Total body washout for the treatment of endotoxin shock. An experimental study.

In order to study the therapeutic effects of total body washout (TBW) in experimental endotoxin shock, we used the following procedure. Seventeen rabbits (controls) received Escherichia coli endotoxin (5 mg/kg) intravenously and were observed for 12 hours. Shock developed in 14 rabbits; they died in 5.2 +/- 1.0 (mean +/- SD) hours, with a survival rate of 18%. Seventeen rabbits were subjected to TBW only. Muscle temperature was lowered to 25 C with a pump oxygenator circuit and the animals were exsanguinated. After residual blood was flushed out with cold, lactated Ringer solution, the animals were rewarmed with another circuit that was primed with homologous blood. Fourteen animals survived (82%). Two hours after E. coli endotoxin was injected intravenously 17 animals were treated with TBW. The survival rate (53%) of this group was significantly higher than in the control group (18%) (P less than .005). Eight nonsurvivors showed hypotension and acidosis even after TBW treatment, thus indicating the irreversibility of their endotoxin shock. This study indicates that endotoxin shock may be reversed by TBW if it is instituted before irreversible cellular damage.


[Pathological anatomy, etiology and pathogenesis of remote sequelae of radiation dust exposure].

Thorium (Th) is one of the widely spread radioactive substances in nature, and together with other aerosols it can penetrate into the organism. The article considers relationships between various histological forms of neoplasms induced by a radioactive dust agent containing Th and the power of this etiological factor (total body dose and its power). In experiments with 3000 white rats a 150 rad dose accumulated in the lungs during 15--21 months led to an increase in the number of cases of reticulosarcoma, a 300 rad dose produced no such effects. Doses as big as 1000--2700 rad brought about multicentrical adenomas and glandular cancerous tumours against the background of pneumocirrhosis. But no reticulosarcomas of the lung were noted. Adenocarcinomas and squamous-cell carcinomas were distributed most often in the neighbourhood of accumulated radioactive dusts. The question is raised about the need for taking into account the microdistribution of energy in the lung tissue in exposure to alpha-active aerosols with subsequent microdosimetry; due account also should be made of detailed registration of forms of tumours and degree of their malignancy, which is not also observed by researchrs in radiobiological investigations.


[Pathomorphology of pulmonary aspergillosis in tuberculosis patients based on according to resection material].

To investigate various manifestations of broncho-pulmonary aspergillosis and to ascertain its association with a previous disease there were carried out histotopographic, histological and mycological studies of specimens of the lung tissue resected in 37 patients in connection with aspergillomas of the lungs and broncho-pulmonary aspergillosis. The investigations showed that aspergillosis was one of the forms of dysbacteriosis in the human organism and could develop against the background of post-tuberculous, tuberculous and nonspecific changes. The development of aspergillosis following post-tuberculous changes should be considered as a second nonspecific disease pathogenetically associated with tuberculosis, but with an etiological factor ot its own. There are the following forms of broncho-pulmonary aspergillosis: aspergillomas, isolated aspergillosis of the bronchi, extensive aspergillosis of the bronchi and cyst-like cavities, aspergillous pneumonia.


Clinical effects of myotonic dystrophy on pregnancy and the neonate.

The article describes pregnancies and labors of five women with myotonic muscular dystrophy and their four severely involved infants, and reviews the pertinent literature. Three of the four neonatal cases died by 3 weeks of age of respiratory failure or aspiration; the fourth infant is now 4 months old and has respiratory and swallowing difficulties. The symptoms of myotonic dystrophy worsen during pregnancy. A high rate of fetal loss occurs due to spontaneous abortion, prematurity, and neonatal involvement with the disease. Prolonged labor has been described as a consistent complication, but the evidence does not justify this conclusion. Although many neonates with myotonic dystrophy are asymptomatic, severely affected newborns have a recognizable disorder unrelated to the severity of the maternal disease. The most common clinical manifestations in the neonate are arthrogryposis involving predominantly the lower extremilies, generalized hypotonia and weakness, and pharyngeal weakness. Less constant features include polyhydramnios, facial diplegia, diaphragmatic paralysis, respiratory failure, decreased motility of the gastrointestinal tract, congenital cataracts, and electrocardiographic abnormalities.


Lattice corneal dystrophy.

The clinical and histologic aspects of 34 primary and six reoperative keratoplasties for lattice corneal dystrophy are evaluated. Surgical and postoperative complications were minimal and not considered specific for lattice corneal dystrophy. Visual results were highly favorable in a long-term evaluation. No lattice figures were seen to recur in any of the 34 primary or six reoperative grafts; however, opacities did occur in several grafts. Amyloid was definitely found in one reoperative graft specimen. A nonamyloid material was also found in the subepithelial and superficial stromal area in both the lattice dystrophy and reoperative specimens.


Aberrant bacterial forms from various ocular sites.

Patients may harbor cell-wall-deficient organisms or other aberrant bacterial forms (ABFs) in their eyes. In this survey of 400 cultures, we found the incidence of ABFs isolated from various ocular sites to be 13.2%. The rate of isolation of these forms from the eyes of patients with suspected bacterial infection differed greatly from that for noninfected eyes. We will describe the microbiological techniques employed and will present our analysis of the data obtained.


The "lazy-T" correction of ectropion of the lower punctum.

The "lazy-T" technique consists of a surgical horizontal and vertical shortening of the involved portion of the lower eyelid. As a result of the surgical procedure, the lid margin is restored to its normal relationship with the globe. The punctum is reestablished into its normal anatomical position in the proximity of the lacrimal lake. The cosmetic blemish resulting from the surgical scar is insignificant.


Correction of entropion from Stevens-Johnson syndrome: use of nasal septum and mucosa for severely cicatrized eyelid entropion.

The resistant entropion, especially of the upper lid, that results from the persistent contraction of the conjunctiva after Stevens-Johnson syndrome is difficult to correct. Grafts of buccal mucosa have relieved this condition for only a few weeks or months. Because of the rigidity of the nasal septum, a sector of this structure, with the perichondrium and mucosa intact on one side, has been grafted into the posterior layer of the upper lid at the margin to turn the lashes and skin away from the globe. For more than two years postoperatively, this graft of septal mucosa has relieved patients of their entropion, and from all indications this correction will be permanent.


Arthroplasty of the elbow in rheumatoid arthritis.

The study consists of 208 elbow arthroplasties performed on rheumatoid arthritic patients. A straight resection of the joint was used in 53 cases and a modified Hass arthroplasty with skin interposition in 155 cases. The average postoperative range of motion in these groups was 100 degrees and 96 degrees respectively. Postoperatively the joint was painless in 81 and 67% of the elbows respectively. The Hass arthroplasty gave a better stability and extension power. The most common complications were paresthesias in the region of the ulnar nerve and bone resorption in the region of the ulnar nerve and bone resorption in the region of the olecranon fossa.


[Clinical problems after the use of polyestercomponents in total hip replacement (author's transl)].

Follow up studies of total hip replacement using polyester as a component of the alloplastic joint have shown that this material causes soft tissue reaction and osteolysis. The problem is demonstrated in this paper.


[Scoliosis, metabolism and growth of the vertebral column (author's transl)].

Modern investigators incline to the opinion, that more biochemical than biomechanical disorders take part in cause of the "idiopathic'' scolioses. It seems, however, that there is not only one cause but more in some subgroups. Idiopathic scolioses, which have symptomes of arachnodactyly, seem to be a big one of these subgroups. These cases allow to state a hypothesis, in which kind a disordered metabolism leads to a deviation of the spine. This hypothesis is basing on the fact, that the enchondral growth in the length and the periostal growth in the width of "long bones'' are not regulated in the same endocrinological kind and that the enchondral growth of the vertebral-bodies-column happens in cranio-caudal direction, the enchondral growth of the vertebral-archies-column, however, in anterior-posterior direction. If the balance between enchondral and periostal growth is disturbed, you  can see typical chances on the long bones, which resemble either an "arachnodactyly" or a "chondrodysplasy". The same disturbance will cause a "kyphosis" respectively a "lordosis" (or scoliosis) on the vertebral spine; either the bodies-column or the archies-column will become longer (higher). The results of metabolism research are suitable to these facts. If the balance between enchondral and periostal growth,--basing on a dysbolism,--is disturbed in such a kind, that the vertebral-bodies-column is growing faster than the vertebral-archies-column, the vertebral spine is forced to change into a lordosis respectively into a scoliosis. If you want to cure an idiopathic scoliosis, you first have to remove or to paralyse the dysbolism. The aim of all research has to be to find an effective chemotherapeutical treatment of mindst a part of all idiopathic scolioses.


Larynx injected with polytef paste.

A patient with inoperable lung cancer developed left-sided laryngeal paralysis. Her dysphonia, difficulty with swallowing, aspiration of secretions, and diminished cough reflex were improved with intracordal polytef injection for the remainder of her life. The foreign body reaction to the implant showed giant cells, few lymphocytes, and no polymorphonuclear leukocytes. This reaction may be described as a bland, chronic type consistent with the age of the implant. No areas of florid, acute reaction were found. Extrusion of part of the polytef through the cricothyroid space was observed. There were no signs of unfavorable tissue reaction, intolerance, or carcinogenicity. In a second case, part of the polytef paste exuded over the thyroid gland and was misinterpreted as a thyroid nodule. Excised 11 months after injection, the foreign body reaction appeared to be of a more acute type.


Rapid detection and isolation of mycoplasmas from cell cultures.

