeric lambda DNA molecules, produced from these sigma-type intermediates, are efficiently packaged into progeny phage, indicating that in the first round of lambda DNA replication, double-branched theta-type molecules are not required for production of viable progeny phage.


The mode of action of griseoviridin at the ribosome level. The antibiotic griseoviridin binds to the larger subunit of Escherichia coli ribosomes blocking the interaction between the 3' terminal end of peptidyl-tRNA and the donor site of the peptidyl transferase centre. Griseoviridin inhibits binding of chloramphenicol, thiamphenicol, lincomycin, erythromycin and streptogramin A to bacterial ribosomes. Moreover griseoviridin protects the ribosomal binding site of gougerotin from the drastic conformational changes taking place in the presence of ethanol. Griseoviridin is also able to interact with eukaryotic ribosomes as shown by its effects on  model systems and on anisomycin, trichodermin and gougerotin binding studies. Nevertheless, griseoviridin affinity for the 80-S type ribosomes (yeast or human) is two orders of magnitude smaller than with E. coli ribosomes. The inhibitory spectrum and mode of action of griseoviridin on ribosomes is compared to that of antibiotics of the streptogramin A group and found to be essentially the same.


Bacteriophage phi X174 DNA synthesis in Escherichia coli HF4704S (dnaHts) cells. The DNA synthesis of bacteriophage phiX174 in Escherichia coli HF470S, a mutant temperature sensitive in the initiation of DNA replication (dnaHts), has been examined. In HF4704S cells, phiX174 can grow normally at 27 degree C whereas the phage cannot grow after the cessation of DNA synthesis of the host cells at 42 degrees C. Upon infection, phiX174 DNA can be injected into the host cell and the parental replicative form can be formed, but the progency replicative form cannot be synthesized at 43 degrees C in the absence of host DNA synthesis. The progency replicative form cannot be synthesized at 27 degrees C in the presence of 30 mug chloramphenicol/ml in the host cell which has been incubated for 74 min at 43 degrees C followed by transfer to 27 degrees C in the presence of 30 mug chloramphenicol/ml. When 30 mug chloramphenicol/ml is added later than 5 min after the temperature shift-down to 27 degrees C, the progency replicative form synthesis is not inhibited. Thus, the host cell function, for which the gene dnaH is responsible, has been shown to be essential to the progency replicative form production.


The ATP-dependent DNAase from Escherichia coli rorA: a nuclease with changed enzymatic properties. The ATP-dependent DNAases from Escherichia coli wild-type and rorA were isolated and purified and their enzymatic properties were compared. The enzymes were found to differ in the amount of ATP that is consumed during DNA degradation. This difference can be influenced by the reaction conditions and the nature of the substrate.


Partial degradation of transfer RNAs by different preparations of snake venom exonuclease. The degradation of yeast tRNASer with eight different exonuclease preparations from four snake venoms was investigated. The reaction products were separated on polyacrylamide gels containing 7 M urea. Patterns of sharp bands were obtained which were more or less similar. Two tRNA fragments were characterized by oligonucleotide analyses, one of which was tRNA degraded by the exonuclease up to the beginning of the T-phi-C-stem. The other one was generated by the additional loss of several nucleotides from the 5'-terminus. The formation of the latter fragment was very probably caused by an endonuclease activity in the exonuclease. The endonuclease contaminant, which was found in all preparations, was further investigated by experiments with modified tRNAs whose 3'-terminus should be resistant to exonuclease (tRNASer-A, tRNASerOX-red). With 3'-AMP as substrate no phosphatase activity was found under the conditions of tRNA degradation. Not only in tRNASer, but also in yeast tRNATyr and tRNAAla as well as in fragments of tRNASer and tRNAPhe, the degradation by exonuclease was inhibited at the beginning of the T-phi-C-stem. The finding of such a retardation site in addition to the general retardation of exonuclease digestion after removal of the C-C-A sequence may indicate that retardation at certain elements of secondary structure is a more general feature of degradations by this enzyme.


30 S pre-ribosomal RNA of Escherichia coli:primary and secondary processing. The metabolism of newly-formed labeled 30 S pre-ribosomal RNA was studied in the Escherichia coli mutant strain AB301-105. Detailed kinetic analysis in rifampicin-treated cells showed that precursors of 23 S and 16 S rRNA are formed from the 30 S RNA species, even in phosphate-limited cultures. To establish the order fo segments along 30 S pre-rRNA, it was allowed to accumulate in replicate chloramphenicol-treated cultures, and pulse-labeled after the addition of rifampicin in segments that progressively contained more 3'-distal label with time. The purified RNAs from the various cultures were then cleaved to a limited extent by RNAase III; the specific activity of the fragments yielded an order from the 5'-end of 17.5S-X-25S, where X refers to some of the sequences released as additional small fragments. More extensive treatment the 25-S and 17.5-S pre-rRNA chains with RNAase III yielded additional fragments and produced RNA species with the mobility of the 23-S and 16-S RNA precursors made in normal cells treated with chloramphenicol. A processing scheme is suggested that distinguishes between early, site-specific cleavage of recognition sites in the RNA itself ('primary processing'), and later, "secondary processing' reactions. The secondary processing reactions are blocked chloramphenicol-treated wild-type cells.


