Intracranial abscess: treatment by continuous catheter drainage.

15 children with brain abscesses were subjected to continuous catheter drainage as the procedure of choice for a solitary encapsulated abscess. Although many still advocate craniotomy and total excision, this series, coupled with the experience of others in the literature, leaves little doubt that catheter drainage alone is sufficient. The simplicity of creating a burr hole under local anesthesia, the ability to irrigate with antibiotics and then outline the cavity with contrast agents are but a few of the advantages of this method.


[Treatment of peptic esophageal stenosis in childhood].

Among a total of 82 children treated conservatively and surgically for hiatal hernia and gastro-esophageal malposition (Lortat-Jacob) 14 cases (17%) of peptic stenosis of the esophagus was observed. In an evaluation of the etiology of peptic esophageal stenosis the importance of anatomical growth and morphological changes of the cardia during infancy is stressed. Analysis of patient data revealed an unusually short period of stenotic manifestation after the first symptoms of reflux had been noted. Fundoplication with or without bouginage on an endless string seems to constitute the most beneficial form of therapy.


[Critical notes on the transport of patients with cervical spinal cord injuries].

Patients with a cervical spine injury who cannot be taken to a special centre immediately after the accident should be transferred to such a centre only when one is sure that no additional transportation trauma will be produced. The fracture cervical spine must be adequately stabilized during transportation. The partial oxygen pressure must not be lower than 60 mm Hg and one should wait at least 1 week post removal of tracheostomy tube. An acute stress ulcer (melaena) also has to be excluded.


[Contribution to the surgical and cytostatical treatment of multiple abdominal and hepatic echinococcosis].

This is a report on 5 patients with multiple liver and abdominal echinococcosis. Besides surgical treatment the cytostatic Cyclophosphamide (=Endoxan) has been used in all cases. With this combined treatment there has been no recurrence. At this time the cytostatic-surgical combination treatment represents the therapy of choice in multiple echinococcosis. It remains to be seen whether a specific drug therapy will displace these methods in the near future.


[Comparative strength analysis of selected osteosynthesis materials for pertrochanteric femoral fractures].

Four commercially available hip-fixation devices for stable fixation of pertrochanteric fractures were selected for a biomechanical evaluation, in order to determine, which construction would best support the load of weight-bearing in postoperative period. Intramedullar nailing by Lezius, Küntscher or Ender showed less bending moments than trifin nail-plates or extramedullar plate attachments with plate angles of 130 degrees or 95 degrees, so that bending tension of Lezius-nails were reduced compared to other implants in a proportion of 1.1,6:1,8:2,7. Whenever possible intramedullar nailing should be preferred. For extramedullar fixation of pathologic or unstable pertrochanteric fractures a double-T-plate was constructed, which shows a 3-fold load resistance of a Lezius-nail, 5-fold load-bearing of a 130 degree plate or a trifin nail combination and a 8-fold higher stress tolerance than a 95 degree-plate.


[Results of the fixation of pertrochanteric fractures by condyle nails using Ender's and Simon-Weidner methods].

2 year-results are reported after intramedullary fixation of pertrochenteric fractures with the so-called "feather nails" of Ender and Simon-Weidner. The procedure is discussed with special references to possible complications. This method performed with the necessary experience represents a real progress in the treatment of pertrochenteric fractures.


Partially sighted Swedish schoolchildren.

812 visually handicapped children aged 7-21 years and educated by black print are investigated with regard to the degree and cause of visual impairment. The rate of visually handicapped children amounted to around 0.7 o/oo, boys being more frequently afflicted than girls. The causes were most often prenatal; 30% of genetic origin, 15% caused by pre- or perinatal damage and 40% of prenatal origin which could not be specified. Some of the more common clinical entities are discussed. The importance of preventing an additional functional amblyopia in the visually handicapped child is stressed.


[Tolerance and immunogenicity of an attenuated live influenza virus vaccine in man (author's transl)].

The attenuated influenza-A-virus strain "Alice" (H3N2) - A Recombination from A2/England/72 and A/PR8 Mount Sinai/34 - was given intransally in drops (10(7,2) ID50 per dose), twice seven days apart. In a similar fashion, 25 healthy persons received placebos. Local antibodies were determined before and three weeks after the first vaccination from nasal irrigation fluid. The fluid was concentrated and adjusted to an IgA amount of 50 mg/l. Blood samples were obtained at similar intervals. Systemic antibody formation was comparable to that obtained with inactivated virus vaccine. There was good antibody formation against retrospective and prospective virus strains of the actual drift period. The conversion rate of antineuraminidase antibodies was comparable to that obtained with inactivated virus vaccine. The locally induced antibodies behaved predominantly in a strain-specific manner. However, there were also reactions with the virus strains Port Chalmers and Hong Kong. Live vaccine should in future be used in "drift" times, while inactivated virus vaccine should be used in shift periods (occurrence of new virus strains).


[Nomogram for the relation between plasma renin activity and fasting urinary sodium creatinine ratio (author's transl)].

Based on repeat measurements in 11 male and 12 female normal subjects, aged 20 to 35 years, on normal, low and high sodium intake a nomogram for the relation between fasting urinary sodium creatinine ratio and plasma renin activity (PRA) is presented. The use of fasting urinary sodium creatinine ratio as index of sodium intake and hydration of the subject results in a better prediction of PRA as compared with 24-hour urinary sodium excretion. Although fasting urinary sodium creatinine ratio in normal subjects on a normal sodium intake varies between 0.2 and 1.6, a uniform increase of basal PRA is found only at a ratio of below 0.3. A ratio greater than 0.5 excludes any stimulation of PRA by low sodium intake and (or) dehydration of the subject. The shape of the nomogram should make it applicable independent of the method used for PRA assay, as soon as the variation of PRA in normal subjects with a urinary sodium creatinine ratio above 0.5 is established.


Low-dose clonidine: a review of its therapeutic efficacy in migraine prophylaxis.

The demonstration that long-term administration of relatively low doses of clonidine decreased the responsiveness of blood vessels to vasodilator and vasoconstrictor drugs in animals led to its investigation in the prevention of migraine in man. Results of placebo-controlled and open therapeutic trials have shown that clonidine in low dosages (75 to 150 mug daily) is useful in preventing migraine headaches in about 30%-50% of patients. A 50% or greater reduction in headache frequency or headache indices has been reported in 40% of patients in controlled and open studies. Thus clonidine, like other drugs used in the interval therapy of migraine, can be expected to be effective in only a proportion of patients. Although clonidine has not been compared directly with other drugs used in the prophylactic treatment of migraine, the general clinical impression is that it is less effective then pizotifen or methysergide. Because it is relatively well tolerated at dosages of 75 to 150 mug daily it is worthy of a trial, particularly in patients considered to need prophylactic  migraine therapy for the first time, and when migraine occurs in association with hypertension. At the dosages used in migraine prophylaxis, which are almost invariably lower than used in hypertension, clonidine does not cause hypotension and can be used in patients with cardiovascular disease. The principal side-effects are drowsiness and dry mouth which tend to diminish as treatment continues.


Sodium chloride suppression of renin release in the unanesthetized rat.

Previous studies have shown that increased sodium chloride in drinking water failed to suppress serum renin activity (SRA) in the etheranesthetized rat. In the absence of anesthesia, sodium chloride decreased SRA by 76% and serum aldosterone by 72%. Deoxycorticosterone acetate, when given alone, produced similar results but was more effective in reducing SRA (96%) when combined with sodium chloride. However, if "light" ether anesthesia was administered prior to blood collection, there was a 10-fold increase in SRA and the effects of sodium chloride were blocked. Thus, in the absence of anesthesia, the renin-angiotensin-aldosterone system of the rat responds, as in other species, to a sodium load.


Site of iodination in the rat thyroid gland deduced from electron microscopic autoradiographs.