An improved medium for the isolation of Mycoplasma species from contaminated cell cultures is reported. A modification of the mycoplasma detection method using the uridine/uracil uptake ratio method is described. Results obtained using this method with mycoplasma contaminated cell cultures and with contaminated cell cultures treated with the antibiotic Lincomycin are presented.


Studies in infant behavioural audiometry. III. Six-month-old infants.

The behaviour of 95 clinically normal 6-month-old babies previously studied as neonates and as 6-week-old infants was video-recorded while they were presented with sound stimuli. The stimulus series varied in sound pressure level (SPL) (30 and 60 dB), band with (BW) and rise time (RT), and included two voice signals and a no-sound (control) trial. Video-records were made both for the whole body aspect and for a 3 1/2 X 'close-up' of the head. Later, the video-records were shown to 6 observers who were allowed to see the babies for 13 s at each trial. The first 5 s was a pre-stimulus observation period, and the second 8 s usually contained a sound stimulus. Between the trials, the observers were given 20 s in which to record pre-stimulus activity, confidence in response, and movement details. Data for movement details are not reported here. Different segments of the baby's body were observed on different occasions as detailed in our previous reports. The confidence ratings for response to tonal stimuli were analyzed using aspects of signal detection theory for differences amongst various body segments (n.s.), SPL (p less than 0.001), BW (p less than 0.05), and RT (p less than 0.01). A 60-dB broad-spectrum noise band (BSN) and the 2 voices were by far the most effective stimuli. They elicited detectablities which differed between segments. The response to different sound stimuli was affected by the baby's pre-stimulus activity state. The results are discussed in relation to our previous studies.


Antibody response in sows following intramammary or intramuscular immunization with a killed Escherichia coli vaccine.

Pregnant sows were immunized either intramuscularly or intramammarily with a killed E. Coli vaccine. Samples of blood and mammary secretion were assayed for antibody to E. Coli cells and E. coli enterotoxin. The association of each class of immunoglobulin with antibody to the bacterial cells was also estimated. It was found that immunization by either method increased direct agglutination titres and antiglobulin enhancement titres in serum and mammary secretion. There were no major differences in these parameters between the two groups of sows. In serum and mammary secretion from both groups of sows IgG was quantitatively the most important class of immunoglobulin associated with antibody to E. coli cells. Immmunization had the effect of increasing the percentage of IgG and IgM associated with antibody in blood serum from about 5% to about 20%. In contrast, the percentage of IgA in serum with antibody activity against E. coli was around 20% prior to immunization and did not alter significantly after immunization. Samples of blood and mammary secretion from sows immunized intramammarily were more effective in neutralizing E. coli enterotoxin than corresponding samples from sows immunized intramuscularly; the biological significance of this finding is at present uncertain.


Salmonella enteritidis infection in rats: antigens involved in cell-mediated immunity.

When rats are sub-lethally infected with S. entertidis by the intravenous route, there is a period of at least 14 days in which peritoneal exudate cells are unable to migrate from capillary tubes on to glass surfaces; thereafter they migrate as do macrophages from normal animals. The migration of peritoneal cells from rats infected 18-21 days previously is inhibited by lipopolysaccharide (LPS) prepared from homologous and heterologous strains of Salmonella. The apparent non-specificity of LPS activity in the macrophage migration inhibition test has been resolved by demonstrating that lipid A, containing less than 0.01% protein amino acids, is also capable of eliciting this test of cell-mediated immunity.


Vibrio cholerae infection and immunity in mice.

Lyophilized cultures of V. cholerae 569B slowly lose their virulence for neonatal and adult mice during long term storage. Following a single passage in orally infected 6-day old mice, a highly virulent strain (designated 569B/MP) was isolated. This organism causes rapidly fatal intestinal infections in 6-day old mice; large numbers can be isolated in pure culture from the intestinal fluid. Freezing and storage at -60 degrees of dead animal provides a simple means of maintaining the high virulence of the culture over a period of at least 9 months. This strain produces choleracic symptoms and death in approx. 50% of adult mice following oral infection. Large numbers of viable organisms may also be isolated from the small intestine over a period of at least 40 h. These criteria have been used as a basis for assessing protection against cholera infection induced by immunization with living or heat-killed V. cholerae given orally or intraperitoneally.


Antilymphocyte serum: studies of the specificity of the rosette-inhibiting antibody in rabbit anti-mouse ALS.

In an investigation into the characteristics of the rosette-inhibiting antibody of antilymphocyte serum (ALS), rabbit anti-mouse ALS were assayed for antibodies against lymphoid cells from the spleen, lymph nodes and thymus of Quackenbush (Q) mice, and against spleen cells from athymic nude mice. The lymphagglutinating and lymphocytotoxic titres were similar with both Q and nude cells, and the titres showed no significant correlation with skin graft survival times in mice receiving the corresponding ALS. A significant depletion of peripheral blood lymphocytes occurred in animals receiving both an immunosuppressive and a non-immunosuppressive ALS, suggesting that the antibodies involved in this depletion are not significant in determining the immunosuppressive activity of ALS. In contrast, the rosette inhibition titres showed a significant correlation with graft survival times in the results obtained in tests using Q spleen, lymph node and thymus cells. No inhibition of rosette formation was obtained when using nude spleen cells. However, the numbers of spontaneous rosettes formed by both the mixed B and T cell populations from Q spleen were not significantly different from the numbers formed by the largely B cell population from nude spleen. These results confirm that the antibodies of ALS which are responsible for both the in vitro inhibition of spontaneous rosette function and the in vivo suppression of graft rejection are specifically directed against a T cell population, and it is suggested that the antibodies are related if not identical.


Radiosensitivity of suppressor cells in newborn rats.

The resistance of neonatal rats to sustaining adoptive immune responses against heterologous erythrocytes following the transfter of normal thoracic dust lymphocytes was examined. Irradiation of the neonatal rat at levels as low as 350 rad was found to be effective in overcoming this resistance, although preliminary exposure to antigen could interfere with facilitation of adoptive responses by irradiation. It is suggested that the failure of the neonate to sustain adoptive immune responses is explicable on the basis of an active suppression and, as a corollary, unresponsiveness resulting either from macrophage immaturity or the transfer of maternal antibody is discountered as a likely explanation for the immunological behavior of the newborn rat towards the antigens examined.


Role of the thymus in suppression of immune responses in newborn rats.

The role of thymus-derived cells in controlling the process of immunological maturation in the neonatal rat has been examined. Thymectomy performed on the day of birth resembled irradiation given at this time in that it permitted adoptive immune responses to sheep erythrocytes to be initiated in the neonatal host. Whereas the consequences of irradiation could be demonstrated if antigenic challenge was given of the following day, adoptive responses were not sustained unless an interval in excess of 24 h had elapsed after thymectomy, suggesting that extra-thymic suppressor cells persist for at least this period. Suppression of adoptive responses was achieved in irradiated hosts with thymus cells obtained from rats on their day of birth but not with cells from older donors. Using the transfer of allogeneic lymphocytes as a stimulus, antibody-forming cell precursors were shown to be plentiful in the newborn rat and it was accordingly suggested that thymic suppressor cells are reponsible for the poor responsiveness of newborn rats to antigenic challenge.


Effect of denervation of pyloric antrum on the gastric secretory response to food.

The effect of selective denervation of the pyloric antrum on secretory response to three types of food was determined in 3 dogs with innervated and 4 with denervated stomach pouches. With the antrum in situ this procedure has surprisingly little effect on the total meal responses in innervated pouches, on the variation in response induced by altered buffering capacity of the meals, or on the effect of subsequent vagotomy. However, the antrum appears less sensitive to local stimuli after denervation, as evidenced by the reduced meal response from the denervated indicator pouch after denervation of the antrum.


Cytotoxic mechanisms detected in vitro following sheep renal allografts.

Cytotoxic effects of lymph and blood components, removed from sheep following renal allografting were determined by in vitro assay using 51Cr labelled donor sheep lymphocytes as target cells. Lytic antibodies were detected in lymph efferent from the node draining the graft site and at lower concentrations in blood serum and in lymph leaving the graft. The cytotoxic antibodies had both complement-dependent and leucocyte-dependent functions; the complement-dependent antibodies were fractionated and shown to be of IgM and IgG1 subclasses. Cytotoxic cells were found in lymph leaving the graft but not in blood or in lymph efferent from the draining node; those detected in the renal lymph were non-specific in action and their appearance correlated with increased numbers of macrophages.


Low dose heparin and early kidney transplant function.

A prospective randomised double blind trial was carried out to assess the effect of subanticoagulant doses of subcutaneous heparin (Calciparine) compared to placebo on early renal allograft function. Thirty-six patients were entered into the trial, 18 received placebo and 18 heparin. There was no significant difference between the patients in either group in respect to early graft function, the number of rejection episodes or the response of graft function to treatment of rejection. There were four patients with haemorrhagic complications who had received heparin, whereas no patients in the placebo group had bleeding combinations. It is concluded that subanticoagulant doses of heparin have no demonstrable effect on early graft function and may cause haemorrhagic complications.


Inhaled and oral bronchodilator therapy in exercise induced asthma.