Purification of biosynthetic threonine deaminase from Escherichia coli. Biosynthetic threonine deaminase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) was purified to apparent homogeneity from cell extracts of Escherichia coli by chromatographic procedures using valine-Sepharose, isoleucine-N-hexamethyleneamine-Sepharose, and hydroxyapatite with an overall yield of 40%. Analytical ultracentrifugation shows a molecular weight of 214 000. In sodium dodecyl sulfate gel electrophoresis, the enzyme migrates as a single band corresponding to a molecular weight of about 50 000. These data confirm that the enzyme is a tetramer. The sedimentation coefficient, s-020,w, determined by differential sedimentation experiments is 9.2 S. The enzyme shows absorption maxima at 415 and 280 nm. Determination of pyridoxal phosphate by three indenpendent methods shows the presence of two molecules of pyridoxal phosphate per enzyme molecule, the different methods being in excellent agreement equilibrium dialysis experiments establish the presence of two isoleucine binding sites. The Scatchard plot suggests non-cooperativity of these sites. The association constant for isoleucine is 1.2 - 10(5)M-1.


Anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyl-transferase from Salmonella typhimurium. Inactivation of glutamine-dependent anthranilate synthetase by agarose-bound anthranilate. Exposure of the anthranilate synthetase-anthranilate phosphoribosyltransferase enzyme complex (chorismate pyruvate-lyase (amino-accepting) EC 4.1.3.27 and N-(5-phosphoribosyl)-anthranilate pyrophosphate phosphoribosyl-transferase, EC 2.4.2.18) from Salmonella typhimurium to agarose-bound anthranilate led to the slow inactivation of glutamine-dependent anthranilate synthetase activity, an activity dependent on protein-protein interaction in the enzyme complex. Region I of phosphoribosyltransferase, the location of the enzyme complex glutaminase activity, is the site of alteration. Phosphoribosyltransferase and NH3-dependent anthranilate synthetase activities and trypto phan regulation of phosphoribosyltransferase were unaffected by the anthranilate matrix. The molecular weight (280 000) and subunit molecular weight (62 000) of the enzyme complex eluted from an anthranilate matrix were identical to those of enzyme complex purified by classical methodology. The enzyme complex could be partially protected against inactivation by storiing in 0.1 M L-glutamine or 30% glycerol and completely protected by storing in 50% glycerol at -18 degrees C. Evidence is presented that the anthranilate matrix acts as a hydrophobic matrix and may be binding to and altering a hydrophobic region in the enzyme complex. The anthranilate matrix provides a convenient tool for altering a specific region of an enzyme complex without covalent modification. At the same time, the results demonstrate that affinity matrices are not necessarily innocuous but may subject macromolecules to an adverse environment not previously recognized.


Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12. The thiol-oxidizing agent "diamide" (CH3)2NCON equal to NCON(CH3)2 was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic process. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in gamma-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide rang of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.


Type III procollagen and collagen in skin. A form of collagen, containing three alpha chains of type III, can be extracted from foetal calf, calf and rat skin under physiological conditions. This native collagen was purified by DEAE-cellulose chromatography and then was analysed by polyacrylamide gel electrophoresis which showed it consisted of several high molecular weight components, the size of gamma components and larger species. Prior reduction in dithiothreitol dissociated these large polymers into two components: the minor one migrated between the alpha1 (I) and alpha2 chains while the predominant one migrated between the alpha and beta chains. These two monomers were isolated by CM-cellulose chromatography. The minor one, which eluted between the alpha1 and alpha2 chains, had a molecular weight of approx. 95 000; its amino acid composition was similar to that of alpha1(III). The major one eluted in the alpha1 region and had a molecular weight of approx. 120 000; its amino acid composition, while similar to that of the alpha1(III) chain, differed in detail, and it is presumed to be a pro-alpha1(III) chain. Following pepsin digestion, the native collagen remained as a disulfide-bonded trimer which dissociated into only one component, a1(III), when denatured in dithiothreitol. These results suggest that the original, extracted protein consisted primarily ofa precursor form of type III collagen. This procollagen did not polymerize when heated at 37 degrees C and did not form the usual segment long spacing aggregates under suitable conditions. It was not modified by incubation with a purified procollagen peptidase preparation. This appears to be the first example of the isolation of type III (pro)collagen by extractive methods, without resorting to tissue digestion by proteolytic enzymes.


Reversibility of the affinity labelled-biotin transport system in yeast cells. Transport of biotin by Saccharomyces cerevisiae is inhibited by biotynyl p-nitrophenyl ester. Conversion of the inhibited cells to spheroplasts or simple treatment with thiols results in a total restoration of vitamin transport. Biotynyl p-nitrophenyl ester-induced inhibition is not due to an intracellular accumulation of the vitamin and consequent regulation, but appears to be due to specific labelling  of the transport system.


Proteins of the camel erythrocyte membrane. Electrophoresis on polyacrylamide gels containing dodecyl sulphate has revealed that the major proteins of the camel erythrocyte membrane are similar to those of the human and bovine species in both electrophoretic mobility and relative abundance. The major difference lies in the major intrinsic membrane protein of molecular weight approx. 100 000. In the camel, this protein has a higher apparent molecular weight than in the human and bovine species. The very high molecular weight water-soluble protein "spectrin" appears to be very tightly bound to the camel erythrocyte membrane, and is only partially extracted after prolonged low ionic strength dialysis. Total release of spectrin is only achieved by means of more drastic treatment such as incubation with urea, guanidine hydrochloride, sodium hydroxide of p-chloromercuribenzoate. Concurrent with the total release of spectrin, the camel cells undergo a shape change from flat ellipsoids to spheres, suggesting an important shape-maintaining role for spectrin in the erythrocytes of this species.