The site of iodination of protein in the thyroid gland (whether intracellular  or intraluminal)  was ascertained by autoradiographic studies using iodide-125I. In tissue fixed within about 40 sec after intravenous injection of radioiodide the silver grains of autoradiographs were concentrated over the follicular lumen generally as a ring of grains close to the apical border of the follicular cells. The zone of grains was sharply limited toward the cells. No concentration of silver grains was detected associated with any intracellular organelle. The autoradiographic ring which had a minimum width of about 2 mum was continuous along the apical plasma membrane of the follicle cells but there was a drastic reduction in grain density along the plasma membrane of the distal portion of pseudopods. Tissue was fixed so soon after radioiodide injection that it appeared likely that a negligible fraction of radioiodoprotein, if formed in the cell, could have been transferred to the lumen. The observations strongly indicate that the iodination of thyroglobulin occurs in the follicle lumen, probably at the apical surface of the follicle cells. Since in the TSH-treated animals the distribution of the labeling along the apical plasma membrane agrees well with the reported histochemical distribution of thyroperoxidase in this membrane, it is further concluded that iodination may well be catalyzed by peroxidase in the apical plasma membrane.


Surface modifications evoked by estradiol and diethylstilbestrol in isolated endometrial cells: evidence from lectin probes and extracellular release of lysosomal protease.

Endometrial cells were isolated from the uteri of ovariectomized rats. The inhibitory effect of alpha-methyl-D-mannoside on fluorescein-labeled concanavalin A (Con A) binding to these cells indicates that they possess specific binding sites for Con A. The lectin also mediates adsorption of homologous erythrocytes to these cells. Both Con A binding and Con A-mediated hemadsorption to endometrial cells are depressed at 4 C compared with these functions in cells maintained at 22 C. Gross elevations in lectin-mediated hemadsorption to endometrial cells are evident following prior exposure to 1 X 10(-9)M concentrations of diethylstilbestrol (DES) or estradiol-17beta, but not to the physiologically inactive 17 alpha-epimer, at 22 C. The enhancement of hemagglutinability cannot be attributed to a corresponding increase in lectin binding at 22 C. Although estrogen treatment elicited significant increments in Con A binding as early as 5 min after addition of estrogen to cell suspensions, the increment in agglutination attributable to hormone treatment consistently ranged from 1.5-3 times greater than the increase in lectin binding. These estrogenic effects were reduced by incubation of the endometrial cells at 4 C or when cortisol, 3 X 10(-6)M, was present with estradiol-17beta. In parallel experiments, treatment with DES and estradiol-17beta, but not estradiol-17 alpha, also enhanced the release of cathepsin B 1 and acid phosphatase from uterine segments into the particle-free extracellular media in which the tissues had been incubated for 30-60 min. The marked increment in the extracellular activity of the lysosomal hydrolases induced by estrogen treatment was suppressed in cells incubated at 4 C or when cortisol was present concomitantly. These and related data suggest the hypothesis that acute increments in lysosomal hydrolase activity may contribute to cell surface alterations which have been described in both normal and aberrant processes of cell growth.


Studies on luteolysis: effect of antiserum to luteinizing hormone on sterols and steroid levels in pregnant hamsters.

The effect of luteinizing hormone antiserum (LH A/S) on sterol and steroid levels in luteal and non-luteal ovarian compartments, and on serum steroid levels of pregnant hamsters, was studied. Injection (ip) of 0.1 ml of LH A/S on day 7 caused termination of pregnancy and morphological regression of the corpus luteum (CL) within 18-20 h of treatment, whereas LH-free non-immune serum had no effect. Within 3 h of administration of LH A/S the luteal progesterone levels fell from a control value of 36.0 +/- 2.4 to 15.6 +/- 3.4 ng/mg, and estrogen from a control value of 20.8 +/- 3.4 to 12.2 +/- 1.5 pg/mg. By 12 h progesterone had dropped to 7.3 +/- 0.3 ng/mg whereas estrogen was undetectable by radioimmunoassay. Serum progesterone fell in 3 h from 5.53 +/- 0.39 to 3.15 +/- 0.26 ng/ml whereas estrogen fell from 172 +/- 26 to 123 +/- 9 pg/ml. The progesterone content of non-luteal ovarian tissue dropped in 3 h from 1.06 +/- 0.01 to 0.47 +/- 0.06 ng/mg and estrogen from 115.0 +/- 1.5 to 11.0 +/- 0.65 pg/mg. Both progesterone and estrogen were undetectable at 24 h. In the CL, free cholesterol concentration did not change significantly after A/S treatment, while esterified cholesterol increased from 2.82 +/- 0.14 to 4.14 +/- 0.25 mug/mg within 6 h and 5.52 +/- 0.17 mug/mg by 12 h. The results suggest that LH plays a critical role in the maintenance of CL of early pregnancy in the hamster and that deprivation of LH, even for periods as short as 3 h, significantly influences steroid production. The accumulation of cholesterol esters following LH-deprivation suggests that loss of cholesteryl esterase activity could be one of the events leading to luteolysis.


Effects of a non-steroidal antiandrogen on sexual behavior and pituitary-gonadal function in the male rat.

An investigation was conducted on the effects of the non-steroidal antiandrogen flutamide (F; alpha-alpha-alpha-tri-fluoro-2-methyl-4-nitro-m-propionotoluidide) on two neuroendocrine mechanisms in the male rat, androgen-dependent sexual behavior, and LH regulation. F was administered in the dose of 50 mg/kg/day SC. In intact, sexually experienced adult males, no quantitative or qualitative behavioral effects were noted. In long-term castrates, F completely suppressed the effects of 100 mug testosterone propionate (TP) per day on accessory sexual glands and penes, but only partially inhibited the marked stimulatory effects of this moderate TP dose on mating. Although the incidence of testosterone (T)-activated ejaculatory behavior was markedly diminished, there was no statistically significant effect on occurrence of mount and intromission behavior. The rapid and profound elevations of circulating LH and T in intact males indicate an effective antagonism of the negative feedback effect of endogenous androgen, and suggest the usefulness of F as a provocative test of pituitary-testicular function. Pituitary LH response to exogenous LHRH was markedly enhanced, as previously found in castrated rats. The administration of F did not affect circulating T levels in T-treated or untreated castrates, indicating lack of interference of circulating F in the T assay. It was concluded that, like the steroidal antiandrogen cyproterone, non-steroidal F shows a divergence between its effects on peripheral androgen-dependent and central feedback mechanisms on the one hand, and sexual behavior on the other. It was not determined whether the inhibition of ejaculatory behavior following F treatment is centrally mediated or results from failure of the peripheral, androgen-dependent structural or functional elements.


Comparison of results from in situ and in vitro perfusions of rabbit uterus with labeled estrone and estradiol.

The interconversion of labeled estrone (E1) and estradiol (E2) in rabbit uterus was studied in vivo and in vitro, in order to evaluate the influence of incubation conditions upon the preferred direction of the reversible conversion of E2 to E1. In a typical experiment, one uterine horn was perfused intraluminally in situ with a mixture of [3H]E2 and [14C]E1, while the other horn was dissected and used for in vitro perfusions with the same tracers, both intraluminally and on tissue slices. The ratios of rate constants corresponding to the oxidative and reductive reactions were found to be similar under the three perfusion conditions, as calculated from the isotopic data. The rate constant of conversion of E1 to E2 was 10-20 times larger than the rate constant of the opposite reaction. Equal E2 nuclear saturation levels were obtained by perfusion of the uterine tissue with excess [3H]E2 in situ and in vitro after slicing.


Blockade of testosterone-induced mounting behavior in the male rat with intracranial application of the aromatization inhibitor, androst-1,4,6,-triene-3,17-dione.

Propylene glycol (glycol) solutions containing either testosterone (T) or estradiol (E2) were infused directly into the preoptic area (POA) of longterm castrated rats in order to reinstate male copulatory behavior. In addition, castrated males were administered T or E2 in the POA in combination with a steroid that has been shown to block the aromatization of testosterone to estradiol, androst-1,4,6-triene-3,17-dione (ATD). The facilitatory action of testosterone on mounting behavior was blocked when it was given in combination with ATD. Animals treated in the POA with glycol +T, glycol +E2 or ATD + E2 all showed significant increases in mounting behavior over preimplant levels. There was no significant rise in the number of intromissions or ejaculations in any of the hypothesis that, at least for mounting behavior, aromatization is necessary for the stimulation of male sexual behavior by testosterone.


Bone resorbing activity of vitamin D metabolites and congeners in vitro: influence of hydroxyl substituents in the A ring.