Measurements of peak expiratory flow rate during serial exercise tests were used to assess the efficacy of the beta adrenergic stimulants oral terbutaline (5 mg) and fenoterol aerosol (400 micrograms), in preventing post-exercise bronchoconstriction in 18 asthmatic subjects. While both compounds significantly elevated resting levels of peak expiratory flow rate for at least four hours, there were qualitative and quantitative differences in the effects of the two drugs on exercise induced bronchoconstriction. Oral terbulation did not significantly alter the pattern of response to exercise. However, the peak expiratory flow rates before during and after exercise were significantly higher compared with placebo at two, four and six hours after the drug. The importance of examination of actual values for peak expiratory flow rates as well as percentage changes when testing oral bronchodilators is emphasised. Aerosol fenoterol completely blocked post-exercise bronchoconstriction immediately after inhalation and for up to four hours in most subjects. Bronchodilatation occurred in all subjects during exercise in the presence of both the active and placebo drugs. It is postulated that the superiority of the aerosol in blocking exercise induced bronchoconstriction may be function of its activity at the surface of the bronchial mucosa.


Marijuana smoking and cold tolerance in man.

Ten men who were marijuana users served as subjects in a study of the effects of marijuana smoking on response to cold. Cold water (28 degrees C for 60 min) and cold air (20 degrees C for 120 min) mediums were utilized with three exposures in each medium. The three exposures followed smoking marijuana, smoking placebo, and a no-smoking control period. Additionally, a breathhold experiment preceded and followed the four smoking periods. Marijuana and placebo smoke were inhaled from a spirometer with each man receiving the smoke of 0.739 g of marijuana and placebo. Smoking marijuana did not greatly modify body heat content, since rectal temperature and most peripheral temperatures were not altered. However, temperatures over voluntary muscles likely to be involved in shivering were elevated. Heat production also greatly increased after marijuana, suggesting that it had stimulated shivering. Marijuana also produced tachycardia and abolished apneic bradycardia. The mechanism of this action is not clear, but some sympathetic involvement is indicated.


[Osteolathyrism. Quantitative-morphological studies on the experimental rat skeletal disease].

In young growing rats, fed the lathyrogenic agent aminoacetonitrile for 3 weeks, characteristic skeletal changes developed resembling the chronic state of osteolathyrism. In both lathyritic and untreated control rats, the femoral head, the distal end of the femur, the proximal tibial end, the thalocalcanean joint and the first lumbar vertebra were investigated using undecalcified thin sections and ground sections. The following methods were employed: quantitative morphometrical analysis of bone structure and bone remodelling of the lumbar vertebra, histochemical demonstration of the composition of polysaccharides, detection of the course of collagenous fibres in cartilage and bone by the polarizing microscope, tetracycline-labelling and fluorescence microscopical determination of epiphyseal longitudinal growth and bone formation activity, photometrical quantification of microradiographies and computer-aided determination of the mineral content of bone.


Correlation between the immunoadjuvant activities and pyrogenicities of synthetic N-acetylmuramyl-peptides or -amino acids.

A total of 14 different N-acetylmuramyl-peptides or -amino acids with or without configurations inherent to bacterial cell wall peptidoglycans were synthesized and their pyrogenicities on intravenous injection into rabbits were tested. N-Acetylmuramyl-peptides, and especially N-acetylmuramyl-L-alanyl-D-isoglutamine and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-lysine, which were previously shown to be adjuvant-active in both induction of delayed-type hypersensitivity and stimulation of increased serum antibody levels to ovalbumin in guinea pigs, exhibited distinct pyrogenicity at as low dose as 16 mug per rabbit. However, none of the adjuvant-inactive analogues or diastereomers of the above N-acetylmuramyl-dipeptide or related compounds caused any significant febrile response in rabbits, even at a dose of 250 mug per animal.


An unusual precursor of 50S ribosomes in a mutant of Escherichia coli.

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein ("47S") particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.


Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.


Physiochemical characterization of proteolytic cleavage fragments of bovine colostral immunoglobulin G1 (IgG1).

Normal bovine colostral immunoglobulin G1 was subjected to enzymic digestion (pepsin, papain and trypsin) and the resulting fragments separated by a combination of molecularsieve and phosphocellulose chromatography. Fragments F(ab')2 derived from peptic digestion, fragment Fab from papain digestion and fragment Fab(t) from tryptic digestion showed complete antigenic identity with each other. Although fragment F(ab')2 (peptic digestion) had a sedimentation coefficient (S2o,w) of 5.3S, those for fragments Fab' (peptic digestion), Fab (papain digestion) and Fab(t) (tryptic digestion) were found to be 3.9S, 3.7S and 3.7S respectively. The mol.wts. calculated for the various fragments from the sedimentation equilibrium data were: F(ab')2, 104000 +/-200; Fab', 51900+/-340; Fab, 50900+/-230; Fab(t) 50900+/-300. Fragment Fc' (peptic digestion) had an S20,w of 3.2S and a mol. wt. of 42900+/-650; fragment Fc (papain digestion) had an SI0,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-300; fragment Fc(t) had an S20,w of 3.7S and a mol.wt. of 50800+/-450.


Fb'2, a new peptic fragment of human immunoglobulin G.

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb'2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb's was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb'2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb's; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb'2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.


A cyanogen bromide fragment of beta-galactosidase from Escherichia coli with alpha-donor activity in complementation of the enzyme from mutant M15.

Aminoethylated beta-galactosidase from Escherichia coli was cleaved by CNBr. The fragment C4a was purified by gel filtration and ion-exchange chromatography. The molecular weight of the fragment C4a was determined to be 9000 +/- 600. The N-terminal amino acid was found to be isoleucine. Qualitative examination of homogeneity was carried out by disc-gel electrophoresis. The fragment C4a was shown to be active as an alpha donor in complementation of beta-galactosidase activity in vitro with E. coli mutant M15, which has a deletion in the alpha region of the z gene. The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400.


The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar.

1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.


The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide 'mapping'.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.


Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.


[Examination of pyrogens in vitro by means of the limulus test/2. Results (author's transl)].

Rabbit and Limulus test were studied comparatively in regard to applicability of water for injections. The examinations included the auxiliary material water and water bacteria, which have to be considered potential pyrogens. With freshly distilled water conformity of 95%, with water germs of 80% was reached.


[In vitro effect of cephacetril and colistin combinations on Klebsiella pneumoniae and Proteus Strains (author's transl)].

In 14 out of 43 Klebsiella pneumoniae strains (33%) the combination cephacetril-colistin showed a synergistic, in 11 strains (25%) an additive effect. In Proteus strains no synergistic action was found. The combination was more likely to be synergistic or additive in strains requiring higher minimal inhibitory concentrations of cephacetril and colistin. Most highly resistant Klebsiella pneumoniae strains were synergistically inhibited by concentrations of both drugs, which can easily be obtained in serum by usual clinical doses.


[Medical problems in the practical execution of drug testing].

Different experimental designs for a clinical trial have to be applied for every individual substance even if it, though closely related to some other, shows minor differences in its effects. Difficulties which often occur during phase I trials are: the choice of the volunteer, dosage and route of application of the substance, especially in the first trials in humans, the instructions to the volunteer after intake of the drug, increase in dosage, the number of repeating applications and the interval between uptake. Problems arising during phase II, III and IV trials are concerned mainly with controlled clinical trials: choice of the standard treatment or of a placebo, search for the influencing factors, etiology of the symptoms, stage of the illness, control of patient's compliance etc. In contrast to common opinion, clinical trials cannot be considered to be especially dangerous as data show.


Predominance of T cells in the lymphocytic infiltrates of synovial tissues in rheumatoid arthritis.

Synovial tissues from 5 patients with rheumatoid arthritis (RA) were examined with immunofluorescence microscopy for the presence of lymphocytes with either bone marrow-derived (B) or thymus-derived (T) surface markers. Five synovial tissues with severe to mild lymphocytic infiltrations by bright field microscopy were examined in parallel with immunofluorescence. B cells were identified with a pepsin-digested fluoresceinated anti-F (ab')2 antiserum and T cells were detected with a specific rabbit anti-T lymphocyte antiserum. By these techniques 75-90% of the lymphocytes in these frozen sections were identified as T cells. Cell suspensions were also prepared by collagenase digestion of two of the five synovial tissues. The lymphocytes in these cell suspensions were predominantly T lymphocytes (78-85%) as shown by their ability to form spontaneous rosettes with sheep erythrocytes (E rosettes).


On the structure-activity relationship of antiviral substances.

The significance of the study on structure-activity relationship in the antiviral substances is considered for: 1. directed synthesis of highly active and selective antivirals; 2. elucidation of the mechanism of action of antiviral substances by revealing the chemical structures which determine the inhibition of a given step in the virus growth cycle. The basic trends in conducting directed synthesis of antiviral substances are pointed out: synthesis of new inhibitors with a broad antiviral spectrum, as well as improvement by structural modifications of known ones. The basic methodological approaches in the structure-activity studies are considered. A classification of the available antiviral synthetic substances is presented.


Controlled trial of cyclophosphamide in rheumatoid arthritis.

Twenty-four patients with severe progressive rheumatoid arthritis were randomly assigned to cyclophosphamide or placebo in a double-blind crossover trial. Eleven patients who completed 9 months on cyclophosphamide (average dose: 1.8 mg/kg/day) demonstrated significant decrease in painful joints, swollen joints, and morning stiffness and increase in grip strength when compared to 11 patients on placebo. After crossover, significant improvement was observed in patients switched to cyclophosphamide, and deterioration within 2 months was observed in most patients changed from drug to placebo. Serum immunoglobulins and rheumatoid factor titers decreased with cyclophosphamide but antibody response to Vi antigen was unaffected. Primary delayed immune response to 2,4-dinitrochlorobenzene was markedly depressed. Adverse effects were troublesome--hemorrhagic cystitis affected 4 patients and amenorrhea occurred in 3. Despite striking beneficial effect, cyclophosphamide should be prescribed cautiously and only in severe resistant cases of rheumatoid arthritis.