[Individuality of mannonate and altronate hydro-lyases in Escherichia coli K 12]. In Escherichia coli, mannonic and altronic hydrolyases act, respectively, on mannonate, the intermediate aldonate of the glucuronate branch, and on altronate the intermediate aldonate of the galacturonate branch of the hexuronate pathway, yielding 2-keto-3-deoxy-gluconate. Our results demonstrate that the two hydrolyases are two distinct proteins. First, each hydrolyase shows a different induction pattern. In addition, separate constitutive mutants for either hydrolyase have been obtained. Second, single mutants negatively affected for one of the activities but not the other have been isolated in each case. Third, comparative heat inactivation of both activities at 59 degrees C shows mannonic hydrolyase to be clearly more thermosensitive than altronic hydrolyase. Furthermore the two enzymes also react differently to various effectors. Fourth, the two enzymes could be resolved on a DEAE cellulose column into two neighbouring but distinct peaks of activity, and a further purification yielded two pure hydrolyase fractions each being devoid of the activity of the other.


[Purification of beta-lactamases by affinity chromatography]. Affinity columns able to purifie beta-lactamases have been prepared either by linking covalently reversible inhibitors or substrates to agarose beds. The enzyme is eluted with a gradient of sodium chloride or released with the substrate. This method is a pertinent one for the purification of these enzymes and for the study of bacteria harbouring more than one beta-lactamase.


[Polarization state of the light source and determination of attenuated total internal reflection spectra in intact microorganisms]. The problems are considered of the interaction of linear polarized light current with undestroyed microorganisms while recording the spectra of attenuated total refraction(ATR) and repeated ATR (RATR). It is shown that proper allowance should be made of dichrosim of absorption bands of microorganisms during quantitative studies. Peculiarities of obtaining RATR spectra of the linearly polarized light source with polarization azinut other than 0 and 90 degrees are considered.


[Determination of the optical constants of intact microogranisms by the method of attenuated internal reflection spectroscopy]. The problems are considered of the determination of the optical constants (dispersion of refraction, n2, and absorption, K, indexes) of strongly dispersing biological objects on undestroyed cells of microorganisms (E. coli, strain C-85) by the method of repeated attenuated total refraction. The volume of unpacked intercellular space and the effect of repeated propagation of the light current through measuring elements with high refraction index were taken into account. Relationships R, s (n1, n2, O) K were obtained for determining the optical constants on the elements with the angle of light current incidence 0=45 degrees, made of infrared glass, IRG-24, IRF-24 and of Si and Ge as well. The optical constants of bacteria in the region of absorption bands 1660, 1550 cm-1 were also determined.


tRNAs undermethylation in a met-regulatory mutant of Saccharomyces cerevisiae. A study of in vivo and in vitro methylation of tRNAs in regulatory mutants affected in methionine-mediated repression (eth2, eth3, eth10) has led to the following results: 1) The eth2-2 carrying strain presents a great undermethylation of its tRNAs of the same order of magnitude as observed during methionine starvation of methionine auxotrophs. 2) This undermethylation leads to a shift of the tRNAIII met peak on a BD cellulose column, while tRNAIII met peak is unchanged. 3) The study of a double mutant strain carrying eth2 and met2 mutations has shown that this undermethylation is a consequence of the high internal pool of methionine. 4) Undermethylation unequally affects the different bases and the different tRNA species.


Sequence diagrams and the presentation of structural and evolutionary relationships among proteins. Protein sequences mapped on two-dimensional diagrams show characteristic patterns that should be of value in visualising sequence information and in distinguishing simpler structures. A convenient map form for comparative purposes is the alpha-helix diagram with aminoacid distribution analogous to the surface of an alpha-helix oriented so that an alpha-helix structure corresponds on the diagram to a vertical band 3.6 residues wide. The sequence diagram for an alpha-keratin, high-sulphur protein suggests a new form of polypeptide helix based on a repeating unit of five which may be an important component of alpha-keratin fibres.


The application of nuclear magnetic resonance spectroscopy to the study of natural and model membranes. This review article outlines some potentials and limits of the recent application of high resolution Nuclear Magnetic Resonance technique--coupled to the Fourier transformation methods--to the study of biological membranes. Molecular arrangement and dynamical structure characters can be assessed at the level of individual chemical groups in lipid bilayer regions of natural and model membranes, through the determination of physical parameters like chemical shifts, spin-lattice (T1) and spin-spin (T2) nuclear magnetic relaxation times. The results of some significant experiments carried out on single-wall lecithin vesicles as well as on intact natural membranes, are summarized and discussed. Useful information can be obtained on the lipid fatty-acid chains thermal transition, by comparing two lecithin vesicles of the same size, formed by the same host lecithin, but incorporated with different molecular components. In particular, T1 and T2 measurements, interpreted in terms of a two- (or more-) correlation time theoretical models, are able to demonstrate different degrees of motional anisotropy in bilayers formed by mixed lecithins or by mixtures of lecithin and fatty acids, possessing moderately different chain lengths [13]. Chromophore-containing molecules, like chlorophyll [12] or fluorescent probes [14] can be located, within few Angstroms, in a lipid bilayer through proton chemical shift measurements; in addition the perturbation of the lipid membrane structure, as induced by the incorporated probe, is assessed mainly in terms of the intramolecular dynamical structure of the host lecithin molecules, by means of T1 and linewidth studies. The comparison of the n.m.r. relaxation behaviour in intact membranes and in vesicles formed by their extracted lipids may, finally, provide indirect information on the lipid-protein intermolecular interactions and relative mobility, besides indicating the intramolecular mobility characters of the lipid bilayer regions of the membrane.