The vitamin D3 derivatives, 1alpha-hydroxyvitamin D3; 1alpha-hydroxy-3-deoxyvitamin D3; 5,6-trans-vitamin D3; and 5,6-trans-25-hydroxyvitamin D3 were tested for bone resorbing activity in vitro. 1alpha-Hydroxy-3-deoxyvitamin D3 and 5,6-trans-vitamin D3 were inactive at concentrations as high as 10(-6)M. 1alpha-Hydroxyvitamin D3 had significant effects on mineral and matrix resorption at concentrations of 2.5 X 10(-8)M and above, and was 2-3 orders of magnitude less potent than 1,25-dihydroxyvitamin D3 in this system. A concentration of 2.5 X 10(-7)M of 5,6-trans-25-hydroxyvitamin D3 was required to stimulate 45Ca release. The results indicate that 1) in the vitamin D3 series of compounds the presence of only one hydroxyl group in the molecule in either the 1alpha or the 3beta position in the A ring results in a compound with very limited or no direct effects on bone, and 2) the direct effects of vitamin D3 congeners on bone resorption do not require the presence of a 25-hydroxy group, but their activity is markedly enhanced by the 25-hydroxy group.


Effects of 1alpha,25-dihydroxyvitamin D3 and Solanum glaucophyllum on intestinal calcium and phosphate transport and on plasma Ca, Mg and P levels in the rat.

The responses elicited by a single dose of either S. glaucophyllum (SG) or 1alpha,25-dihydroxyvitamin D3 (1alpha, 25-(OH)2D3) in vitamin D-deficient rats were qualitatively identical. Both compounds stimulated duodenal absorption of calcium and phosphate 3 h after administration, and the effects persisted for 96 h. The SG effects on calcium transport were maximal at 6 h and exceeded the greatest response to 1alpha,25-(OH)2D3 which occurred at 48 h. While the early SG effects on phosphate absorption exceeded those of 1alpha,25-(OH)2D3, the latter compound had a much greater effect at 48 h. Both SG and 1alpha, 25-(OH)2D3 produced marked hyperphosphatemia that was accompanied by hypocalcemia and transient hypermagnesemia at 3-6 h. The phosphatemia increased for 48 h in the 1alpha,25-(OH)2D3 group and fell toward baseline between 48 and 96 h, while this parameter was maximal in the SG group at 3-12 h and returned to baseline values by 96 h. Plasma calcium levels tended to vary inversely with phosphorus concentrations and did not exceed baseline values until 48 h for the SG group and 96 h for the 1alpha,25-(OH)2D3 group. We suggest that the hypocalcemia is due to calcium phosphate precipitation. The quantitative differences in the effects on calcium and phosphorus metabolism may be dose-related. The responses to SG suggest that a gram of dried leaf has approximately the same activity as 1 mug of 1alpha,25-(OH)2D3. It appears that the water-soluble SG factor has the same biological effects on Ca and P metabolism as does 1alpha,25-(OH)2D3.


The intratesticular localization of cytochrome P-450 and cytochrome P-450-dependent enzymes in the rat testis.

Following separation of the seminiferous tubules from the interstitial cells in the rat testis, the amount of cytochrome P-450 and the activities of the cytochrome P-450-dependent enzymes, the 17alpha-hydroxylase and the C17-C20 lyase, were measured in the microsomes of the separated fractions. The amount of cytochrome P-450-dependent enzymes recovered in the microsomal fraction of the interstitial cells ranged from 71 to 86% of the whole testis. However, in some experiments lower recoveries of the activities of the enzymes were attributed to the breakdown of cytochrome P-450 to cytochrome P-420. In all cases, less than 10% of the testicular cytochrome P-450 and the cytochrome P-450-dependent steroidogenic enzymes were found in the tubular microsomes. Moreover, the specific activities of the 17 alpha-hydroxylase and the C17-C20 lyase were found to be 10 to 30 times higher in the interstitial tissue than in the seminiferous tubules of the rat testis. From these results, we have concluded that cytochrome P-450 and the activities of the cytochrome P-450-dependent enzymes in the rat testis are predominantly, if not sole, located in the interstitial cells.


Prolactin restores plasma testosterone levels and stimulates testicular growth in hamsters exposed to short day-length.

In male hamsters, light deprivation was reported to reduce both testis weight and prolactin (PRL) levels. Therefore, we decided to examine the effects of PRL on testicular function in hamsters exposed to short day-length. Adult hamsters were exposed to 5 h of light per day to induce gonadal atrophy, and, starting two months later, were injected daily with 300 mug PRL, 20 mug LH or 150 mug FSH daily for 2 1/2 weeks. In animals treated with PRL the concentration of testosterone in peripheral plasma, as well as the weights of the testes and the accessory reproductive glands, were significantly greater than in the controls. Treatment with LH or FSH had no effect on any of the parameters examined. It is suggested that changes in the rate of PRL release may mediate the effects of light on testicular function in the hamster and possibly, in other species.


Evaluation of gossyplure, compared with hexalure, for monitoring pink bollworm infestations in cotton fields of Israel.

Gossyplure used to bait field traps at a dose of 50 micrograms admixed with 4-16 milligrams of an antioxidant attracted and captured male pink bollworm moths early in the cotton-growing season (early May) in Israel, whereas 20 milligrams of hexalure plus antioxidant was completely inactive under identical conditions. Although gossyplure without antioxidant was inactivated through decomposition within ten days, the mixtures remained attractive for at least two months; the antioxidant also increased moth catches 2.5 fold. Monitoring data obtained with 3000 gossyplure-baited traps, at one trap per 50 dunams, in cotton fields in the Beth-Shann region proved that capture thresholds of 5 moths/trap/night to the end of July and 8 moths/trap/night afterward were completely satisfactory for maintaining a low 2.8% average boll infestation for which only 5.8 insecticidal treatments were required during the entire growing season; results were even better in other areas of the country.


Cadmium, lead and zinc accumulation in soils near a smelter complex.

Concentrations of nitric acid extractable Cd, Pb and Zn in surface soils, predominated by partially decomposed litter, from 15 sites within 9.7 km of the lead-zinc smelter complex at Trail, British Columbia, Canada, attained 36.1, 12123, and 1394 ppm but averaged 17.8, 2607, and 571 ppm, respectively. Although underlying mineral soils contained smaller concentrations of all three metals, the relative immobility of Pb and mobility of Zn were observed. A close association between Cd and Zn concentrations was reflected by significant correlations but variation of Pb concentration in surface soils was independent from concentrations of the other metals.


Solvent extraction of cadmium from alkaline cyanide solutions with quaternary amines.

Quaternary amines are unique in their ability to extract metal cyanides from highly alkaline solutions. Cadmium cyanide extracts essentially quantitatively at both the subnanogram and macro levels. Regeneration of the amine solvent is achieved by stripping the cadmium with sodium hydroxide, sodium hypochlorite, or alkaline or acidic formaldehyde solutions. Because they readily extract free cyanide as well as anionic metab cyano complexes, the high-molecular-weight quaternary amines show considerable promise for industrial pollution abatement applications.


Chlorination in dilute aqueous systems; 2,4,6-trichlorophenol.

Chlorination of wastewaters can cause extensive transformations of organic contaminants. In the case of phenol, the reaction path is only partially understood. In an effort to elucidate this reaction, 2,4,6-trichlorophenol in dilute aqueous solution was treated with excess hypochlorous acid at pH 3.5-6. The major products were 2,6-dichloroquinone and 2,4,4,6-tetrachloro-2,5-cyclohexadienone. Additional polychlorinated species are formed in minor amounts by the addition of chlorine and/or hypochlorous acid to the aromatic ring.


Charged aerosol particles and air pollution.

Possible environmental effects and the importance of charged aerosol particles are examined. Some calculations of the evolution of the charge distribution of the atmospheric aerosol are presented. Several possible environmental effects of particle charge are noted including effects on particle coagulation, dry deposition, and deposition in the lung. Finally some problems in aerosol charging are reviewed as they pertain to various technical operations in air pollution control.


Particle sizing of arc smokes by forward light scattering.