Effect of prophylactic colchicine therapy on leukocyte function in patients with familial Mediterranean fever.

Patients with familial Mediterranean fever (FMF) who were part of a double-blind trial of daily colchicine as prophylaxis for their disease had leukocyte functions studied while receiving colchicine or placebo. Leukocytes taken from these patients while on prophylactic doses of colchicine produced normal quantities of leukocytic pyrogen, ingested bacteria normally, and migrated normally in chemotatic chambers. In addition these patients had normal numbers of circulating T and B lymphocytes as well as normal blastogenic reponses of their peripheral lymphocytes to mitogenic stimuli. The patients on colchicine, however, had significantly fewer neutrophils and monocytes accumulating at skin-window sites 24 hours after the initial abrasion. Because the early phase of the skin-window response was normal in these patients, the decreased late response may be related to a failure to amplify the initial inflammatory reaction. The reduced capacity to generate a normal inflammatory response may account for the failure of these patients to develop full attacks while taking colchicine.


Antibacterial activity of ribostamycin on Enterobacteriaceae.

The study of the inhibitory activity of ribostamvcin (Vistamycin), an antibiotic derived from Streptomyces ribosidificus, on 161 strains of Gram-negative bacilli shows that the antibacterial spectrum of this antibiotic is identical to that of kanamycin. If controlled clinical studies confirm that ribostamycin is less toxic than kanamycin on the otovestibular system, this antibiotic will constitute a real therapeutic advance.


[Spontaneous peripheral proteolysis/2nd communication: Effect of peripheral proteolysis (author's transl)].

With the modified Astrup-plate method a spontaneous peripheral proteolysis was often found in chronical infections of the skin and mucous membranes. The origin of this proteolysis and its effects on the bacterial growth and wound healing were discussed.


Di-n-propylacetic acid--profile of anticonvulsant activity in mice.

The anticonvulsant effect of di-n-propylacetic acid (n-DPA) was studied in mice and compared to those of phenobarbital, trimethadione and ethosuximide. n-DPA  was only weakly active in the maximal electroshock test, but had an ED50 of 420 mg/kg orally in the pentetrazole seizure threshold test, which corresponds rather well to the activity of trimethadione and ethosuximide. The duration of action was only short, and the first signs of neurotoxicity--inability to perform in the chimney test--appeared well below the anticonvulsant ED50 against pentetrazole. n-DPA proved to be most active against convulsions induced by picrotoxin (ED50 200 mg/kg orally), which might indicate a role of the elevation of the central levels of gamma-aminobutyric acid in the anticonvulsant effect of the drug.


Safety evaluation of chemicals in food: toxicological data profiles for pesticides. 1. Carbamate and organophosphorus insecticides used in agriculture and public health.

The sources of the scientific information used over the past several years by the Joint FAO/WHO Meetings on Pesticide Residues in carrying out toxicological evaluations are classified systematically according to compound and subject for the first time in this paper. It is hoped that those engaged in the toxicological assessment of pesticide chemicals, for the purpose of standardizing pesticide tolerances or for developing criteria of acceptability, will profit from this classification.


Clinical patterns and results of radioallergosorbent test (RAST) and skin tests in penicillin allergy.

Seventy-nine patients with acute or former reactions to penicillin were investigated by a benzylpenicilloyl (BPO)-specific RAST and/or by skin tests with penicilloyl-polylysine (PPL), benzylpenicillin and penicilloic acid and the results were correlated with the different clinical pictures. Positive RAST and skin test results could be found in patients with anaphylactic shock, urticaria and serum sickness-like reaction and sometimes in a special group of exanthems, which are characterized by the existence of many different lesions at the same time, therefore called 'polymorphic exanthems', and often observed after high-dosage penicillin therapy. In cases of scarlatiniform or morbilliform exanthems no positive results were found. The BPO-specific RAST showed an overall correlation of 95-I% with skin tests using PPL. However, some patients with positive skin tests to benzylpenicillin and penicilloic acid did no have detectable circulating IgE antibodies to BPO. This emphasizes the need for including these antigens in in vitro methods. The RAST was informative even at the allergic reaction or in the first 15 days afterwards and seems to be very valuable for early diagnosis of penicillin allergy especially in cases when many drugs have been given.


Failure to produce warts on human skin grafts on 'nude' mice.

Split-thickness human skin was exposed to a suspension of human papilloma virus containing 10(11)-10(12) particles/ml, by immersion for varying lengths of time. It was then grafted onto the backs of congenitally athymic 'nude' mice. All grafts were accepted, but no warts developed during an observation period of up to 9 weeks.


Pemphigus controlled by dapsone.

Three uncomplicated cases of pemphigus were clinically controlled by dapsone. Improvement was associated with decreasing titres of circulating intercellular antibodies. Interestingly, the sera from one case of pemphigus foliaceus contained intercellular antibodies found in the Malpighian and basal cell layers using the fluorescent technique and in the granular layer using the peroxidase technique. These findings suggest that the intercellular antibodies in pemphigus vulgaris and pemphigus foliaceus are similar but bind at different anatomical sites.


Activation of pepsin (EC 3.4.4.1) by heavy-metal ions including a contribution to the mode of action of copper sulphate in pig nutrition.

1. Kinetic experimetns were done with pepsin (EC 3.4.4.1) using haemoglobin as a substrate in the presence of different metal cations. 2. The activation of peptic hydrolysis with higher concentration of cupric ions added to the reaction mixture was determined from turnover-rate curves in experiments with constant substrate concentration. With a Cu2+ concentration greater than 1.67 X 10(-4) M activation was obtained. 3. Nickel ions at a concentration of 8-33 X 10(-4) M and at higher concentrations also increased pepsin activity. Additions of ferrous ions and zinc ions had no effect. 4. Experiments were done using variable substrate concentrations in the presence of different Cu2+ concentrations. The concentrations of haemoglobin ([S]) at half maximum velocity were determined. The double-reciprocal plots of [S] v. reaction velocity (v) (i.e. 1/[S] v. 1/v) had no common intersection point. Therefore the kinetics did not correspond to any of the known kinetics. The activation brought about by Cu2+ cannot easily be explained by the study of the kinetics. Certain simple explanations of the phenomenon can be eliminated.


Use of dimethyl suberimidate and novel periodate-cleavable bis(imido esters) to study the quaternary structure of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Two new symmetrical bis(imido esters), N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride and N,N'-bis(2-carboximidomethyl)tartarimide dimethyl ester dihydrochloride, have been synthesized. Tests with the tetrameric enzyme, fructose diphosphate aldolase, show that these reagents closely resemble dimethyl suberimidate in their ability to cross-link protein subunits. However, identification of the cross-linked species, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is greatly facilitated since the cross-links can be broken by a simple treatment with sodium periodate. The periodate cleavage step can be introduced between the two dimensions of a diagonal gel electrophoretic separation, the contributors to a cross-linked species then moving off the diagonal formed by uncross-linked proteins and reverting to the positions in the gel that correspond with their regenerated monomeric form. When the pyruvate dehydrogenase multienzyme complex of Escherichia coli was treated with dimethyl suberimidate or N,N'-bis(2-carboximidoethyl)tartarimide dimethyl ester dihydrochloride, cross-links rapidly formed between the subunits of the transacetylase and lipoamide dehydrogenase components. On the other hand, cross-links failed to form between the subunits of the decarboxylase component themselves, or between the decarboxylase and the other two types of subunit in the complex. Cross-linking experiments with the isolated lipoamide dehydrogenase were compatible with the accepted dimeric structure of this enzyme is free solution, whereas the isolated pyruvate decarboxylase component also failed to cross-link when treated with dimethyl suberimidate in free solution. The cross-linking experiments with the intact multienzyme complex provide evidence for the existence of the lipoamide dehydrogenase dimer in the assembled enzyme and show the need to interpret such experiments with care since, from other evidence, the pyruvate decarboxylase component is known to be bound to the transacetylase "core" of the complex.


The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting.