Lymphoreticular aggregates in the newborn lung and their relation to intrauterine INFECTION and neurospinal dysraphism. A survey has been carried out of lymphoreticular aggregates in the lungs of 200 children, stillborns and dying within 15 days of birth and in a further 21 children with anencephalus, and controls. Lymphoreticular aggregates occur in response to amniotic infection and are found in the 'intrauterine infection syndrome'. They are not found in children showing early hyaline membranes. The time interval required for the production of lymphoreticular aggregates in the human child appears to be at lease 48 h and is probably 72 h before the production of well-formed aggregates and this probably applies to both the intrauterine and the postnatal state. There is a significant increase in the number of lungs showing lymphoreticular aggregates at birth in children in whom there is neurospinal dysraphism. In such children there is necrosis of nervous tissue into the amniotic cavity and it would seem likely that this material has been antigenic to the lung in utero.


Number fluctuation spectroscopy of motile microorganisms. A random-walk model of motility is used to predict the dynamics of fluctuations in the number of particles in a small observation volume. The results show that number fluctuations provide a measure of the mean swimming speed as well as the persistence length. Experimental light-scattering results are presented for three strains of Escherichia coli whose motion appears random-walk in nature. For the strain with th elongest persistence length, excellent agreement is found that theoretical predictions. For the more erratic strains, however, the shape of the measured scattered light intensity correlation functions indicates the presence of a contribution due to orientational fluctuations.


Quantitative assessment of thymus-dependent (t)-cell function in human peripheral blood. A new micromethod for evaluating lymphocyte responses to phytohemagglutinin (PHA) has been developed and used for the survey of T-cell function in human peripheral blood. The results of lymphocyte responses to PHA stimulation in whole blood culture were expressed in terms of unit volume of blood (50 mul). This new method has eliminated the major uncontrollable factors of conventional methods, and appears to be more correlated with the cell-mediated immunity in vivo.


Some characteristics of human rosette-forming cells. Some evidence for identification of human rosette-forming cells as thymus and thymus-dependent lymphocytes (T cells) is presented. Application of the rosette assay to several clinical conditions including primary immunodeficiency diseases indicates the usefulness of this assay in quantitating the number of T cells in peripheral blood.


Selective IgA deficiency---some perspectives. The clinical spectrum of IgA deficiency varies from good health to respiratory, gastrointestinal, autoimmune and malignant disorders. Evaluation of B lymphocytes with surface IgA receptors in this disease reveals cap formation, decreased to normal density of IgA receptors, and IgA secretion following pokeweed stimulation. Further study of antigen-induced B-lymphocyte phenomena is needed. The presence of decreased numbers of T-cell markers in some individuals with IgA deficiency suggests a T-"helper" cell defect.


T-cell deficiency in diGeorge syndrome.

T-cell deficiency in diGeorge syndrome. DiGeorge syndrome is broadly defined as absence or hypoplasia of the thymus due to dysmorphogenesis of the third and fourth pharyngeal pouches in early embryonic life. Various tests of thymic function have been evaluated in some 19 infants and young children with this syndrome. Deficiency of T lymphocytes has been shown to occur in the presence of as much as 10 to 20% of the normal amount of histologically normal thymic tissue in the partial DiGeorge syndrome.


Immunodeficiency and autoimmunity.

Immunodeficiency and autoimmunity. The following aspects of autoimmunity and immunodeficiency will be discussed: 1) Autoantibodies and autoimmunity and their relationships to human disease and particularly to the rheumatic diseases. 2) Relationships between human immunodeficiency diseases and autoimmunity including: a) autoimmune disease in patients with immunodeficiency; b) arthritis in patients with immunodeficiency; c) lupus-like illness in carriers of chronic granulomatous disease; d) occurrence of autoantibodies and autoimmunity in relatives of patients with immunodeficiency. 3) Immunodeficiency as a basis for human rheumatic or autoimmune diseases. The role of immunodeficiency in NZB mouse diseases and human SLE. 4) The role of viral infections in autoimmune disease.


Immune deficiency, autoimmunity and aging. Immune deficiency states in man and animals have been observed at all ages---in the very young, in adult life and the very old. At all ages there appears to be a correlation between immune deficiency and propensity to produce autoantibodies and malignancy. Cellular replacement in immunologically deficient mice results in correction of immune function and slightly increased survival while selected hybridization produces dramatic increases in survival and a delayed onset of autoimmune symptoms. Aging is defined in terms of primary and secondary events where a hypothetic rate of decline of function, called optimum functional longevity, is arbitrarily assigned to the long-lived population. Optimal functional longevity is genetically based and controlled by a hypothetic longevity homeostasis gene complex which includes Ir genes and genes that control endocrine balance.


T Cells in immunodeficiencies as evaluated by an antihuman T-cell serum. An antihuman T-cell has been prepared in rabbits by infecting lymphocytes from a patient with Bruton-type agammaglobulinemia and absorption with cultured B-lymphoblast cells. Cytotoxicity of the antiserum was assayed with peripheral blood lymphocytes in the presence of rabbit complement and its specificity for T vs B cells was verified. This anti-T-cell serum killed 66% (range 49-78) of normal blood lymphocytes. In patients with Bruton-type agammaglobulinemia the percentage of cells sensitive to the antiserum was above normal (77-89%), whereas it was decreased in some patients with lepromatous leprosy or under antilymphocyte globulin therapy.


Subacute sclerosing panencephalitis (SSPE). Subacute sclerosing panencephalitis (SSPE) is a degenerative disease of the central nervous system occurring in children and adolescents. The measles virus, or a virus closely associated, plays an important role in the disease. SSPE appears years after these children have had the measles but still a measles-like virus can be found in their brain and in other organs. A certain deficit, still to be defined of the cell-mediated immunity as a chronic stimulation of the antibody response against the measles virus is documented in SSPE pointing to a possible immunologic imbalance in these patients. Studying the possibility of the presence of immune complexes in SSPE, immunofluorescent studies of kidney biopsy in SSPE patients have been performed. Complement deposits in the glomerular basement membrane have been found in most patients. The possible role of immune complexes in SSPE is still to be defined.