The polarization ratio method similar to that proposed by Kirker et al. has been applied to obtain size distribution for several types of metallic smoke particulates generated by a dc electric arc. Results are presented for carbon, tungsten and aluminum arc smokes. These results are compared with the average size of the particulates measured by the two-angle ratio method of Hodkinson and are found to be consistent and within experimental error. The optical system was calibrated with standard latex spheres in both cases. It is believed that the parameters of the arc smoke particulate systems should be useful for environmental particulate research.


Placental transfer and teratology of pentachlorophenol in rats.

Pentachloro[U-14C]phenol was administered orally to Charles River CD strain pregnant rats on day 15 of gestation. Concentrations found in the placentas and fetuses up to 32 hr remained very small indicating that the amount that passes through the placental barrier is negligible. Unlabeled compound was administered on days 8, 9, 10, 11, 12, and 13 of gestation. The incidence of resorptions in the treated animals was not significantly greater than that in the controls. Although malformations were observed, the number was minimal and could have been due to the toxic effects of the compound on the maternal rat.


Investigation of the transport of metals and orthophosphate away from a sewage treatment plant outfall.

The distribution of O2, chlorophyll a, alkalinity, phosphate, Ca, Mg, Mn, Fe, Cu, Zn, Cd and Pb has been measured in the discharge plume of the large sewage treatment plant on Back River, Maryland. The concentrations of phosphate and the trace metals decrease downstream more rapidly than can be accounted for by conservative dilution. Solubility calculations suggest that saturation with respect to phosphate or oxyhydroxide phases could limit the mobility of phosphate, Fe, Mn, and Pb. For Cu, Zn, and Cd, saturation is not a feasible control mechanism, and other processes such as biofixation or adsorption are probably involved.


Use of the electroretinogram to measure the quality of vision of the fruit fly.

The electroretinogram (ERG) technique was used to measure the visual sensitivity of Caribbean Fruit flies Anastrepha suspensa (Loew), reared at 3 laboratories on 5 artificial diets. These ERGs were compared with those of flies reared from field-infested fruit.  Flies reared on artificial diets differed greatly in visual sensitivity, and those reared on fruit were at least 10 times more sensitive than certain strains. Since ERGs can be obtained 3-4 days before the Caribbean fruit fly is sexually mature, the use of visually substandard flies in behavioral studies or field releases could be avoided. The results have stimulated further studies of the effects of nutrition and handling procedures on the vision and behavior of the Caribbean fruit fly in the laboratory and the field. A simple, easy to operate, and inexpensive ERG system is being developed that can be used at any rearing facility to monitor the quality of visual receptors of the insects being produced.


Arsenate ion reduction in non-living biological materials.

The reduction of arsenate ion to arsenite ion by a variety of tissues was widely distributed, and not a property of any particular tissue. The nature of the reduction was investigated by using various chemical inducers and inhibitors and is believed to be chemical in nature.


A new mechanical disruption technique for organic enrichment of hi-vol samples.

A mechanical disrupting technique capable of extracting the organic content from a high volume fiber glass filter is described. The method is rapid, inexpensive and reproducible.


Improvement of the attractiveness of spodoptera littoralis sex pheromone, and its possible use in safety belts around cultivated areas to control the pest in Israel.

A comparison of the field attractiveness of five samples of American-synthesized and one sample of Japanese-synthesized (Z,E)-9,11-tetradecadien-1-01 acetate (9,11-TDDA), the sex pheromone of Spodoptera littoralis Boisd. and S. litura (F.), showed that traps baited with one of the American-produced samples and the Japanese product were equally effective in attracting and capturing S. littoralis males in cotton and alfalfa fields of Israel. Optimum captures were obtained for periods up to three months with 4800 mug of 9,11-TDDA plus 8 mg of an antioxidant; higher  doses were not advantageous. Paper squares were superior to two types of rubber septa and to closed polyethylene vials as dispensers for the pheromone; the polyethylene vials were least effective. In terms of numbers of moths captured, open polyethylene vials were as effective as paper dispensers when both were baited with 9-11-TDDA plus antioxidant. Admixture of (Z,E)-9,12-tetradecadien-1-o1 acetate (9,12-TDDA) with 9,11-TDDA was antagonistic at ratios of 1:20 and 1:50, but ratios from 1:500 to 1:1600 were synergistic, increasing moth catches up to 2.5-fold. Pheromone traps placed in Caesarea sand dunes located at least 3 km from the nearest host area captured larger numbers of males than traps placed in cultivated areas. It is therefore recommended that safety belts of pheromone traps, about 500 m apart, be used around cultivated areas to prevent invasion of the pest from outside and to attract males present inside such areas, thus reducing both the male population and mating through mass trapping and disruption of sex communication.


Field studies on ozone inactivation of a Gymnodinium breve toxin.

Water samples were collected from Boca Ciega Bay (St. Petersburg) during the April, 1974, red tide that occurred on the Florida west coast. The causative agent of this phenomenon was the toxic dinoflagellate, Gymnodinium breve. The toxic red tide samples were treated with ozone gas and rendered nonlethal, as measured by mouse injection.


The effects of 5,5-diphenylbarbituric acid on experimental seizures in rats: correlation between plasma and brain concentrations and anticonvulsant activity.

Diphenylbarbituric acid was administered by gavage to male albino Sprague-Dawley rats for evaluation of anticonvulsant activity and analysis of blood plasma and brain drug concentrations. A method for gas-liquid chromatographic analysis of blood plasma and brain for diphenylbarbituric acid is described. The drug was effective in the maximum electroshock test and as an antagonist to clonic seizures and death produced by pentylenetetrazol. Brain concentrations associated with 50 to 70% protection in the maximum electroshock seizure test were of the order of 2 to 6 mug/g of brain and are thus equal to values for phenobarbital in this test. Large doses of diphenylbarbituric acid (up to 2,800 mg/kg) failed to produce signs of neurotoxicity. The results of the present investigation continue to indicate that diphenylbarbituric acid exhibits promise as a potential antiepileptic agent.


Diphenylhydantoin serum levels, toxicity, and neuropsychological performance in patients with epilepsy.

The neuropsychological effects of diphenylhydantoin were assessed in 70 adults having primarily psychomotor and major motor seizures. All patients were stabilized on diphenylhydantoin alone, and assessment of this drug by gas-liquid chromatography provided a basis for dichotomizing the group into High and Low drug groups, both with and without toxic patients. In addition, patients showing signs of toxicity were contrasted with those not showing such signs. In each of these comparisons, control was maintained with respect to the variables of age, education, age at onset of disorder, and duration of disorder. Neuropsychological assessment was by means of the test battery originated by Halstead and developed by Reitan. Results showed that the High and Toxic Groups rather consistently did more poorly on all tasks than the Low and Nontoxic Groups. However, differences which were statistically significant appeared only in connection with tasks which had a heavy motor component. Possible reasons for the lack of statistically significant differences on the higher level mental tasks were presented.


The covalent structure of collagen. The chymotrypsin, trypsin and hydroxylamine peptides derived from alpha2-CB4 of calf-skin collagen.

The cyanogen-bromide-derived peptide alpha2-CB4 from calf skin collagen, consisting of 321 amino acid residues, has been fragmented in order to obtain peptides suitable for automated sequential analysis. Digestion with chymotrypsin liberated six unique peptides consisting of 12, 17, 19, 54, 63 and 156 amino acid residues. Treatment of alpha2-CB4 with hydroxylamine yielded four peptides with 24, 87, 96 and 114 residues. No unspecific cleavage by hydroxylamine was encountered. All of the trypsin-derived peptides of alpha2-CB4 were isolated and characterized by their amino acid compositions. Most of the peptides isolated were ordered along the peptide chain of alpha2-CB4. Ordering of the peptides was greatly assisted by the isolation of double peptides from the chymotrypsin, trypsin and hydroxylamine-derived peptide mixtures.


Incorporation of L-tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine as single units into rat brain tubulin.

The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit.


Stabilization of energy charge, generation of oscillations and multiple steady states in energy metabolism as a result of purely stoichiometric regulation.