It has been proposed that the rate-limiting step in the synthesis of aminoacyl-tRNA is the rate at which the product dissociates from the enzyme. The experimental evidence supporting this hypothesis comes from work at low pH and low temperature (although the reaction has been argued to have the same mechanism under physiological conditions). We have reexamined the binding assay by which M. Yarus and P. Berg (1969) (J. Mol. Biol. 42, 171-189) measured the kd for dissociation of Enz-(Ile-tRNA). We find that when overall reaction and dissociation are measured under identical conditions the two rates are not the same. Moreover, while an increase in ionic strength greatly stimulates dissociation, the same increased ionic strength slows aminoacylation. Spermine accelerates overall aminoacylation without affecting dissociation. Because any change in a rate-limiting step must, by definition, cause a parallel change in the overall reaction, these observations prove that under these conditions the synthesis of Ile-tRNA is not limited by the rate of dissociation of Enz-(Ile-tRNA). Entirely similar observations were made for the dissociation of Enz-(Val-tRNA) and the overall synthesis of Val-tRNA at 0 degrees C, PH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacylation was shown not to be saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged substrate tRNA and with product Val-tRNA. E. W. Eldred and P. R. Schimmel ((1972) Biochemistry 11, 17-23) report that the formation of Ile-tRNA proceeds at two rates: (a) k = 2 X 10(-2)S(-1) until the enzyme is saturated with the first mole of product, and (b) k = 2 X 10(-3)S(-1) for subsequent cycles. We did not observe this behavior at any pH or temperature with four different amino acid:tRNA ligases. Because aminoacylation proceeds more rapidly than "dissociation" under some conditions, we believe that the binding assay measures not only enzyme-product dissociation but also other slower reactions such as aggregation or disaggregation of Enz-(AA-tRNA). In conjunction with recent studies from other laboratories, this work makes it unlikely that enzyme-product dissociation is the rate-limiting step in the synthesis of aminoacyl-tRNA either at low temperature and pH or under more nearly physiological conditions. From the effect of salt, it would appear that the rate of aminoacylation of tRNA is largely limited by the rate or extent of formation of Enz-(tRNA) (Loftfield, R. B., and Eigner, E. A. (1967), J. Biol. Chem. 242, 5355-5359). Using the binding assay of M. Yarus ((1972) Biochemistry 11, 2050-2060), we find the Kass for Enz-(Ile-tRNA) varies linearly with the Debye-Hückel function at ionic strengths of 0.1-0.4 from 10(8) to 10(6).


Initiation of synthesis of N-terminal acetylated histones with methionine.

The studies presented were undertaken to clarify the unsettled question whether the synthesis of histones with a N-acetylserine residue at the amino terminal end is initiated with methionine. Histones were synthesized in vitro in a rabbit reticulocyte lysate, primed with a mRNA preparation from ascites cells. Initiation of polypeptide synthesis was investigated by using N-formyl[35S]met-tRNAfMet from yeast to label the N-termini. N-Formylmethionine was incorporated into histones H1 and H4 whose N-terminal amino acid is alpha-N-acetylserine. By comparison of tryptic peptides derived from these two histones labeled either with methionine or formylmethionine and from Edman degradation it is shown that N-terminal acetylated histones are initiated with methionine, as is the case for other eukaryotic and bacterial proteins.


Regulation of membrane phospholipid synthesis by the relA gene: dependence on ppGpp levels.

A series of experiments using a pair of isogenic rel+ strains of Escherichia coli differing only in the spoT locus has demonstrated a quantitative correlation between the inhibition of phospholipid synthesis and the intracellular level of ppGpp. The conditions examined were (1) amino acid starvation; (2) release from amino acid starvation; and (3) balanced growth. We also have been shown the presence of a third gene (in addition of relA and spoT) concerned with ppGpp metabolism and have been found that the level of ppGpp during amino acid starvation is unaffected by an increase in the dosage of the relA gene.


Molecular mechanism of the rifampicin -RNA polymerase interaction.

Equilibrium and kinetic studies of the interaction of rifampicin with RNA polymerase of Escherichia coli were performed by exploiting the quenching of intrinsic fluorescence of the protein by the drug. Fluorimetric titrations show that rifampicin binds stoichiometrically to the core and holoenzyme with an apparent Kd of less than or equal to 3 x 10(-9) M. Neither the addition of template nor the formation of the initiation complex in the presence of dinucleotide and nucleoside triphosphate prevents the rifampicin-enzyme interaction. Although the equilibrium binding constant for the rifampicin-RNA polymerase complex is about the same for the core and holoenzyme and the holoenzyme-T7 DNA complex, stopped-flow studies indicate that the rates at which rifampicin interacts with these enzyme forms are different. In all three cases, the kinetic data can be interpreted in terms of a mechanism in which the rapid bimolecular binding of rifampicin to RNA polymerase is followed by a relatively slow isomerization of the drug enzyme complex: (See article). While the values of dissociation constant K1 = (k-1/k1), for the first binary complex (ER) are similar, the rate constant for the forward isomerization, k2, decrease in the order of core enzyme greater than holoenzyme greater than the holoenzyme-T7 DNA complex. The fact that this order is parallel to the relative rates of inactivation of the enzymes and the enzyme-DNA complex suggests that the inactivation may be due to the rifampicin-induced isomerization (conformational change) of the enzyme. This is supported by our observations that an enzyme complex which is in the process of elongating RNA chains can still bind rifampicin, although the enzyme activity is not inhibited by such binding. The values of overall binding constants calculated from the kinetic parameters, 1-2 x 10(-9) M, are in good agreement with the values of the apparent Kd obtained from fluorimetric titrations and Ki determined by enzymatic assays. In addition, the observations that the formation of an initiation complex leads to a significant but not complete rifampicin-resistant RNA synthesis and the recent finding that rifampicin only partly inhibits the formation of the first phosphodiester bond in an abortive initiation of RNA chains are consistent with our kinetic mechansim, i.e., the existence of two forms of the rifampicin-RNA polymerase complex, only one of which is able to initiate the RNA chains.


Membrane changes in Escherichia coli induced by colicin Ia and agents known to disrupt energy transduction.

The addition of colicin Ia to a suspension of intact Escherichia coli in the presence of the hydrophobic fluorescent probe N-phenyl-l-naphthylamine causes dramatic changes in the fluorescence of the probe. The fluorescence intensity increases several fold, the emission spectrum shifts to the blue, the fluorescence lifetime approximately doubles, and the polarization increases. These changes do not appear to result from an increase in membrane microviscosity, as has been previously postulated to be the case for the N-phenyl-l-naphthylamine fluorescence changes seen with colicin El-treated cells (Helgerson, S.L., Cramer, W.A., Harris, J.M., and Lytle, F.E. (1974), Biochemistry, 13, 3057); rather, they result from an increased binding of the dye to the cell envelope. A variety of agents have been used to demonstrate that a very similar fluorescence response results whenever the cells are "deenergized." These agents include electron transport inhibitors (malonate, amytal, cyanide) as well as the uncouplers CCCP and azide. In addition, depleting the cells of either endogenous substrates or oxygen results in the same fluorescence response. In these cases, the fluorescence response is reversed upon addition of an oxidizable substrate or oxygen. It is clear that there are significant changes in the Escherichia coli envelope as energy transduction processes are disrupted and restored. The changes reported by the fluorescent probe may prove useful indeciphering structure-function relationships in the Escherichia coli envelope.


Comparison of isotope labeling patterns of purines in three specific transfer RNAs.

Purine C-8 tritium-labeling rates have been measured at specific sites in Escherichia coli tRNAIle and tRNA2Tyr. The results are compared with those obtained for yeast tRNAPhe (preceding paper(Gamble et al., 1976)). The tRNAIle and tRNAPhe fall into the same general class of tRNA structures, while tRNA2Tyr is in a differint class; in particular, the latter is characterized by a large extra loop. In each of the three tRNAs the 3'-terminal A has the same labeling rate and, on a relative basis, appears to be the most rapidly labeled site. Bases in cloverleaf helical sections have markedly retarded labeling rates that collectively fall within an approximately threefold range of time constants. At some of the common purines, believed to be essential for the construction of a general system of tertiary interactions, exchange rates for yeast tRNAPhe are significantly different than those for the two Escherichia coli tRNAs. these differences may arise from variations among the tRNAs in the relative stabilities of specific tertiary interactions, or from other factors as well. In the case of tRNA2Tyr, labeling rates for bases in the large variable region are sufficiently retarded to suggest some structural organization for this part of the molecule. In addition, since exchange rates are similar for some of the bases common to Escherichia coli tRNAIle and tRNA2Tyr, it is likely that the large variable loop of tRNA2Tyr does not interact with or perturb these common sites. Finally, for all three tRNAs, structure formation (e.g., base pairing, base stacking) invariably decreases the labeling rate, even though the variety of base environments in the three-dimensional structures of these tRNAs might be expected to affect the acidity of C-8 and other chemical properties in diverse ways. Although these chemical effects no doubt bear influence, in these studies the dominant influence on exchange may be the effect of structure on the accessibility of solvent molecules, i.e. water.


The synthesis of 3'-dATP and its use as an inhibitor of ATP-dependent DNA synthesis in toluene-treated Escherichia coli.

A structural analogue of ATP, 3'-deoxyadenosine triphosphate (3'-dATP), has been synthesized from cordycepin (3'-deoxyadenosine), characterized, and determined to be an inhibitor of ATP-dependent DNA synthesis in Escherichia coli cells which have been reduced permeable to nucleoside triphosphates by treatment with toluene. The analogue is a competitive inhibitor of ATP and it inhibits replicative DNA synthesis 50% at concentrations of ca. 0.15 mM in the presence of 1.0 mM ATP and 4 x 10(8) cells/ml. The degree of inhibition of a given amount of 3'-dATP is inversely related to the cell concentration in the reaction mixture. The analogue interferes with some function of ATP which is continuously required during the course of the reaction and does not irreversibly inactivate the cells' DNA synthesis apparatus. 3'-Deoxyadenosine triphosphate may prove useful in elucidating the roles of ATP in DNA synthesis in more purified replicating systems.


Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template.

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either  magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.


Fluorescence energy-transfer measurements between coenzyme A and flavin adenine dinucleotide binding sites of the Escherichia coli pyruvate dehydrogenase multienzyme complex.