Immunodeficiency in leprosy.

Immunodeficiency in leprosy. It is known that the impairment of cell-mediated immunity (CM) exists in lepromatous leprosy patients. This was shown by 1) delayed skin reaction to various test antigens, 2) decreased transformation of lymphocytes by various antigens, 3) delayed rejection of skin allograft, 4) decreased migration inhibitory fa-tor (MIF) formation, and 5) histologic study of the lymph node. In addition to these previous works, we have studied T and B lymphocytes in peripheral blood of leprosy patients; T cells by the technic of rosette formation, and B cells by staining surface immunoglobulins. The result of our study shows decreased numbers of T cells and increased numbers of B cells in peripheral blood of lepromatous patients. A the same time, we found that allogenic leukocyte infusion treatment reversed T and B-cell abnormalities. We know that repeated infusions of allogenic leukocytes dramatically brought about improvement of the clinical status of the patients, resolution of skin lesions, subsidence of erythema nodosum leprosum reaction, clearance of bacteria from the skin and lymph nodes, and reconstitution of peripheral lymph nodes.


Ataxia-telangiectasia: some historic, clinical and pathologic observations. Although an isolated clinical case report was published in 1926 and another in 1941, ataxia-telangiectasia (A-T) was not established as a distinct entity until 1957, when it was first delineated clinicopathologically. Susceptibility to sinopulmonary infection was identified as the main cause of death and as the third major component of the syndrome; its heredofamilial nature was documented, and it was designated "ataxia-telangiectasia." In a later review of 101 published cases, lymphoreticular malignancy emerged as the second most frequent cause of death. Although the thymus was found to be absent in the first reported autopsy in 1957 and the serum IgA deficiency was first recorded in 1961, A-T was not established as an immunodeficiency disease until 1963. Thymic abnormality and dysgammaglobulinemia explain the 2 main causes of death, sinopulmonary and neoplastic, but the immunodeficiency is probably not the central defect. It does not appear to explain either of the 2 main clinical diagnostic keys, the ataxia and the telangiectasia, or any of the other seemingly unrealted multisystemic facets of this complex disorder. Some of our most provocative long-term clinical observations and recent pathologic findings in our series of 9 autopsies are discussed.


immunodeficiency in ataxia-telangiectasia. Ataxia-telangiectasia is characterized by endocrine, neurologic, hematologic, hepatic, cutaneous and immunologic abnormalities. The immunologic deficiencies vary considerably from patient to patient, and in each patient with respect to time. The most frequent deficiencies of humoral immunity are diminished or absent serum and salivary IgA, diminished or absent serum IgE and impaired antibody responses to a variety of bacterial and viral antigens. Deficiencies of cellular immunity are commonly found by both in vivo and in vitro analyses. Histologic confirmation of these immunodeficiencies is readily observed in the lymphoid tissue. The thymus, which may be the seat of the primary abnormality in the immunodeficiency syndrome, regularly shows morphologic characteristics of an embryonic thymus.


A spectrum of B-cell differentiation defects. The nonparallel relationships between development of classes of B lymphocytes and of serum immunoglobulins during ontogeny and in some patients imply: 1) that B-cell differentiation is discontinuous and 2) that class-specific-controls for terminal B-lymphocyte differentiation exist. Three separate defects in B-cell differentiation in vivo have been identified using pokeweed mitogen stimulation of lymphocytes. B lymphocytes may fail to develop, may develop but lack the capacity to differentiate further, or may be normal in both respects. The latter result suggests involvement of other cell types in antibody deficiency.


Development of T and B cells and their functional interactions. The development of IgM-, IgG- and then IgA-bearing B lymphocytes is an orderly process occurring independently of antigens and T cells. T cells enhance antigen-induced B lymphocyte to plasma cell differentiation according to the hierarchy: IgA greater than IgG greater than IgM. T cells also exhibit antigenindependent and dependent stages of differentiation and a functional diversity, perhaps attributable to different classes of T cells. New evidence indicates the presence of a receptor on T cells for IgM antibody produced by B cells.


Assessment of the B-lymphocyte population in agammaglobulinemia. The population of B lymphocytes was assessed in the blood of 16 patients with agammaglobulinemia using immunofluorescence and EAC1423 reactivity as B-cell markers. Lymphocytes were fractionated on gradients of bovine serum albumin which are capable of separating lymphocytes into B-cell-rich and T-cell-rich populations. There was complete absence of B lymphocytes in the majority of patients with X-linked agammaglobulinemia. Normal or increased numbers of B lymphocytes were found in all patients with common variable agammaglobulinemia.


Use of transfer factor in patients with depressed cellular immunity and chronic infection. Two patients with chronic mucocutaneous candidiasis and a defect in cellular immunity received a single injection of dialysable transfer factor from Candida-positive donors in an effort to reconstitute immunologic function. The transfer of cellular hypersensitivity was successful in one of the two patients and was monitored by skin tests and MIF production; however, the effect was temporary and did not change the clinical course of the patient's infection. The other patient did not respond eitherimmunologically or clinically to transfer factor at this time, although she did respond subsequently to repeated doses of transfer factor and amphotericin B therapy (Pabst and Swanson: Brit. med. J. 2:442, 1972). In another instance transfer factor from tuberculin-positive donors was used successfully to eradicate an infection in a patient with progressive primary tuberculosis and an acquired defect in cellular immunity. The patient had not responded clinically or bacteriologically after 7 1/2 months of antituberculous therapy, although the organism was shown to be sensitive in vitro to the drugs she was receiving. She received 6 doses of dialysable transfer factor over a 3-month period and during this time she responded clinically, bacteriologically and roentgenographically.