A simple kinetic model of cell energy metabolism with autocatalytic reaction sequences has beepn analysed. The model accounts for the fact that part of energy produced in the form of ATP, or any other equivalent form, is utilized in "sparking" reactions to activate initial substrates. Analysis of the model shows that energy metabolism, in the absen-e of all non-stoichiometric (i.e. isosteric, cooperative, and allosteric) regulations, is capable of (a) stabilizing, to a high degree of accuracy, the relative concentration of the "charged form" of the energy-transferring cofactor (ATP); (b) alternating between two stable stationary states by means of hysteretic transitions; (c) generating self-oscillations in energy production. It is proposed that energy metabolism can be a source of very slow, in particular circadian (of about a one-day period), oscillations which may serve as the basis for temporal organization of the cell.


Mitochondrial protein synthesis in a mammalian cell-line with a temperature-sensitive leucyl-tRNA synthetase.

The temperature-sensitive Chinese hamster ovary cell mutant tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 degrees C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 degrees C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34 degrees C is not inhibied by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 degrees C is inhibited by tevenel, (b) Protein synthesis by isolated mitochondria from tsH1 cells is not significantly inhibited at 40 degrees C. (c) At 40 degrees C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [14C]leucine at 40 degrees C into mitochondrial proteins of tsH1 cells is inh-bited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsH1 cells at 40 degrees C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40 degrees C and at 34 degrees C in the presence of cycloheximide have been compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40,000 to 20,000 and a second with an apparent molecular weight range from 20,000 to 10,000.


Studies on the intracellular location of enzymes of the photosynthetic carbon-reduction cycle.

Homogenates of dark-pretreated leaves yield two particulate fractions in density gradient centrifugation: one contains chlorophyll (chloroplasts) while a second fraction contains ribulose-1, 5-bisphophate carboxylase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and catalase. Addition of a microbody-rich pellet to chloroplasts isolated from dark-pretreated plants largely enhances both oxygen evolution and CO2-fixation into organic compounds. The pathway of CO2 reduction may be part of a membrane system which, under suitable conditions, may separate from the chloroplast as a distinct cytoplasmic entity, having physical properties similar to those of microbodies.


Enzyme-bound intermediates in the biosynthesis of bacitracin.

1. Bacitracin synthetase, a three-component enzyme complex which catalyzes synthesis of the dodecapeptide bacitracin A, has been prepared from Bacillus licheniformis strains ATCC 10716, AL and SB 319. During synthesis of bacitracin, the amino acids (smaller amounts) and peptides are covalently bound to the enzyme complex. The nature of the bindings suggest that the amino acids and peptides are thioester linked. 2. The peptides, identified by thin-layer chromatography after performic acid liberation were Ile-Cys, Ile-Cys-Leu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu, Ile-Cys-Leu-Glu-Ile, Ile-Cys-Leu-Glu-Ile-Lys-Orn, Ile-Cys-Leu-Glu-Ile-Ile-Orn-Ile, Ile-Cys-L-EU-Glu-Ile-Lys-Orn-Ile-Phe, Ile-Cys-Leu-Glu-Ile-L-YS-Orn-Ile-Phe-His-Phe-His and Ile-Cys-Leu-Glu-Ile-Lys-Orn-Ile-Phe-His-Asp. 3. The labelled peptides covalently bound to bacitracin synthetase were intermediates in bacitracin synthesis. 4. Chain growth is initiated on one enzyme component (A) by the addition of isoleucine and cysteine. The sequential addition of the other amino acids proceeds in the C-terminal direction until the pentapeptide is formed. Further addition of amino acids and production of bacitracin are obtained by adding the other enzyme components (B and C) to the incubation mixture.


Characterization of the component, which controls the transformation between the kinetic forms of the b cytochromes.

1. In the presence of KCN and a saturating concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA-the active form which was reduced in the rapid phase, and bS-the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, names "y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N1,N1-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107-116] 2.  Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "y" and thus permits the observation of the biphasic phenomen, but that it is not essential for the operation of this dynamic control mechanism.


Alcohol oxidase and catalase in peroxisomes of methanol-grown Candida boidinii.

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3%. As shown by electron microscopy the peroxisomes were 0.4-0.6 mum in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria.


Studies on the role and mode of operation of the very-lysine-rich histones in eukaryote chromatin. The conformation of phi1 histones from marine invertebrate sperm.

Proton magnetic resonance, circular dichroism and infrared spectroscopy are used to investigate the secondary and tertiary structure of three very lysine-rich histones from marine invertebrate sperm. At high ionic strength both Arbacia lixula and Holothuria tubulosa histone phi 1 are observed to contain 25-30% alpha-helix, no beta-structure and to form specific folded structures. Both phi 1 proton magnetic resonance spectra have perturbed methyl resonances at chemical shifts close to those observed for calf thymus H1, suggesting analogies in tertiary structure. Mytilus edulis histone phi 1 however, shows no spectroscopic evidence of secondary and tertiary structure on salt addition.


The sex-factor-dependent exclusion of coli virus T7.

The cause of T7 exclusion by the F episome was investigated. Extracts from neither normal nor infected F+ cells contained an inhibitor of gene expression in vitro. The protein synthesizing systems prepared in vitro from these cells supported T7 early and late protein synthesis with normal efficiency. The content of translational initiation factors in F- and F+ cells, both noninfected and infected, was almost identical. The episome-dependent block of T7 gene expression was observed only in intact cells and detailed kinetics of gene expression in vivo revealed a stop of all transcription and translation at or just before 11 min after T7 infection. The mechanism of F+-dependent T7 exclusion involves both episomal and viral gene products. The data indicate that a T7-induced membrane alteration of the F+ cell membrane leads to cessation of T7 development as well as to the death of the host cell ('suicide').


Interactions between prostaglandin analogues and a receptor in bovine Corpora lutea. Correlation of dissociation constants with luteolytic potencies in hamsters.

The dissociation constants for the interactions between some prostaglandin analogues and a prostaglandin F2 receptor in bovine corpora lutea were determined. These values were compared to the antifertility potencies of these compounds in hamsters and the rates of metabolism by 15-hydro-syprostaglandin dehydrogenase. The most active analogues with regard to both affinity for the receptor and luteolytic potency were 17-phenyl-18, 19, 20-trinorprostaglandin F2alpha and 15-methylprostaglandin F2alpha. The alkyl side chain of prostaglandins could be modified considerably without altering the affinity for the receptor. In this way metabolism by 15-hydroxyprostaglandin dehydrogenase could be blocked. Some of these compounds -ad greatly increased luteolytic effects. Substitution of a phenyl group for the 3 terminal carbon units of the alkyl side chain of prostaglandins increased both the affinity for the receptor and the luteolytic activity in vivo. 7-oxa-13-prostynoic acid, an antagonist of the luteolytic effect of prostaglandin F2alpha in vivo was a weak competitive inhibitor of the interation between prostaglandin F2alpha and the receptor.


Molecular characterization of an insect genome: Chironomus thummi.

DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.


The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents.

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.


Metabolism of [4-14C] testosterone in the rat uterus in vitro.

In view of the uterine action of androgens we have investigated in vitro the metabolism of [4-14C]-testosterone in uterine tissue of ovariectomized rats. After purification of the extracts on Amberlite XAD-2 the metabolites have been isolated by gel. Five metabolites were isolated and identified during these incubation studies: 4-androstene 3,17-dione, 17beta-hydroxy-5alpha-androstan-3-one, 5 alpha-androstane-3alpha17beta-diol, 4-androstene-3 beta, 17beta-diol and 4-androstene-3alpha, 17beta-diol. Furthermore, two polar C19O3-metabolites and one isopolar to 5 alpha-androstane-3, 17-dione have also been detected. The metabolites were characterized by radioactive gas chromatogrphy, and determination of the relative specific activity in the eluates of Sephadex column chromatography. The identification of allylic alcohols was complemented by their oxidation to 4-androstene-3,17-dione. The present data show that activity of 17beta,3alpha- and 3beta-hydroxysteroid-oxidoreductase and 5alpha-ring-reductase are involved in the metabolism of testosterone in vitro in the rat uterus. The very low 5 alpha-reductase activity under the experimental conditions used in this work explains the formation of allylalcohols as the principal metabolites of testosterone in the rat uterus.


Methylation of ribosomal-precursor RNA, synthesized in vitro, by isolated rat-liver nucleoli.