The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 1,N6-etheno-CoA (epsilonCoA) and coenzyme A (CoA) has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. A procedure for the resolution of the pyruvate dehydrogenase multienzyme complex into the pyruvate dehydrogenase enzyme and the transacetylase-flavoprotein subcomplex also is given. Direct binding studies with epsilonCoA indicate that 25 bound epsilonCoA molecules/multienzyme complex can be readily displaced by CoA, while approximately 21 bound epsilonCoA molecules/transacetylase-flavoprotein subcomplex can be displaced by CoA. The dissociation constant for the CoA displaceable epsilonCoA is 57.8 muM for the complex and 126 muM for the subcomplex in 0.02 M potassium phosphate (pH 7.0) at 5 degrees C. The kinetic behavior of epsilonCoA as a substrate was investigated and compared with that of CoA under a variety of conditions; the apparent Michaelis constants for epsilonCoA are considerably larger than those for CoA, while the corresponding maximal velocities are smaller. Fluorescence energy transfer measurements between bound epsilonCoA on the dihydrolipoyl transacetylase enzyme and flavin adenine dinucleotide on the dihydrolipoyl dehydrogenase enzyme either in the complex or subcomplex indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 50 A apart.


The "phosphoryl-enzyme" from phosphoglycerate kinase.

The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may well be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.


Studies on secondary structure in chicken egg-white lysozyme after reductive cleavage of disulfide bonds.

Native lysozyme and three reduced derivatives (carboxymethyl, carboxamidomethyl, and triphenylethyl-phosphonium) were examined by circular dichroism, and fractions of the protein chain present as alpha-helix, beta structure, and unordered structure were estimated by a computeradapted curve-fitting program. The alpha helix ranged from 2 to 23% in the three products, while native lysozyme exhibited 26 to 31%. However, beta structure in the reduced samples occupied 23-62% of the chain length (the latter in 50% methanol), consistently in excess of the native content(11-16%). Secondary structure in the reduced samples increased with pH, while that of the native protein remained nearly constant. In 8 M urea alpha helix was mostly eliminated from the reduced protein, while beta structure was nearly unaffected. Qualitatively, a partial loss of beta structure appeared to result from peptic digestion of the reduced samples, with further loss on exposure of the digest to urea. Stability of the observed beta structure indicates its existence prior to the oxidation of SH groups, which is concomitant with development of the native conformation. This structure could, therefore, constitute at least part of a precursor conformation in the formation of native structure.


Positional specificity of cyclopropane ring formation from cis-octadecenoic acid isomers in Escherichia coli.

An unsaturated fatty acid auxotroph of Escherichia coli was grown with a series of cis-octadecenoate isomers in which the location of the double bond varied from positions 3 to 17. Each of these fatty acid isomers was incorporated into the cellular lipids, but cyclopropane derivatives were formed to at least a 3-fold greater extent from the cis-9 and cis-11 isomers than from any other positional isomers. The extent of cyclopropane acid formation was observed to be highly dependent on the rate of shaking of the culture. A culture shaking at 340 rev./min converted 8.7% of its oleate to the cyclopropane derivative at stationary phase, whereas a parallel culture shaken at 110 rev./min converted 66% of the oleate to a cyclopropane acid. The inability to observe selectivity or form derivatives from isomers other than the cis-9 and cis-11 isomers seems to be due to enzyme specificity rather than a secondary affect of the abnormal unconverted fatty acids on the cell, because the cis-9 isomer is converted to its cyclopropane derivative even in cells grown with abnormal unreactive positional isomers. The preferred substrates for cyclopropanecarboxylic acid formation contained a cis ethylenic bond at either the 9 position or the (n-7) position. In combination with results of previous studies the specificity reported here supports a concetpt that two different enzymes may participate in cyclopropane ring synthesis. One enzyme activity may recognize its substrate by the distance from the pi-bond to the carboxyl group and the other by the distance to the methyl group.


[Phospholipid composition in thermosensitive mutants of Escherichia coli (author's transl)].

Quantitative phospholipid composition was investigated on various thermo-sensitive mutants of Escherichia coli K 12. Phosphatidylethanolamine was the major component. Three mutants (T1, T46 and T5) grown at 40 degrees C, had more anionic phospholipids (phosphatidylglycerol and diphosphatidylglycerol) and less bipolar phospholipids (phosphatidylethanolamine and lysophosphatidylethanolamine) than the wild type. Two mutants strains T46 and T83 which have the same Dna A mutation and identical membrane proteins alterations, have different phospholipid composition. Other mutants, such as T46, exhibit a blocking o? THE INITIATION OF DNA synthesis or morphological alterations under restrictive growth conditions (40 degrees C); however, the phospholipid composition of these mutants is normal and differs from the phospholipid composition of T46.T1 and T5 mutants have the same alterations in the phospholipid composition as T46 although they do not possess Dna A and Div A mutations of the T46 strain. Mutations leading to the alteration of the phospholipid composition are unrelated to other mutations leading to changes in the morphology and cellular division.


Function of phosphatidylglycerol molecular species in membranes. Activation of membrane-bound sn-glycerol 3-phosphate acyltransferase in Escherichia coli.

SN-Glycerol 3-phosphate acyltrasferase (EC 2.3.1.15) bound to the elaidate enriched membranes of an unsaturated fatty acid auxotroph of Escherichia coli had a lower specific activity than the acyltrasferase associated with the wild-type membranes. The 1-saturated-2-cis-unsaturated and 1,2-di-cis-unsaturated molecular species of phosphatidylglycerol activated this enzyme. However, these molecular species did not change the original temperature profile obtained by Arrhenius plots of the enzyme activity bound to the elaidate-enriched membranes.


Collagen heterogeneity in pig heart valves.

For the first time, it has been possible to solubilize a significant amount of heart valve collagen from 6 month old pigs by pepsin treatment. Chromatographic and electrophoretic analysis of this pepsin-soluble collagen gives evidence for the presence of two types of collagen molecules: type I collagen and another type which has properties similar to those of type III collagen. For example, this collagen, present as a gamma component, gives rise, when reduced by dithiothreitol, to two bands by SDS-acrylamide gel electrophoresis: a beta band and an alpha band. Furthermore, the collagenic web of this tissue is highly polymerized, which explains the inability to solubilize all of the collagen molecules and the presence of high-molecular-weight molecules in the pepsin-soluble fractions.


Microcalorimetry of thermal denaturation of pepsin and trypsin in the crystal state.

Heating of pepsin and trypsin crystals was studied by scanning microcalorimetry. A sharp decrease in temperature, halfwidth and heat of transition with a decrease in heating rate was discovered. It was shown that thermal transition is connected only with the denaturation of protein molecules in the crystal and not accompanied by the crystal disintegration into separate molecules.


Activity of stringent protein in ribosomes of Escherichia coli during the growth cycle.

To investigate whether the stringent protein in Escherichia coli was lost from ribosomes at certain phase of growth as in other organisms (Bacillus subtilis, mouse embryo), cells growing at various phases of the growth cycle were harvested and ribosomes tested for activity. The results showed that in E. coli, stringent protein was associated with the ribosomes throughout the growth cycle. The peak activity for the synthesis of guanosine tetra- and pentaphosphates appeared around midlogarithmic phase.


Proinsulin biosynthesis by pancreatic islets of the rat and the study of alloxan cytotoxicity in vitro.

Incorporation of L-[4,5-3H] leucine into proinsulin plus insulin by isolated pancreatic islets was shown to be severely inhibited by previous brief exposure to 1.25 mM alloxan, but incorporation into other islet proteins was not affected. The system proved valuable for the study of the prevention of alloxan cytotoxicity by various carbohydrates and carbohydrate derivatives. D-glucose, 3-O-methyl-D-glucose, D-mannose, 2-deoxy-D-glucose effectively prevented the injury by alloxan. D-Mannoheptulose, D-glucosamine, 1-thio-beta-D-glucose, 5-thio-D-glucose, 2,6-deoxy-D-glucose, D-glyceraldehyde, and dihydroxyacetone had either insignificant or no protective effect.


Methylated guanine derivative as a minor base in the DNA of phage DDVI Shigella disenteriae.

7-Methylguanine has been identified in the DNA of phage DDVI, which replicates in Escherichia coli B cells. The amount of this minor base is 0.27 mol per 100 mol of nucleotides. In the DNA of DDVI phage there is no 6-methylaminopurine which is usually a minor component in the DNA of E. coli B and phage T2, yet the DNA of DDVI phage is readily methylated during incubation in vitro with the B-specific methylase and adenosyl[3H]methionine with the label found only in 6-methylaminopurine. An extract of E. coli B cells infected with DDVI phage showed activity of a novel methylase which transfers the [3H]methyl group from S-adenosylmethionine to guanine, leading to the appearance of 7-methylguanine in the acceptor DNA.


Undermethylated transfer RNA does not support phage RNA-directed in vitro protein synthesis.

A cell-free protein synthesis system from Escherichia coli Q13 was depleted of mRNA and tRNA so that restoration of maximum activity was dependent of the addition of these components. Protein synthesis, directed by either MS2 or Qbeta phage RNA, was stimulated significantly by the addition of normal tRNA from either E. coli Q13 or B. In contrast, undermethylated tRNA from methionine-starved E. coli RCrel did not cause this stimulation. It is concluded that undermethylated tRNA Lacks sufficient base modifications to function in protein synthesis.


Nucleotide clusters in deoxyribonucleic acids. XIII. Sequence analysis of the longer unique pyrimidine oligonucleotides of bacteriophage S13 DNA by a method using unlabeled atarting oligonucleotides.