Transfer factor as an approach to the treatment of immune deficiency disease. Use of transfer factor in the treatment of chronic mucocutaneous candidiasis is discussed. The clinical experience in treating 2 patients with different clinical expressions of the syndrome and their different responses to treatment with repeated injections of transfer factor given in conjunction with amphotericin-B are reported. Results indicate that this form of therapy is a safe and effective way of restoring cell-mediated immunity to Candida and successfully treating some patients with chronic mucocutaneous candidiasis.


Restoration of cell-mediated immune responses with transfer factor. Five anergic patients with chronic mucocutaneous candidiasis were given transfer factor from donors with positive delayed reactions to Candida. In each recipient, the delayed skin reactions of the transfer factor donors appeared in the recipients and no recipient developed reactivities not possessed by the donor. Prior to injection of transfer factor, in vitro stimulation of the patients' lymphocytes with antigens did not result in MIF production, however, after transfer factor this response was positive. Therapy with transfer factor alone did not have therapeutic benefit, however, in 2 patients treatment with amphotericin-B followed by transfer factor has produced cutaneous remissions of 18 months.


Transfer factor II: results of therapy.

Transfer factor II: results of therapy. Transfer factor is a dialyzable extract of sensitized leukocytes, which transfers reactivity from skin test-positive donors to skin test-negative recipients. Transfer factor supplied by our laboratory has been used therapeutically to induce cellular immunity in 78 patients around the world. Many patients received multiple doses of transfer factor ranging from 1 unit given every 6 months for 3 years to 1 unit every week for 6 months to as much as 8 units per week for a brief period. A total of 299 units of transfer factor have been given. Diseases in which transfer factor appeared to cause improvement include the Wiskott-Aldrich syndrome, severe combined immunodeficiency disease, mucocutaneous candidiasis, chronic active hepatitis, coccidioidmycosis, dysgammaglobulinemia, Behcet disease, aphthous stomatitis, linear morphea, familial keratoacanthoma and malignancy.


Relation of lymphoid system and hormones to aging. Congenital deficiencies of developmental hormones, such as growth hormone and thyroxine, are responsible for the thymus-dependent immunodeficiency disease observed in dwarf mice. Such an immunodeficiency state is associated with early aging phenomena. This fact suggests that lymphoid cells and primarily T-derived cells are involved in the control of aging processes.


Potentiating effect of fetal thymus on fetal liver cells for the promotion of recovery from the radiation injury in murine allogenic radiation chimeras or a little bit of thymus goes a long, long way. Lethally irradiated CBA (H-2k) mice were treated with small numbers of liver cells from strain-A (H-2a) fetal donors. Recovery from the radiation injury was dependent upon the dose of liver cells administered. Giving very small numbers of fetal thymus cells, in addition to liver cells, had a highly significant and salutary effect on survival. The results suggest that clinical transplantation of liver cells from a single fetus might succeed if thymus cells are added.


Developmental immunity in the piglet.

Developmental immunity in the piglet. Germfree colostrum-deprived piglets are immunologically "virgin" and extremely susceptible to microbial infection due to lack of passive maternal immunity. They are, however, highly immunologically competent as determined by their excellent immune response to various antigenic stimulation. The results suggest that antigen is the sole inducer of specific antibody formation and support the hypothesis that the initial step in the "true" primary immune response involves priming of multipotential uncommitted immunocompetent cells ("virgin" X cells) to committed monopotent cells (Y cells), and that Y cells proliferate and differentiate into antibody-forming cells (Z cells).


Congenital neutropenia: impaired maturation with diminished stem-cell input. Children with congenital neutropenia (Kostman disease) present a syndrome of recurrent and eventually fatal bacterial infections. The disease is uniformly lethal and the mean age at death is 24 months. To attempt to better understand the nature of this inborn error, we have studied marrow and peripheral blood of 3 children with the disease. Along with classic morphologic analysis we have used the technic of soft agar bone marrow culture in vitro. All 3 patients showed marked deficiency of numbers of neutrophils in the peripheral blood, intermittent monocytosis especially with infection, and predominant nitroblue tetrazolium (NBT) dye staining in monocytes during infection. The marrow of all 3 patients showed evidence of maturation arrest at the promyelocyte stage and very few or no neutrophils. Two cases showed no neutrophils in the Rebuck skin window inflammatory study. The 3rd case, however, developed small numbers of neutrophils in the peripheral blood, up to 6%, but the first 2 patients, although studied on many occasions, did not show neutrophils above 1%. Evidence from soft agar culture of bone marrow in 1 patient was compatible with the view that development of committed neutrophil stem cells is defective. This patient did not develop neutrophils in soft agar culture and showed loose colonies developing only to promyelocytes. In the 2nd case, where depression of neutrophil development was somewhat less severe clinically, culture of the marrow in soft agar yielded normal neutrophil colonies and none of the loose colonies showing arrested maturation were seen. These findings suggest that the basic mechanism underlying the congenital neutropenias may differ from patient to patient.


Human thymus-derived rosette-forming cells and immunologic diseases. This paper describes some studies done on the human lymphocytes that bind to normal sheep red blood cells in a rosette formation. On the basis of human fetal studies and investigations of markers for B cells, it is demonstrated that rosette-forming cells are valid markers for T cells. These cells were looked at in various immunologic diseases. A modification of the basic technic, one that detects subpopulations of lymphocytes, was mainly used. Results correlate closely with the cellular immune status of the patient.