Nucleoli isolated from rat liver were incubated for synthesis of RNA in vitro in the presence or absence of S-adenosyl [3H] methionine. The results obtained indicate that neither the rate of RNA synthesis not the processing of pre-ribosomal RNA was changed if methylation was allowed to take place. The methylation process acts on the RNA most recently synthesized, rather than on the bulk of the RNA already present in the nucleoli. The reaction seems to occur faithfully both quantitatively and qualitatively. It is calculated that 104 mol methyl groups were incorporated per mol of newly synthesized 45-S RNA. Methylation of the ribose rather than the bases predominated. The pattern of alkali-stable oligonucleotides of RNA methylated in vitro was analyzed and found to correspond closely to that of ribosomal RNA labelled in vivo.


Nucleotide sequence of 5-S RNA from Bacillus licheniformis.

The complete nucleotide sequence of 5-S RNA from Bacillus licheniformis was determined by analysis of complete and partial digests obtained with either T1 or pancreatic ribonuclease. The molecule was found to have a length of 116 nucleotides and may possess a minor sequence heterogeneity. There is a large degree of homology between the sequence of B. licheniformis 5-S RNA and those published for 5-S RNA from B. megatherium and B. stearothermophilus. The difference between the three 5-S RNA species are limited mainly to the two terminal and one internal sequence. B. licheniformis 5-S RNA contains the sequence U95-G-A-G-A-G100, which in B. subtilis has been implicated in the processing of precursor 5-S RNA. Possible models for the secondary structure of prokaryotic 5-S RNA are discussed on the basis of the results of limited digestion of B. licheniformis 5-S RNA by ribonuclease T1.


Chemical synthesis of partially and fully phosphorylated protamines.

Chromatographically purified components Z and YI of clupeine from herring have been phosphorylated by a chemical method. To enhance solubility in trimethylphosphate, the protamine capronate salts were used and phosphorylation was performed with POCl3. Both serine and threonine residues were phosphorylated; however, besides monophosphate esters considerable amounts of polyphosphate esters were obtained. Its nature was identified by 31P nuclear magnetic resonance spectroscopy and by chemical methods, such as comparison of the hydrolysis rate with that of ADP and phoshocreatine, respectively. The pyrophosphate bonds were cleaved by controlled hydrolysis with HCl. The phosphorylated protamine species were purified by gel filtration and chromatography on a Sephadex CM column. Fully phosphorylated clupeine Z (3 serine residues and 3 phosphates) and YI (3 serine, 2 threonine residues and 5 phosphates) were obtained as well as partially phosphorylated fractions; in the case of clupeine Z the resolution into mono, doubly and triply (fully) phosphorylated fractions was excellent. The overall yield of phosphorylated clupeine was better than 50%.


New useful reagents for peptide synthesis. Insoluble active esters of polystyrene-bound 1-hydroxybenzotriazole.

Insoluble 1-hydroxbenzotriazole-bound polystyrene was prepared through a series of chemical modifications of commercially available polystyrene. Reaction of 3-nitro-4-chlorobenzyl alcohol or of 3-nitro-4-chlorobenzyl bromide with polystyrene in the presence of aluminium trichloride yielded (3-nitro-4-chloro)benzylated polystyrene. Upon reaction with hydrazine it was converted to (3-nitro-4-hydrazine) benzylated polystyrene which was cyclized, by acidolysis, to yield 1-hydroxybenzotriazole-bound polystyrene. This was coupled, using N, N' -dicyclohexylcarbodiimide as the coupling agent, to many N-blocked amino acid derivatives, yielding polymeric polystyrene-bound active esters. Such derivatives are highly reactive and their efficacy in the synthesis of several peptides, including that of the tetrapeptide Boc-L-Leu-L-leu-L-Val-0bzl-L-Tyr-0Bzl and of thyrotropin-releasing hormone was demonstrated.


Investigation of the symmetry of oligomeric enzymes with bifunctional reagents.

The symmetry of proteins composed of identical polypeptide chains has been investigated by means of cross-linking with bifunctional reagents and subsequent sodium dodecylsulfate-polyacrylamide gel electrophoresis. The majority of the investigations were performed with diimidates of different chain lengths (C3-C12), which react exclusively with amino groups. Aldolase, catalase, fumarase, pyruvate kinase, tetrameric proteins with identical polypeptide chains, reveal a D2 symmetry, i.e. they appear to be composed of two pairs of polypeptide chains. The validity of this conclusion is demonstrated with lactate dehydrogenase. This enzyme, shown by X-ray analysis to have a D2 symmetry, yields after cross-linking and subsequent polyacrylamide electrophoresis the band pattern expected for a protein with this quaternary structure and similar to the pattern obtained with the above enzymes. 2. The influence of the experimental conditions on the cross-linking reaction has been investigated. The selectivity of the bifunctional reagent for the different contact domains within the quaternary structure of a protein depends on the reaction time, the chain length and on the concentration of the reagent. In general the D2 symmetry becomes more obvious with increasing chain length and with increasing concentration of the diimidate. Diethylpyrocarbonate showed very little selectivity.


The pyruvate-dehydrogenase complex from Azotobacter vinelandii.

The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.


Hydrogen exchane at the beta-carbon of amino acids during transamination.

The hydrogen exchange at the Beta-carbon of L-alanine, L-glutamate and L-asparate with water has been examined during transamination catalyzed by glutamic-oxaloacetic transaminase and by glutamic-pyruvic transaminase. A significant hydrogen exchange at the Beta-carbon has been demonstrated during incubation of L-[3-3H]alanine + glutamic-pyruvic transaminase, L-[3-3H]alanine + alpha-oxo-glutarate + glutamic-pyruvic transaminase, L-[3-3H]glutamate + glutamic-oxaloacetic transaminase, L-[3-3H]glutamate + oxaloacetate +glutamic-oxaloacetic transaminase, and L-[3-3H]glutamate + pyruvate + glutamic-pyruvic transaminase as shown by the appearance of 3H2O. No hydrogen exchange at the Beta-carbon of L-glutamate occurred during incubation of L-[3-3H]-glutamate with glutamic-pyruvic transaminase alone. The hydrogen exchaned at the Beta-carbon of L-glutamate coincides with transamination as demonstrated by nuclear magnetic resonance studies of 2H2O-L-glutamate exchange during transamination by glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase. No hydrogen exchange at the Beta-carbon occurred during transamination of L-aspartate by glutamic-oxaloacetic transaminase as shown by nuclear magnetic resonance spectroscopy and confirmed by nuclear magnetic resonance simulation studies. The results are discussed with special reference to the different equilibria between the pyridoxal form and the pyridoxamine form of glutamic-oxaloacetic transaminase and of glutamic-pyruvic transaminase.


Pt(CN)2-4 and Au(CN)-2: potential general probes for anion-binding sites of proteins. 35Cl and 81Br nuclear-magnetic-resonance studies.

Nuclear magnetic quadrupole relaxation appears to be a general method for studying the binding of anions to proteins. This is shown by the increase in transverse quadrupole relaxation rate of 35Cl- and 81Br- in the presence of horse liver alcohol dehydrogenase, lysozyme, trypsin, alpha-chymotrypsin, human carbonic anhydrase, fructose-1,6-bisphosphate aldolase and human serum albumin. Of the many possible binding sites at the surface of a protein (e.g. positively charged amino acid side-chains) only a few account for the main part of the relaxation enhancement. This is shown by the decrease in 35Cl- and 81Br- relaxation rate on addition of functional ligands. Large, kinetically inert, complex anions like Pt(CN)2-4 and Au(CN)-2 are found to act as strong competitors towards halogen ions for the high-affinity anion binding sites of a number of proteins. Titrations with complex anions following the 35Cl- or 81Br- relaxation rates are found to be helpful in attempts to elucidate binding mechanisms. Especially, the complex anions may be useful probes for the discrimination between general and metallic anion binding sites in proteins and they also permit correlation of information from X-ray investigations of crystals with that from physical measurements in solution. From the change in halide ion quadrupole relaxation rate on addition of strongly binding ligands the quadrupole coupling constants of the high affinity Cl- and Br- binding sites are estimated using certain assumptions. It is found that for several proteins, comprising the metal-free proteins but also alcohol dehydrogenase and Escherichia coli alkaline phosphatase, the 35Cl quadrupole coupling constants have approximately the same values. For some other metallo-proteins like carbonic anhydrase and a zinc - serum-albumin complex considerably greater quadrupole coupling constants were obtained. The estimated quadrupole coupling constants are used as a basis for a discussion of the interactions involved in anion-protein interactions.