A method has been designed for sequence analysis of unlabeled oligodeoxynucleotides of chain length up to 20 nucleotides with no restriction on base composition. The unlabeled oligonucleotide preparation, is partially degraded with spleen exonuclease to give a series of products each differing in size by one nucleotide. The oligonucleotides in the digest are 5'-32 P terminally labeled with [psi-32] P ATP and T4 polynucleotide kinase, the excess ATP removed by chromatography on Sephadex G-25 then the oligonucleotides fractionated according to change length on DEAE-Sephadex. Each isostich fraction is analyzed for base composition and the nucleotide at the 5' terminus determined by its 32P label, resulting in direct read off of the sequence up to the penultimate 3'- terminal nucleotide. The 3'-terminal dinucleotide is analyzed by DEAE-cellulose chromatography of the Sephadex G-25 dinucleotide fraction. The method has been demonstrated by sequence analysis of the unique longer pyrimidine oligonucleotides C5T6, C2T8, C6T4 and C6T3 from S13 DNA. The sequences have extensive internal sequence homology.


An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of lysyl-tRNA synthetase from Escherichia coli K-12. I. Influence on subunit composition and molecular weight distribution.

Lysyl-tRNA synthetase was purified to 70-90% of homogeneity from Escherichia coli K-12. The enzyme was purified from wild-type cells grown in minimal medium, or minimal medium containing either 20 mM L-alanine or 3 mM glycly-L-leucine. The synthetase was similarly purified from a mutant strain grown in minimal medium plus 20 mM L-alanine. Results based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and trypsin inactivation studies indicate (A) that the presence of L-alanine of glycyl-L-leucine in the culture medium alters the properties of the wild-type enzyme; (B) that the alteration of the synthetase by l-alanine and glycyl-L-leucine is different; and (c) that the molecular weight of lysyl-tRNA synthetase is at least 135000--140000. The results suggest that most likely the metabolites modify the structure of lysyl-tRNA synthetase, but the possibility that the metabolites induce the synthesis of a new lysyl-tRNA synthetase cannot be completely eliminated.


An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of the lysyl-tRNA synthetase from Escherichia coli K-12. II. Kinetic evidence.

Wild-type Escherichia coli K-12 was grown in minimal medium alone or with the addition of 20 mM L-alanine or 3 mM glycyl-L-leucine. A lysyl-tRNA synthetase mutant strain was grown in minimal medium containing 20mM L-alanine. The lysyl-tRNA synthetase from these strains was purified to 70-90% of homogeneity. Kinetic studies comparing the effect of thermal and urea inactivation on these different lysyl-tRNA synthetase preparations and measurement of the Michaelis constant for lysine and transfer RNA indicated that growth of Escherichia coli in the presence of alanine and glycyl-L-leucine induces an alteration in the properties of the synthetase. Measurement of the apparent Km for ATP at pH 7.25 indicates lysyl-tRNA synthetase has two two binding sites for this substrate, and further studies indicated a dependence of the apparent Km for lysine on the ATP concentration.


The biosynthesis of the thiazole moiety of thiamine in Salmonella Typhimurium.

The mechanism of biosynthesis of 4-methyl-5-beta-hydroxyethyl thiazole, the thiazole moiety of thiamine was studied in Salmonella typhimurium. Using the adenosine derepression technique the incorporation of various 14C-labeled precursors was determined. We found that;e1Me-14C]methionine, [2-14C]methionine, [U-14C]alanine, and [2-14C]glycine were not incorporated whereas [2-14C]tyrosine was incorporated. Degradation of the 4-methyl-5-beta-hydroxyethyl thiazole obtained after [2-14C]tyrosine incorporation revealed that all of the activity was located on carbon-2. These findings are discussed and compared with previous findings concerning 4-methyl-5-beta-hydroxyethyl thiazole biosynthesis.


Purification and properties of a debranching enzyme from Escherichia coli.

The debranching enzyme (EC 3.2.1.-) from Escherichia coli K12 was purified 312-fold with a 21% yield, DEAE-cellulose and DEAE-Sephadex chromatography were used for purification. The preparation was homogeneous and showed only a single band of protein and activity upon polyacrylamide gel electrophoresis. The enzyme hydrolyzed 1,6-alpha-glucosidic linkages in phosphorylase and beta-amylase limit dextrins prepared from glycogen and amylopectin. Small branched oligosaccharides were also hydrolyzed. Amylopectin was also completely hydrolyzed but the enzyme showed only a very low activity with glycogen as the substrate. The enzyme cannot be classified as a pullulanase because it has practically no activity with pullulan. But it also differs from the bacterial isoamylases described in other studies because of its inability to hydrolyze glycogen. The optimal pH is about 5.6. The optimal growth conditions for the synthesis of the enzyme by E. coli were also examined in the present studies.


Peptide substrates for chymosin (rennin). Kinetic studies with peptides of different chain length including parts of the sequence 101-112 of bovine k-casein.

Kinetic parameters have been determined for the reaction between milk-clotting chymosin (EC 3.4.23.4) and a series of peptides (or their methyl esters) including the amino acid sequence around the enzyme-sensitive Phe(105)-Met (106) bond the bovine k-casein. In particular, the influence of the substrate's chain length on the kinetic parameters has been studied. Evidence is presented that in the model peptides studied the sequence -Ser-Phe-Met-Ala with a further residue added to either end (in casu Leu(103) or Ile(108)) is necessary to induce any cleavage by the enzyme. When both the Leu(103) and Ile(108) residues form part of the peptide chain, a marked improvement of the substrate properties is observed. It is suggested that prolyl residues on either side of the sensitive peptide bond form additional sites for secondary enzyme-substrate interactions.


Chromatin structure and function in proliferating cells.

The conclusions that we would like to draw from this review are the following: (a) Chromatin structure and function are exceedingly sensitive to changes in the proliferative state of a cell. Differences can be detected between cells in mitosis, G1 and S, and even between G0 and G1 cells. (b)These differences are very unlikely to be artifactual, since similar changes can also be demonstrated in intact nuclei. (c) Some of these differences can be abolished by extraction of chromatins with low concentrations of salt. (d) Differences between chromatins of normal and neoplastic cells can also be detected, but they are largely related to differences in the extent of cell proliferation. (e) A number of laboratories have been very busy in trying to elucidate chromatin structure with different technologies. Sometimes a change in a macromolecule cause by a physiological stimulus can tell us as much about its structure as a thousand instruments. The changes occuring in chromatin of proliferating cells could perhaps be profitably used to know more about chromatin structure.


Cell surface proteins and malignant transformation.

Many of the altered properties of malignant cells are thought to involve alterations in cell surface functions. In order to understand these alterations it is necessary to know more about the molecular structure of the surface. Methods for analyzing surface proteins are discussed and their application to normal and transformed tissue culture cells are reviewed. A number of surface proteins are observed to be altered by transformation. Most of the alterations are reductions in amounts of particular species, although a few proteins do increase. Evidence concerning the reasons for these alterations and the possible functions of some of the molecules is reviewed. Working hypotheses arising from these data are presented and prospects for understanding the physiological changes in terms of molecular effects are discussed. Particular emphasis is placed on the idea that surface molecules are associated in specific non-covalent complexes which are important for their functions.


Attempts to purify a membrane attached chromoid of bacteriophage lambda.

Using methods which proved successful for the isolation of E. coli chromosome in a folded form (the E. coli chromoid), we have attempted to purity the "native" form of bacteriophage gamma chromosome from gamma infected cells. Upon sedimentation of lysates we find that phage DNA separates into two fractions, one of which cosediments with the bacterial chromoid ; the other sediments nearer to the top of gradient. Both fractions probably contain membrane-bound phage DNA, and both support the in vivo synthesis of phage DNA. The heavier fraction contains more closed circular parental DNA molecules than the lighter fraction. Formation of the latter is blocked by certain phage mutations. Being relatively free of bacterial DNA, the lighter fraction is suitable for further analysis.


L-tryptophan in depression.

L-tryptophan, the amino acid precursor of serotonin, was administered to 16 depressive patients in a double-blind study of its potential antidepressant efficacy. Antidepressant responses were observed in one of ten unipolar patients and in three of six bipolar patients. These results are discussed in the context of possible interactions of amines with electrolyte systems in the etiology of affective illness.


Auditory attention in process and reactive schizophrenia.

The present data indicate that, based on level of premorbid adjustment (process-reactive), schizophrenic subjects differ in auditory attention processes. Generally speaking, poor premorbid adjustment is associated with more attention dysfunction. Results also indicate that attentional deficits improve for all schizophrenic subjects after a 28-day period of neuroleptic therapy.


Visual agnosia and cognitive defects in a case of Alzheimer's disease.

A patient with Alzheimer's disease, who is still alive, was investigated using a battery of tests, including design-copying and identification, the Gottschaldt hidden figures, a reading test, picture identification, and identification of tachistoscopically presented figures. The main observations were (i) an inability to handle more than one "element" of visual input simultaneously, (ii) an inability to attain and sustain a correct test performance. This last feature was assumed to be related to a difficulty in establishing and sustaining a set or attitude about how to solve the task. The hypothesis was put forward that both behavioral characteristics were related to a deficient task-related arousal-increasing mechanism.


A comprehensive treatment unit for the respiratory distress syndrome in the newborn.

Various systems have been devised to improve the therapy of idiopathic respiratory distress syndrome in newborn infants. This article describes a new ventilator which provides alternating positive and negative pressure ventilation(APNPV) in addition to the more conventional techniques. Safety aspects, technical performance and cleaning procedures are described. A brief summary of the clinical performance of the machine is given.