Immunoglobulin metabolism in disease.

Immunoglobulin metabolism in disease. Immunodeficiency and hypogammaglobulinemia are shown to occur by a variety of pathophysiologic mechanisms. Hypogammaglobulinemia may be caused by decreased synthesis of all classes of immunoglobulins, or may involve defective synthesis of selective classes of immunoglobulins. Alternatively, hypogammaglobulinemia may be due to a disorder of endogenous catabolic pathways of a single immunoglobulin class, or of all immunoglobulin classes. Finally, hypogammaglobulinemia may be caused by excessive loss of immunoglobulins into the urinary or gastrointestinal tracts.


Platelet size in man.

Platelet size in man. The shape and parameters of platelet size distributions were studied in 50 normal persons and 97 patients in order to test the proposed thesis that platelet size heterogeneity results mainly from aging in the circulation. This thesis was contradicted (1) by size distributions of age-homogeneous, newly-born cell populations which were lognormal with increased (instead of decreased) dispersion of volumes and (2) by the macrothrombocytosis found in some populations with normal age distribution. For these reasons, thrombocytopoiesis appeared to play the major role in determining platelet size. A model was built in which the volume variation of platelet territories due to megakaryocyte growth and membrane demarcation at each step of maturation was a random proportion of the previous value of the volume. This model explains the lognormal shape of both newborn and circulating platelet size distributions. It  also implies that (1) the mean and standard deviation of platelet logvolumes depend on the rates of volume change of the individual platelet territories (growth rate minus demarcation rate) as well as on megakaryocyte maturation time; (2) platelet hyperdestruction causes an increase in the mean and dispersion of the rates of territory volume change; (3) Mediterranean macrothrombocytosis and some hereditary macrothrombocytotic thrombocytopenias or dysthrombocytopoieses reflect a diminished rate of territory demarcation, and (4) platelet size heterogeneity is caused mainly by the variations in territory growth and demarcation and not by aging in the circulation.


The development of vertebral bone marrow of human fetuses. The development of the bone marrow of the thoracic vertebrae in seven human fetuses ranging from 95 to 150 mm in crown-rump length (CRL) was studied using light and electron microscopy. In the 95-mm CRL, hypertrophy of the chondrocytes occurred in the central region of the vertebrae, and blood vessels penetrated there from dorsal and ventral sides of the vertebral body. The primary marrow was represented by liberated cartilage lacunnae, occupied by the thin-walled blood vessels and a few mesenchymal cells and mononuclear cells containing granules or vacuoles (GMC). In the 99-mm CRL, chondroclasts were active in removing the cartilage near the central region of the vertebrae. Consequently, a large cavity was formed and occupied by a dilated sinus. GMC were numerous. Osteoblasts and osteocytes were increased in number. Reticular cells with long processes containing large amounts of glycogen began to appear in the extravascular space and formed the loosely arranged cellular meshwork of the hematopoietic compartment. Bundles of collagen fibrils were scattered in the meshwork. Hematopoietic cells were recognizable only in the 105-mm-CRL fetus and increased in number in the 120-mm-CRL fetus. The sinus endothelium was very thin and continuous without apertures except where blood cells crossed the wall. The developing blood cells lying against the outside of the sinus endothelium indented it. At points, collagen fibrils attached to the outside of endothelial cells and appeared to function as the anchoring filaments of lymphatics. The physiologic implications of the association of stromal cells, vascular sinuses, and hematopoietic cells are discussed in relationship to the microhematopoietic environment of the bone marrow.


Regeneration of blood-forming organs after autologous leukocyte transfusion in lethally irradiated dogs. I. Distribution and cellularity of the bone marrow in normal dogs. Marrow cellularity in adult beagles (1-2 yr old) is highest in centrally located bones, with values between 8000 and 12,000 nucleated cells per sq mm. It decreases gradually towards the peripheral parts of the body, reaching values below 1000 per sq mm in bones distal to the elbow and knee. The first tail segment always contains some active marrow. The fifth segment has only stromal elements. In spongy bones fat cells appear to be distributed at random among the blood-forming elements. In the middle part of the femur the fatty marrow predominates in the center of the cylinder, while the subendosteal area is very cellular. The proximal and distal ends of the femur are more cellular than the middle. The small standard deviation of the cellularity in the spongy bones of the trunk and in the proximal and middle part of the humerus makes these sites areas of choice for quantitative studies of marrow regeneration. The large variations in cellularity of the marrow in the radius and tibia of young adult dogs make these sites unsuitable for such studies. The distribution of active and fatty marrow in dogs is similar to that of humans. The differential count of active marrow is quite constant between different dogs and in the various sites of each animal.