Properties of immobilized flavodoxin from Peptostreptococcus elsdenii. An affinity ligand for the purification of riboflavin 5'-phosphate (FMN) and its analogues.

The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr-activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin-free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively. Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (+/- 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N-10 side-chain of 5 carbon atoms and a C-5' phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12-15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months.


Reversible, covalent immobilization of enzymes by thiol-disulphide interchange.

1. alpha-Amylase and alpha-chymotrypsin have been immobilized by covalent attachment to mercaptohydroxypropyl ether agarose gel. The technique involves two steps: (a) thiolation of the enzymes by methyl 3-mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2'-dipyridyl disulphide. 2. The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities. 3. Immobilized alpha-amylase was used in a packed-bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive uncoupling of the inactive protein and attachment of a new portion of thiolated alpha-amylase.


A urinary pentasaccharide in bovine mannosidosis.

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: alpha-D-Manp-(1 leads to 6)-beta-D-Manp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis.


Effects of external potassium on potassium efflux and accumulation by rat adipocytes.

Effects of external K+ concentration, [K+]o, on K+ transport rate and on relative K+, an index of intracellular K+ concentration, were studied in adipocytes isolated from rat epididymal fat pads. 2. Relative K+ changed upon a step change of [K+]o from normal, but with continued exposure to the second [K+]o  the relative K+ returned to or approached the initial level. 3. Reversal of the initial change in relative K+ was accelerated by restoration of a normal [K+]o. 4. 42K efflux from cells exposed to a single [K+]o was described by a single rate constant for a wash-out period of up to four half-times. 5. The rate constant was bi-stable with respect to [K+]o, with the critical [K+]o between 3-6 and 4-8 mM. 6. Rate constants were independent of relative K+ and uncorrelated with net fluxes. 7. It is suggested that [K+]o exerts its effects only at the outer surface of the adipocyte membrane.


Avian febrile response.

The febrile response of the pigeon (Columba livia) was characterized for comparison with our present day understnading of mammalian fever. 2. When injected with live Pasteurella multocida the birds became febrile and died. 3. Varying doses of dead bacteria produced a complex dose-dependent febrile response. 4. Antipyretics effectively attenuated the febrile response to dead bacteria. 5. The similarities of reptilian, avian and mammalian fever suggest a common origin and perhaps a similar adaptive role of fever in increasing host survival during bacterial infection.


Absorption of amino acids and peptides from a complex mixture in the isolated small intestine of the rat.

Amino acid and peptide absorption from a pancreatic digest of casein at low concentration by an isolated preparation of perfused rat small intestine has been measured. 2. The rate of absorption of each amino acid (free or peptide-bound) is closely proportional to its concentration in the perfusate; this implies a constant Vmax/Km ration for all amino acids in the mixture. 3. There is a high correlation between the compositions of luminal perfusate and secretion into the tissue fluid (apart from the content of glutamic and aspartic acids and alanine). 4. The concentrations of each free amino acid are, on average, 9 times as great in secretion as in lumen; the total peptide-N concentration in secretion is approximately 4 times that in the lumen. 5. The rate of absorption of each free amino acid is highly negatively dependent on the rate of absorption of that amino acid in peptide-bound form, in addition to being positively dependent on the perfusate concentration of free amino acid. 6. While peptide-bound proline appears to be well absorbed, free proline liberated by hydrolysis appears to pass back into the lumen as well as into the tissue fluid. Substantial back flux of hydrolysis products may occur for all amino acids. 7. About one-third of the amino acids appearing in the secretion on the serosal surface are peptide-bound. 8. The rate of absorption of peptides appears to determine the rate of their hydrolysis which probably occurs mainly after entry into the mucosal cells.


Axoplasm architecture and physical properties as seen in the Myxicola giant axon.

A technique is described for extracting axoplasm from the giant axon of a marine worm, Myxicola infundibulum. The operation can be completed in 10 sec. 2. Axoplasm is pulled from the axon of a living worm as a long, clear cylinder, up to 35 cm long and 70 mg wet weight. The worm regenerates a new giant axon in about 4 months. 3. Myxicola axoplasm is a gel, 87% water, held together by protein neurofilaments. It contains small amounts of mitochondria and vesicles, but no detectable microtubules. 4. The internal structure of the gel is superficially similar to that of yarn. Closer inspection with light and electron microscopy, and X-ray diffraction, show it to be organized in a hierarchy of helical forms. Squid giant axons have a similar structure. 5. Initial estimates of the bulk physical properties of extracted Myxicola axoplasm give: breaking strength, 1400 g/cm2; specific gravity, 1-05 g/cm3; birefringence, 1-6 X 10(-4); index of refraction, 1-351; resistivity, 57 omega cm. These average values are shown to be compatible with the observed structure and composition. 6. Despite its mechanical strength, the axoplasm gel is so hydrated that Na+, K+ and homarine diffuse through it at rates approaching those in free solution. Fewer than about 5% of each of these ions are tightly bound to the gel. 7. It is argued that (a) the structure and physical properties of Myxicola axoplasm are representative of those in other axons, (b) the compound helix architecture results from twist of parallel, cross-linked fibrous proteins, and (c) this sturcture serves as a flexible internal skeleton for nerve cell processes.


Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate.

Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.


Biochemistry of development in insects. Stereospecific incorporation of fatty acids into triacylglycerols.

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.


Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.


Tubular polymers derived from Helix pomatia beta-hemocyanin.

Upon trypsinolysis Helix pomatia beta-hemocyanin forms long tubular structures, which appear to be linear polymers of hemocyanin molecules from which the collar structure has been removed. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that only few peptide bonds are hydrolyzed by trypsin. The structure of the polymers has been investigated by electron microscopy, combined with optical diffraction. Preliminary X-ray diffraction data are presented. Functional properties of the polymers are similar to those of the native protein. Both show a calciumion-dependent co-operativity of oxygen binding and a Bohr effect. The results suggest that the collar of a hemocyanin molecule has no special function in the process of (co-operative) oxygen binding, different from that of the wall of the molecule.


Steroid-induced early protein synthesis in rat uterus and prostate.

An early 'induced protein', after exposure of the rat uterus to estradiol, is detected among the soluble proteins with a double-labelling technique and electrophoretic fractionation. Efforts have been directed to establish the subcellular distribution of the induced protein, since such a protein, observable 1 h after hormone administration, may play an important role in the subsequent amplified responses, especially in terms of RNA synthesis. Moreover such an early discrete induced protein was sought in a comparable system responding to another hormone, namely prostate and seminal vesicles under androgens. The induced protein was not found in uterine nuclei of 21-day-old rats after 1 h of estradiol action in vivo and 1 h of tissue incubation with labelled leucine. This negative result summarizes a search among different nuclear protein fractions using various procedures; nor was induced protein observed in mitochondrial and microsomal pellets. Contrary to these negative findings, slight changes of histone labelling were observed under the experimental conditions used to demonstrate induced protein. In addition histone acetylation was increased after 1 h of estradiol action in vivo and 15 min tissue labelling in vitro with radioactive acetate. Furthermore, an increase in total protein synthesis between 0 and 2 h after estradiol action was observed, the relative increase of incorporation of radioactive leucine into protein of estradiol-treated vs non-stimulated uteri being corrected for variations of the acid-soluble radioactive leucine pool. Attempts to obtain an early and discrete induced protein with androgens in prostate and seminal vesicles of immature or castrated rats after different times of exposure to testosterone, androstanolone and estradiol have been unsuccessful. The contribution of both negative and positive findings in steroid-induced early protein synthesis is discussed in the context of the current knowledge of hormone action.


Compartmentation of acetyl-coA in rat-liver mitochondria.

The ratio of the specific radioactivities of 3-hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1-14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5-3.7 were observed. In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetyl-CoA produced from various precursors was tested. It was found that acetyl-CoA produced from L-acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation. These results demonstrate a compartmentation of acetyl-CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3-hydroxybutyrate:citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl-CoA pools.