Effects of hyperosmolality on candidacidal activity of human neutrophil polymorphonuclear leukocytes and on clumping of Candida albicans by human serum.

Hyperosmolalities comparable to those of the renal medulla did not altered the clumping of C. albicans by human serum but diminished ingestion and evenmore the killing of C. albicans by human polymorphonuclear leukocytes (PMN).


Anti-lymphocyte antibodies in systemic lupus erythematosus and in Hodgkin's disease: a comparison by immunofluorescence.

Anti-lymphocyte antibodies of IgM type (cold reactive) in systemic lupus erythematosus and of IgG type (not cold reactive) in Hodgkin's disease have been demonstrated by immunofluorescence to attach the same target cell (T-lymphocyte.) The same behaviour of both kinds of antibodies in "co-capping" experiments confirm that the antigen on the cell surface is the same. Hypothesis about the meaning of this phenomenon are made.


An efficient method of cryopreservation of human lymphocytes for immunological testing.

A reliable technique for cryopreservation of lymphocytes which conserves mitogenic response and marker (E-Rosettes, immunofluorescence and peroxydase) capability is presented. The data which is presented shows no significant difference between tests involving fresh and cryopreserved lymphocytes.


Pressure-jump relaxation studies of the association--dissociation reaction of E. coli ribosomes.

The association--dissociation kinetics of ribosomal particles from E. coli have been studied using a pressure-jump apparatus with optical detection. Experiments on isolated subunits yield two relaxation times of about 10 and 700 ms, respectively. With mixtures of 30 S and 50 S particles an additional relaxation time of about 100 ms is observed, which is assigned to the equilibrium 30 S + 50 S in equilibrium 70 S. The two other times are attributed to reversible equilibria between subunit monomers and subunit homo-associates.


[Hexokinase activity in the soluble fraction of the nucleus and cytoplasm in cell cultures infected with human adenoviruses types 6 and 12].

A study was made of the activity of hexokinase in the soluble fraction of the nucleus and cytoplasm of fibroblasts of rat embryos (primary cultures) during contact with infectious (A6) and oncogenic (A12) human adenoviruses. A high activity of the enzyme was seen in the cytoplasmic fraction in comparison with the nuclear one both in the infectious adrenovirus of type 6 this value differed from the normal and in the experimental samples. With the action of the normal but insignificantly. Marked changes in the enzyme activity were observed in case of cell contact with the oncovirus. They occurred both in the nucleus and in the cytoplasm the first days of the virus inculcation, increasing gradually with the lapse of tiay; these disturbances were much greatert in the cytoplasm than in the nucleus. It is supposed that the changes revealed possibly associated with the impossibility to regulate the cell proliferation process.


[Cytopathogenic action of E. coli strains, containing corresponding ABO type heterogeneous antigens, on transplantable human cells].

A study was made (in vitro) of the interaction of various strains of E. coli, serological type O26 with continuous human cells (HeLa, Tg-33 and RH). Phenomenon of the cytopathogenic action of uropathogenic E. coli strains, containing heterogenous antigens, type O(H) and B, on the human cell strains possessing corresponding isoantigens was revealed on the 6th hour of the interaction. The number of dead cells in these cultures exceeded (1.5--3 times) their count cultures to which E. coli containing no heterogenous antigens or containing different human heterogenous antigens of the ABO type were added. It is supposed that this phenomenon played an important role in the development of chronic forms of colibacterial pyelonephritis.


[Age characteristics of the immune response in rats with multiple administraion of different doses of sheep erythrocytes].

A study was made of the number of antibody-forming cells and of the serum antibody titres in adult and old rats under conditions of a 10-fold immunization with "low" (4 X 10(8) cells) and "high" (4 X 10(10) cells) doses of sheep erythrocytes. The titres of antibodies (hemolysins and hemagglutinins) in the animals of both groups were either close to one another or greater in adult rats (hemolysins - in administration of "high" doses of the antigen). The content of the direct plaque-forming cells was greater in the spleen of old than of adult animals at all the immunization periods, of this period. The data obtained pointed to differences in the maturation of the immune response in the animals of various age.


[Antigen-binding clone cells in hematopoietic spleen colonies].

The antigen-binding cell clones and the Ig-positive cells were found and quantitatively assessed in the primary hemopoietic splenic colonies. The data ogtained were analyzed assuming that the ratio of clone of specialized B-cells should reflect the quantitative ration in the corresponding V-genes in the given lymphocyte populations at definite stages of its development. The colonies differed from one another markedly by the curves of inhibition by DNP-lysine of rosette-formation with DNP-erythrocytes, i.e. by the avidity of B-cells of the given specificity. The colonies differed significantly by the ratio of the volumes of the two clone groups (cell with anti-DNP and anti-BE Ig-receptors) between themselves and the combination of the Ig-positive cells. These quantitaive regularities were incompatible with the view that each B-cell had any conceivable set of V-genes, i.e. with the germ-line hypothesis of the antibody and receptor diversity.


[Histogenesis of sarcomas, induced by plastic films, in murine radiation chimeras].

Sarcomas were induced in radiation chimeras CBA/CBAT6T6 by subcutaneous implantation of plastic films 13 months after the irradiation and donor bone marrow transplantation. Of the 12 tumours examined, 11 possessed host kariotype and one--donor karyotype. Development of connective tissue cells from the bone marrow precursors presumably played no significant role in the histogenesis of plastic sarcomas.


Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality.

A prospective randomized trial of therapy for severe aplastic anemia was designed to compare early bone marrow transplantation with conventional treatments. All patients with a sibling matched at the major histocompatibility region were transplanted. Transplantation was performed with 17-100 (median 33) days of original diagnosis. Conventional treatments included transfusion support with or without androgens. Twenty-four of 36 patients intered on the transplant arm are alive after 4-20 (median 9) mo with full marrow reconstitution. Only two are limited by chronic graft-versus-host disease. In contrast only 12 of 31 conventionally treated patients are alive. Six of these survivors have improved, five incompletely. The 19 nontransplant deaths have occurred within 1-11 (median 3) mo of diagnosis. Compared to nontransplant regimens, early transplantation more effectively restores normal marrow function and decreases the acute mortality of severe marrow aplasia (p = 0.006). Pending longer follow-up, early marrow transplantation appears to be the most effective available treatment for severe aplastic anemia.


Advances in pediatrics and child care in Cuba, 1959-1974.

This article describes the major activities carried out since 1959 in the field of pediatrics and child care in Cuba. In particular, it notes the improvements made through establishment of a national health system and through the participation of community organizations (the Federation of Cuban Women, Committees for the Defense of the Revolution, associations of small farmers, and trade unions) and shows how perinatal, infant, and childhood mortality have been significantly reduced. As of 1973 perinatal mortality had fallen to 27.9 deaths per 1,000 live births, infant mortality to 27.4 deaths per 1,000 live births, preschool mortality to 1.2 per 1,000 children, and school-age mortality, to 0.4 per 1,000 children. This report also cites data on available physical and manpower resources, and outlines a large range of activities linked to a Comprehensive Child Care Program undertaken in 1967. This program, in which newborns are enrolled upon leaving the maternity, seeks to encourage breast-feeding, to promote the activities of well-baby clinics, to provide special examinations for malnourished infants, to provide health care for preschool and school-age children, to promote pediatric medical visits to the home, to assist with camps for asthmatic and diabetic children, to provide pediatric services at pioneer and other camps for schoolchildren, to carry out health education activities, and to combat communicable disease. In particular, activites to prevent communicable disease appear responsible for a good part of the progress achieved to date. As a result of these activities malaria and diphtheria have been eradicated, poliomyelitis has been overcome, and the incidences of tuberculosis, tuberculous meningitis, tetanus (among both newborns and children under 15), and acute diarrheal disease have been substantially reduced.


Microsurgical transfer of the dorsalis pedis neurovascular island flap.

The feasibility of transfer of a free neurovascular dorsalis pedis island flap is demonstrated and its very real advantages, particularly with regard to its thinness are discussed.


Indirect and direct inhibition of dopamine beta-hydroxylase by amphetamine in storage vesicles and synaptosomes.

1 To elucidate the mechanism by which amphetamine produces a functional inhibition of dopamine beta-hydroxylase (DBH), the actions of amphetamine on amine uptake and beta-hydroxylation were examined in isolated adrenal storage vesicles and in whole brain synaptosomes of the rat. 2 Amphetamine produces a competitive inhibition of catecholamine incorporation into adrenal vesicles with a Ki of 279 muM; this action of the drug accounted for slightly less than half of the total inhibition of beta-hydroxylation in the vesicles, indicating that the interference by amphetamine of access of substrate to the enzyme compartment plays an important role in functional inhibition of DBH. The remainder of the effect in vesicles probably represents direct inhibition of the enzyme. 3 In synaptosomes, similar actions on uptake of substrate and on functional enzyme activity were noted, indicating that amphetamine-induced inhibition of the neuronal amine pump can also affect DBH activity if the substrate is supplied exogenously. 4 In addition to the effects of amphetamine in vitro, chronic administration of amphetamine to rats produced an increase in total activity of adrenal DBH. 5 The net effect of amphetamine on DBH activity thus represents the summation of direct inhibition of the enzyme, indirect inhibition via reduced access of substrate and enhancement of activity via trans-synaptic induction.