Myocardial failure with altered response to adrenaline in endotoxin shock. 1 There is a growing concensus that myocardial performance in the early stages of experimental endotoxic and septic shock is relatively normal; however, recent reports have identified an intermediate phase of shock when myocardial dysfunction is clearly apparent.2 The mechanism of dysfunction has become a subject of intense investigation. A current view is that altered myocardial responsiveness to circulating catecholamines may play an important role in the dysfunction observed after endotoxin administration. The present studies, in which an isolated working heart preparation of the dog was used, were designed to test this hypothesis. This particular experimental preparation was selected to provide an adequate interpretation of results; cardiac output, afterload, and concentrations of adrenaline reaching the coronary vascular bed were controlled in all experiments. Responses to infusions of adrenaline were recorded in the ;steady-state' condition. Control (non-shocked) heart responses to adrenaline were highly reproducible in terms of inotropic, chronotropic and coronary vascular behaviour.3 Results from the study document myocardial dysfunction within 4-6 h following an LD(70) endotoxin administration on the basis of increased left ventricular end diastolic pressure (LVEDP), decreased cardiac power and myocardial efficiency, and depressed negative and positive dP/dt parameters.4 Findings suggest significantly altered responsiveness of the myocardium to infused adrenaline at rates of 1, 2, and 5 mug/min with concentrations between 10 and 1 ng/ml blood. LVEDP was elevated while calculated power and efficiency parameters remained significantly below control values during infusion of adrenaline in endotoxin-treated hearts. Depressions of responsiveness were interpreted to occur on the basis of failure to restore positive and negative dP/dt to normal values and depressed coronary blood flow responses during adrenaline administration. Increases in coronary flow were regularly less in experimental hearts than the controls. Heart rate responses to adrenaline in both failing and non-failing hearts were identical.5 In conclusion, it is suggested that myocardial contractile and relaxation characteristics and coronary vascular responses to adrenaline infusion are depressed in endotoxin shock during the period of demonstrated myocardial dysfunction. No distinct causal relationships were observed between the altered myocardial responsiveness and pathogenesis of heart dysfunction since myocardial dysfunction and altered responsiveness to adrenaline were generally observed together. Myocardial oedema formation after endotoxin as previously reported by this laboratory may bear a relationship to the depressed negative dP/dt response to adrenaline.


[Validation of a new method for measurement of pulmonary blood volume (author's transl)]. The pulmonary blood volume (Q) can be measured by dye injection in the pulmonary artery (PA) and in the left atrium (LA) and by sampling in the brachial artery (double injection - single sampling method). We propose to replace the injection in the LA, for which a left heart catheterization is needed, by a pulmonary wedge injection (PW). No significant difference was found between the first and the second method in twenty-four measurements in six anesthetized and artificially ventilated dogs kept in steady hemodynamic state : QPW (1) equals 0.009 + 1.106 QLA (r equals 0.935). The results are independent of the capillary injection site. In man, the indirect validation was made by unilateral occlusion of a main pulmonary artery, allowing blood volume measurement of the occluded lung by two methods, one of them independent of the "capillary" injection. The results of both methods are comparable and highly correlated (r equals 0.86). The range of the values of pulmonary blood volume in sixteen normal supine subjects, at rest, was similar to that of other authors, whichever the technique they used (394 plus or minus 114 ml, 268 plus or minus 59 ml m-2). The reproducibility of the results was comparable. In five subjects, pulmonary blood volume did not change during a moderate muscular excerise in the supine position (57 watts). The pulmonary wedge injection in man can replace the LA injection of the double injection - single sampling method, without loss of precision nor effect on reproducibility.


The natural history of renal and ureteric calculi. In a prolonged follow-up of a series of 416 patients at The London Hospital Stone Clinic it was found that recurrence could still occur even as long as 10 years after the first stone, though this risk decrease slowly year by year. Recurrence is seldom related to hypercalciuria or urinary infection except when infection is caused by or associated with B. proteus in women. Claims for the value of any form of therapy for stone disease must be evaluated against the background of the natural history of lithiasis.


A clinical evaluation of hydrostatic pressure treatment for carcinoma of the bladder. A small clinical series of patients treated by hydrostatic bladder dilatation is reported. The methods used, the complications and technical difficulties encountered are described. 4 patients out of 14, who had a complete course of treatment are free of any further tumour 12 months later, which in the context of their previous progress is a considerable improvement. These 4 patients did not appear to have any particular feature which, on clinical assessment, would have allowed us to be more selective in applying this treatment.


Orthotopic liver transplantation utilizing a vascular stapling instrument. A technique of orthotopic liver transplantation in the pig is described in which the main feature is the accomplishment of four of the five anastomoses by the use of the Vogelfanger NRC vascular suturing instrument. The advantage of this instrument is the rapid accomplishment of safe leak-proof anastomoses.


Renal transplantation in the unimmunosuppressed pig: an abnormal response. A series of 82 kidney allografts and autografts in unimmunosuppressed pigs surviving for longer than 5 days has been compared with a previously reported series of 24 unimmunosuppressed pig kidney allografts. In the previous series 40 per cent survived for longer than 20 days. In the present series only 2 per cent of 55 transplants performed in cross-bred pigs obtained from different farms survived for longer than 20 days. The major causes of death were unrelated to rejection, prinicipally gastric ulcer bleeding. In the further 16 transplants between different pure-bred animals (thereby ensuring genetic disparity) the ulcer incidence was lowered by a gastro-enterostomy, and 12 per cent survived for longer than 20 days. Histological assessment of rejection in the latter group revealed 20 per cent with minimal rejection, was considered insufficient to account for the death of the animals. Based on the evaluation of 95 unimmunosuppressed pig kidney allografts in both series, it is concluded that a wide and unpredictable range of survival time occurs. Therefore the pig kidney allograft model is an unsatisfactory model in which to study immunosuppressive régimes. Also the unimmunosuppressed pig appears to show less evidence of kidney allograft rejection than the dog, irrespective of genetic disparity. It is tentatively suggested that this might be a lesser manifestation of the unexplained phenomenon of minimal rejection of liver allografts noted in unimmunosuppressed pigs.


The use of the vas deferens in inguinal herniorrhaphy. A new method of repair of inguinal herniae is described using the vas deferens as a suture material. It is especially suitable in very large and recurrent herniae. Nineteen cases have been operated on in this way with an average of 38 months' follow-up. In none has recurrence been noted. It is suggested that the vas deferens, so readily to hand, forms a more natural barrier than man-made fibres. It may also, in fact, remain biable though this had not, yet, been proved.