Studies of the distribution of proteins bound to CNBr-activated Sepharose 6B at the electron-microscopic level.

Proteins were covalently attached to Sepharose by the CNBr method. Their distribution across the carrier beads was studied at the electron microscopic level. The approach has been to ferritinstain and to section the gel beads. Ferritin was either coupled directly to the polysaccharide backbone of the carrier or conjugated with pure rabbit anti-aminopeptidase in order to visualize covalently bound leucine aminopeptidase by the immunferritin technique. The results corroborate earlier fluorescence microscopic findings of a uniform protein distribution, provided that a number of conditions are fulfilled.


Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.


Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.


Decreased dissociation of the 3-methylcrotonyl-CoA carboxylase complex from Achromobacter in the presence of 3-methylcrotonyl-CoA. A possible regulatory mechanism for the intracellular degradation of the enzyme.

By inactivation of different concentrations of 3-methylcrotonyl-CoA carboxylase from Achromobacter IVS with a fixed concentration of iodoacetamide, it was demonstrated that the degree of dissociation of the complex is considerably lower in the presence of 3-methylcrotonyl-CoA. ATP did not produce this effect. This property could serve to regulate the intracellular degradation of the enzyme, if the dissociated subunits were attacked preferentially.


Limited trypsinolysis of beta-haemocyanin of Helix pomatia. Characterization of the fragments and heterogeneity of the copper groups by circular dichroism.

A limited trypsinolysis of the tenths of beta-haemocyanin of Helix pomatia was performed at pH 8.2. The absorbance at 346 nm remained constant, indicating a preservation of the oxygen-binding sites. The five tryptic fragments were separated by chromatography on Sephadex G-100 and on DEAE-cellulose. They contained 2 Cu per 50000 daltons and showed different mobilities in agar electrophoresis. The molecular weights indicated that one fragment was constituted of three functional domains of about 50000 daltons, that two fragments were constituted of two domains, and two others of one domain. Twentieths of beta-haemocyanin seemed thus to be made up of 9 domains. The circular dichroic spectra of the fragments indicated the presence of two classes of copper groups according to their positive maximum at 455 or at 500 nm. The circular dichroic spectra also showed that no fragment could have originated from a larger one, confirming the presence of nine domains in the twentieths.


Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.


Biochemical research on oogenesis. Nucleotide sequence of initiator tRNA from oocytes and from somatic cells of Xenopus laevis.

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs. Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.


Translation of honeybee promelittin messenger RNA. Formation of a larger product in a mammalian cell-free system.

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin.


Thermal and solvent perturbation as probes of conformational heterogeneity of haptoglobin 1-1.

The shifts which occur in the ultraviolet spectra of haptoglobin 1-1 (Hp) and a conformational isomer with lower affinity for hemoglobin (Hp), in response to temperature and solvent changes, have been measured by difference spectra. The thermal difference spectra provide evidence for a different participation of tryptophyl residues in the conformational stability of Hp and Hp. Solvent perturbations have allowed an estimation to be made of the number of surface chromophores in the native Hp and Hp. These results are compatible with the existence of two Hp conformational isomers with different free energy, separated by a high-potential barrier.


Dihydrofolate reductase from bovine liver. Enzymatic and structural properties.

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities toward dihydrofolate (26.1-27.0 U/mg) and folate (1.3-2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully acitve enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.


Effect of nucleic-acid-binding compounds on the hydrolytic activity of various ribonucleases.

Studies were conducted on the stimulatory effect that various nucleic-acid-binding compounds have on the hydrolysis of RNA and polyribonucleotides by pancreatic ribonuclease A and by other ribonucleases. The stimulatory activity of chloroquine on tRNA hydrolysis by pancreatic ribonuclease was due to the formation of oligonucleotides of a wide range of sizes and was not due to the formation of very short ( n greater than 5) oligonucleotide fragments of tRNA. The dextrorotatory and levorotatory isomers of chloroquine did not differ in their ability to stimulate the hydrolysis of tRNA by pancreatic ribonuclease A. In addition to chloroquine and primaquine, other nucleic-acid-binding compounds (e.g., quinacrine, lucanthone, and proflavin) stimulated the hydrolysis of tRNA by pancreatic ribonuclease A. Chloroquine did not alter the rate of hydrolysis by pancreatic ribonuclease A of low-molecular-weight substrates (cytidine cyclic 2':o'-monophosphate, uridine cyclic 2':3'-monophosphate, cytidylyl-adenosine, or uridylyl-uridine). Furthermore, chloroquine and primaquine did not affect the hydrolysis of poly(A) by high concentrations of pancreatic ribonuclease A. In studies on the hydrolysis of tRNA by other endoribonucleases, several of the nucleic-acid-binding compounds (e.g., quinacrine and ethidium) exhibited appreciable inhibition of both ribonuclease N1 and ribonuclease T1. None of the compounds tested stimulated the activity of ribonuclease T1, and only chloroquine, and perhaps lucanthone, stimulated the hydrolysis of tRNA by ribonuclease N1.


DNA synthesis in circulating erythroblasts of anemic duck. Isolation and properties of nuclear and cytoplasmic-nonmitochondrial DNA.

In the circulating blood of anemic ducks, 5% of all erythroid cells synthesize DNA. Immature erythroblasts, at all stages of differentiation, synthesize DNA although to a varying degree, while reticulocytes and erythrocytes do not. In the erythroid cell population labeled in vitro 2 h with 32Pi, half of the labeled DNA sediments as small-molecular-weight molecules, suggesting that these molecules fail to integrate into the high-molecular-weight components. Labeled DNA is found in the cytoplasmic postmitochondrial fractions and it is in a form of deoxyribonucleoproteins which cosediment with ribosomes as well as subribosomal particles in sucrose gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosediment with ribosomes as well as subribosomal particles in sucorse gradients. However, fixation with HCHO and centrifugation to equilibrium in CsCl gradient of these particles shows that the deoxyribonucleoprotein bands at the density different than the ribosomes and, thus, not physically linked to them. In EDTA-dissociated ribosomes, the deoxyribonucleoprotein particles cosdeiment with ribosomal subunits in such a way that the larger the particle, the larger the molecular weight of the DNA cosedimenting with it. The specific radioactivity of the cytoplasmic ribosome-derived and postribosomal-particle-derived DNAs and the small molecular-weight nuclear DNA is similar and 10-20-fold higher than that of the bulk nuclear DNA. The former three DNA species sediment between 4-14 S. It is concluded that the cytoplasmic nonmitochondrial DNA species are of the nuclear origin. Less than 0.5% of the total cellular nonmitochondrial DNA can be purified from the nucleus and the cytoplasm as fast-labeled small-molecular-weight components. All of the cellular nonmitochondrial DNA species band at the same mean buoyand density in Cs2SO4/urea gradients. All behave as native structures in hydroxyapatite and contain less than 5% of their length as single-stranded regions.


On the control of arginine metabolism in chicken kidney and liver.

Arginases have been found to be located on the external side of the inner mitochondrial membrane of chicken kidney and liver. Transamidinase has been detected within the liver mitochondrial matrix space. Arginases and transamidinase act upon two different intracellular arginine pools. Penetration of arginine into the matrix space occurs only in respring mitochondria and in the presence of anions such as acetate and phosphate; D-arginine, L-ornithine, D-'ornithine and L-lysine penetrate with the same modalities. L-Histidine penetrates only kidney mitochondria. Because of transamidinase compartmentation, the rate of creatine synthesis is influenced by the rate of penetration of arginine into the mitochondria.


Properties of the ribose-ring-opened adenine nucleotide, 2,2'(1-(9-adenyl)-1'-(tri-,diphosphoryl-oxymethyl))-dihydroxydiethylether in mitochondrial adenine-nucleotide translocation.

(Adenine-14C) or (gamma-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl]-dihydroxydiethylether (rroANP) was obtained from ANP by cleavage of the C-2--C-3' bond by sodium periodate oxidation and subsequent borohydride reduction. Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki = 9.3 X 10(-5) M. Experimental rroANP confirmed that an intact adenine base and three anio.nic charges of the phosphate chain are essential for the recognition between ANP-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system.